阅读说明：本技术 核酸修饰和鉴定方法 (Nucleic acid modification and identification method ) 是由 S.L.埃姆瑞斯 B.赖希霍尔夫 V.A.赫索格 J.朱伯 M.穆哈尔 于 2018-04-13 设计创作，主要内容包括：本发明提供了鉴定多核酸(PNA)的方法,其包括以下的步骤：提供PNA；通过添加或除去氢键键合配偶体来修饰PNA的一个或多个核碱基,从而改变一个或多个核碱基的碱基配对能力；将互补核酸与PNA进行碱基配对,包括与至少一个经修饰的核碱基进行碱基配对；至少在与至少一个经修饰的核碱基互补的位置处鉴定互补核酸的序列。(The present invention provides the methods of identification polynucleotide (PNA) comprising following step: providing PNA；One or more nucleobases of PNA are modified by adding or removing hydrogen bonding gametophyte, to change the base-pairing abilities of one or more nucleobases；Complementary nucleic acid and PNA are subjected to base pairing, including carry out base pairing at least one modified nucleobase；The sequence of complementary nucleic acid is identified at least at the position complementary at least one modified nucleobase.)

1. the method for identifying polynucleotide (PNA) comprising following step: providing PNA；Matched by adding or removing hydrogen bonding Even body modifies one or more nucleobases of the PNA, to change the base pairing energy of one or more of nucleobases Power；Complementary nucleic acid and the PNA are subjected to base pairing, including carry out base pairing at least one modified nucleobase； The sequence of the complementary nucleic acid is at least identified at the position complementary at least one modified nucleobase.

2. the method for claim 1 wherein the base pairing behaviors that the modification causes to change, thus with A, T/U, C and G is selected from Natural nucleobases compare, change A and T/U between and the preferential base pairing between C and G.

3. method according to claim 1 or 2, wherein the step of modification by the inclusion of the nucleobase modified through mercaptan into Row.

4. according to the method in claim 3, further comprising will be described with the alkylating agent comprising the hydrogen bonding gametophyte The alkylation of mercaptan nucleobase.

5. according to the method in claim 3, further comprising aoxidizing the mercaptan nucleobase.

6. method according to claim 1 or 2, wherein the modification includes with the alkylating agent comprising the hydrogen bonding gametophyte In 4 upper alkylations of uridine.

7. method as claimed in one of claims 1-6, wherein the PNA includes one or more 4-thiourdines or 6- sulphur For guanosine.

8. according to claim 1 to any one of 7 method, wherein the PNA changes the base-pairing abilities having It is synthesized in the cell of modification.

9. according to claim 1 to any one of 8 method, wherein by the biosynthesis in cell by modified core alkali Base, the nucleobase preferably through mercaptan modification mix in the PNA, and preferably wherein modifying one or more nucleobases includes pair The modified nucleobase connection removes hydrogen bonding gametophyte.

10. according to claim 1 to any one of 9 method, wherein the base pairing at least one modified nucleobase Cause to be different from the base pairing with the nucleobase not yet modified, wherein the core with the base pairing of another nucleotide Base is identical in other respects.

11. according to claim 1 to any one of 10 method, wherein the PNA includes RNA or DNA, or by RNA or DNA group At.

12. according to claim 1 to any one of 11 method, wherein for be selected from A, G, C, U or T every kind of ucleotides Type, the modified PNA include natural nucleotides more more than modified nucleotide.

13. method according to any one of claims 1 to 12, wherein the PNA include 1,2,3,4,5,6,7,8,9,10 or More and up to 30 modified nucleotide.

14. according to claim 1 to any one of 13 method, be included in cell wherein providing PNA and express the PNA；Institute The method of stating further comprises separating the PNA from the cell；The one of the PNA is modified in the cell and/or after separation A or multiple nucleobases；Wherein one or more cores are added or are removed together in the modification in the cell or after separation or both The hydrogen bonding gametophyte of base, to change the base-pairing abilities of one or more of nucleobases.

15. according to claim 1 to any one of 14 method, wherein being cultivated at least two cultures or growth phase or raw Long one or more cells, one of culture or growth phase include by the PNA of modified nucleotide incorporation biosynthesis In, the PNA is modified by adding or removing hydrogen bonding gametophyte, and another culture or growth phase shortage are such By in the PNA of the modified nucleotide incorporation biosynthesis, or wherein by modified nucleotide with another In culture or growth phase in the PNA of different concentration incorporation biosynthesis；Or in which the method includes by modified core In the PNA of the biosynthesis of the different cells of thuja acid incorporation at least two or in the cell of at least two different groups, wherein preferably The incorporation of more described two difference cells or the cell of two different groups.

16. method according to claim 15, wherein collecting described two cultures or growth phase or at least from the cell The PNA of the biosynthesis of two different cells or the cell of at least two different groups, preferably also mixes, particularly preferably according to institute The origin of cell for stating PNA marks the PNA, and it includes by turning that complementary nucleic acid and the PNA, which are wherein carried out base pairing, Record, preferably reverse transcription generate complementary polynucleotide chain, preferably DNA chain.

17. the method for claim 16 further comprises determining chain sequence described in the sequence of the complementary polynucleotide chain and comparison Column, wherein can by identified compared with the complementary nucleic acid being not decorated by by add or remove hydrogen bonding gametophyte into The complementary nucleic acid of change caused by capable modification.

18. according to claim 1 to any one of 17 method comprising compare at least two cells or in cell at least In two different growth phases at least at the position complementary at least one modified nucleobase the complementary nucleic acid Identified sequence, wherein at least two cell or growth phase at least two cell or the growth phase it Between have differential gene expression, preferably wherein differential gene expression is drawn by the inhibition or stimulation of at least one gene in cell It rises.

19. the kit for the method for carrying out any one of claims 1 to 18, it includes the nucleobase modified through mercaptan and It is suitable at the thiol group making the alkylated alkylating agent of nucleobase modified through mercaptan, wherein the alkylating agent packet Hydrogen bonding donor or receptor are included, preferably wherein the alkylating agent is iodoacetamide.

20. the kit of claim 19, further includes primer, nucleotide, reverse transcriptase selected from A, G, C and T or its Combination, preferably all these components.

Background of invention

Nucleotide analog, such as 4-thiourdine (s⁴) and 6- Thioguanosine (s U⁶G the newborn RNA of incorporation can) be easy In, such as pass through natural enzyme (Tani et al., Genome Res.22,947-956 (2012)).Popular analog includes 5- Broxuridine (5BrU), 5- acetenyl uridine (5-EU), 6- Thioguanosine (s⁶) and 4-thiourdine (s G⁴U), they are easy thin Born of the same parents mix and are further respectively that antibody test, cycloaddition reaction and mercaptan specific reaction and affinity provide unique object Physical chemistry characteristic (Eidinoff et al., Science.129,1550-1551 (1959)；Jao et al. PNAS 105,15779- 15784(2008)；Melvin et al. Eur.J.Biochem.92,373-379 (1978)；Woodford et al. Anal.Biochem.171,166–172(1988)；Et al. RNA 14,1959-1972 (2008)；Rabani et al. Nat Biotechnol.29,436–442(2011)).4-thiourdine (s⁴It U is) that rna expression is dynamic (dynamical) most makes extensively for research Nucleotide analog.It is similar to other nucleotide, s⁴U is absorbed rapidly by cell without electroporation or fat transfection.Thin In born of the same parents, the s of phosphorylation is generated by the phosphorylation of cell uridine kinase⁴The accumulation pond of U, the s of the phosphorylation⁴U is effectively mixed In the newly synthesized RNA in large quantities of cell types including fly, mouse and people's cell (2008, ibid).In addition, fly With in Mice Body transcript cell type specificity label can by with toxoplasma (Toxoplasma gondii) uracil The realization of 4- thiouracil, the uracil phosphorus is applied in combination in the cell type specificity expression of phosphoribosyl transferase (UPRT) The N1 nitrogen of ribose -5- phosphoric acid and uracil (or 4- thiouracil) is coupled to generate in incorporation RNA by sour ribosyltransferase (4- thio -) uridine monophosphate (Cleary et al. Nat Biotechnol.23,232-237 (2005)).It is thio using 4- at present Uridine (s⁴U) dynamic (dynamical) scheme occurs, processes and has enough to meet the need for metabolism RNA label using via s to characterize RNA biology intracellular⁴In U The reversible biotinylated biochemistry separation of thiol group is [such as via N- [6- (biotin amido group) hexyl] -3 '-(2 ' - Pyridyl group two is thio) propionamide (HPDP- biotin) or biotin coupling methanethiosulfonate (MTS- biotin)] (Cleary et al., 2005, ibid).However, basic scheme is time-consuming as any biochemistry separation method, and And due to biotinylation efficiency (especially when being applied to short rna type) and miss the target reactivity (Duffy et al., Mol Cell.59,858–866(2015)；Neymotin et al., RNA 20:1645-1652 (2014)) limitation, typically encounter low The problem of signal-to-noise ratio.

WO 2006/125808A1 describe it is a kind of analysis containing Thiolation RNA the RNA from the beginning transcribed based on micro- battle array The method of column.

WO 2004/101825A1 and WO 2016/154040A2 are related to the biosynthetic labelling and isolated method of RNA.

Miller et al., Nature Methods 6 (6), 2009:439-441, which are described, passes through Drosophila melanogaster Label of the 4- thiouracil food source to RNA in (Drosophila melanogaster).

Schwalb et al., Science 352 (6290), 2016:1225-1228, which are related to one kind, can estimate that total mRNA is closed At the transient transcription object group sequencing approach with degradation.

Hartmann et al., Handbook of RNA Biochemistry volume 2,2014,8.3.3 chapter, 164- Page 166 are related to by modifying 4-thiourdine residue to the RNA modified through 4-thiourdine with iodoacetamide or sulfur-based compound Synthesis after mark.

Testa et al., Biochemistry, 38 (50), 1999:16655-16662 is disclosed to be changed compared with uracil Thiouracil (s²U and s⁴U base pairing intensity).

Hara et al., Biochemical and Biophysical Research Communications 38 (2), 1970:305-311 discloses the 4-thiourdine specificity spin labeling of tRNA.

Fuchs et al., Genome Biology 15 (5), 2014:1465-6906 are related to by determining the 4- sulphur on RNA Full-length genome transcriptional elongation rate is determined for uridine label.This method needs biotinylation and the purifying of the RNA of such label.

In addition, the RNA of label is only capable of analyzing in separation, i.e., not in the back of total serum IgE in reversible biotinylation strategy It is analyzed under scape.Therefore, the dynamic (dynamical) precise measurement of RNA intracellular is needed to analyze three at every point of time by high-flux sequence RNA subset (RNA, total serum IgE and the unlabelled RNA of label), to make that these methods become expensive and downstream analysis becomes not It corresponds to reality.

Therefore, the purpose of the present invention is to simplify the method for detecting modified nucleic acid, being preferably simplified to allows to automate The degree of detection.

Summary of the invention

The present invention is based on nucleotide analog derivatization chemistry, can detect polynucleotides with single nucleotide resolution rate (PNA) modification in type.The inventive process provides expansible, height quantitative, the cost-effective and time is effective For PNA modification quick and full transcript group analysis method.

In a first aspect, the present invention provides the methods of identification polynucleotide (PNA) comprising following step: providing PNA；One or more nucleobases of the PNA are modified by adding or removing hydrogen bonding gametophyte, thus described in changing The base-pairing abilities of one or more nucleobases；By complementary nucleic acid and the PNA base pairing, including at least one through repairing The nucleobase of decorations carries out base pairing；The identification complementation at least at the position complementary at least one modified nucleobase The sequence of nucleic acid.

In preferred embodiments, PNA is synthesized in cell, has especially had modification, its own changes base Pairing ability can be modified further to change base-pairing abilities.Therefore, the present invention can also be defined as identification polynucleotide (PNA) PNA method comprising following steps: is expressed in cell；PNA is isolated from cell；In cell and/or divide One or more nucleobases from rear modification PNA；Wherein the modification in cell or after separation or both is added or is removed together The hydrogen bonding gametophyte of one or more nucleobases, to change the base-pairing abilities of one or more nucleobases；It will be mutual It mends nucleic acid and PNA carries out base pairing, including carry out base pairing at least one modified nucleobase；Identification at least with The sequence of complementary nucleic acid at the position of at least one modified nucleobase complementation.

Invention further provides the kits for carrying out the method for the present invention, especially the core comprising modifying through mercaptan Base and being suitable at the thiol group makes the kit of the alkylated alkylating agent of nucleobase through mercaptan modification, Described in alkylating agent include hydrogen bonding bonding donor or receptor.

It has been described together all embodiments of the invention, and all preferred embodiment party in the following detailed description Case all refers to all embodiments, aspect, method and kit.Such as.Kit or its component method for use in the present invention Or it is suitable for the invention method.Any component used in the method for description can be provided in kit.For given side Method step, about the combination of reagent constituents or its applicability or reagent constituents, the method for the present invention preferably and detailed description It is same to interpret.Unless otherwise indicated, all embodiments can be combined with each other.

Detailed description of the invention

The present invention relates to a kind of methods, wherein modification polynucleotide (being abbreviated as PNA) (is also referred to as passed through with the PNA for creating synthesis The PNA of modification).The presence that PNA is synthesized in PNA sample can be found in the PNA of the sample is sequenced and reads, to identify warp The PNA of modification.It is an advantage of the invention that this identification can be completed in the case where being not necessarily to purifying/separation from unmodified PNA.

In detail, the method for the present invention includes the following steps: modifying PNA by adding or removing hydrogen bonding gametophyte One or more nucleobases, to change the base-pairing abilities (or behavior) of one or more nucleobases；By complementary nucleic acid Base pairing is carried out with PNA, including carries out base pairing at least one modified nucleobase.

Natural nucleobases are A (adenine), G (guanine), C (cytimidine) and T (thymidine)/U (uracil).With A, G, C, T nucleotide are compared in the case where A, G, C, U nucleotide or DNA in the case where RNA, and modification of the invention generates non-natural Nucleobase.The modification leads to the base pairing behavior changed, to change the preferential alkali between A and T/U and between C and G Basigamy is to (passing through Hydrogenbond).This means that with the base pairings of the natural nucleobases as complementary nucleic acid from a kind of natural nucleus Acid changes into another natural acid.Preferably, complementary nucleic acid is DNA, and replaces U using T.The A of change can in conjunction with C or G；The T or U of change can combine C or G；The C of change can combine A or T/U；The G of change can combine A or T/U.Such modification It is known in the art.Modification is usually secondary, and only minimum keeps variation, to change base pairing behavior. Such as.A and G respectively maintains its purine loop system, and C and T/U maintain its pyrimidine ring.For example, Harcourt et al. (Nature 2017,541:339-346) provide the summary and general introduction of such modification.Example sex modification is A to m⁶A, until m¹A, until inosine, until The modification of 2- aminoadenine；C to m⁵C (5-methylcytosine), until hm⁵C (5-hydroxymethyl cytosine), until pseudouridine, until 2- sulphur Cytimidine, until 5- halo cytosine, until 5- propinyl (- C=C-CH₃) cytimidine, until the modification of 5- alkynyl cytimidine；T or U are extremely 2- thiouracil, until s⁴U (4- thiouracil), until 2- sulphur thymidine, until 4- pyrimidone, until pseudouracil, until 5- halogen urine is phonetic Pyridine, such as 5-bromouracil (also referred to as 5- Broxuridine (5BrU)), 5- propinyl (- C=C-CH₃) uracil, 5- alkynyl is urinated phonetic Pyridine, such as the modification of 5-ethinyluracil；G to hypoxanthine, to xanthine, to the modification of isoguanine；A or G is fast to gland The modification of the 6- methyl of purine and guanine and other 6- alkyl derivatives；To the 2- propyl of adenine and guanine and other 2- alkane The modification of radical derivative.Further modification is 6- azo uracil, cytimidine and thymidine, 8- be halogenated, 8- amino, 8- sulphur The adenine and guanine, 5- that alcohol, 8- alkylthio, 8- hydroxyl and other 8- replace are halogenated, especially 5- bromine, 5- trifluoromethyl It is fast with uracil and cytimidine, 7- methyl guanine and 7- methyl adenine, 2-F- adenine, the 2- amino gland of other 5- substitution Purine, guanozola and 8- azaadenine, 7- deazaguanine and 7- denitrogenation adenine and 3- deazaguanine and 3- are de- Nitrogen adenine.Although natural nucleobases are preferably modified into its immediate modified nucleobase as above instructions, principle On, nucleobase can be modified into any modified nucleobase as described above.Correlative factor is the change of hydrogen bonding mode, So that another base pairing gametophyte will combine modified nucleobase compared with unmodified nucleobase.It is bonded gametophyte Variation do not need absolute certainty, be sufficient that change and be naturally bonded the combination certainty of gametophyte, such as change to Few 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100%.Specific nucleobase can incorporate more than a complementary nucleobases (especially wobble base).Determine variation Reference conditions are under the standard conditions of the reverse transcriptase in the isotonic aqueous solution of physiology, and preferred atmosphere is pressed and 37 DEG C.Exemplary item Part is 50mM Tris-HCl, 75mM KCl, the 3mM MgCl of pH 7.5-8.5₂,10mM DTT.Any such variation is ok It is relatively monitored by current detection means, such as sequencing and sequence.Furthermore, it is possible to will be more than that a kind of modify is included in PNA points In son, and each molecule or every multiple molecules only need to detect that one.Certainly, natural from a kind of natural nucleobases to another kind The hydrogen-bonded change rate of nucleobase (in complementary nucleic acid) is higher, and the certainty of detection is higher.Therefore, higher base The variation of pairing rate is preferred, such as changes at least 50% or at least 80%.

" halogenated " refers to halogen, especially F, Cl, Br or I；Br is particularly preferred, such as in 5BrU." alkyl " is Refer to branch or straight chain, substituted or unsubstituted alkyl residue, preferred length C₁-C₁₂Alkyl residue.Preferably length is C1- The alkyl residue of C4, with optional O substituent group and/or optional N substituent group, such as in acetamide or any other alkyl In carbonyl, carbonic acid or amide.

The particularly preferred modified nucleobase of PNA is 5- Broxuridine (5BrU), 5- acetenyl uridine (5-EU), 6- sulphur For guanosine (s⁶G), 4-thiourdine (s⁴U), 5- vinyl uridine, 5- azido methyl uridine and N⁶Allyl adenosine (a⁶A)。

Base pairing behavior is as known in the art, or can be derived from the variation of hydrogen bond donor or receptor Come, including its obstacle for preventing them from matching.Such as.4- pyrimidone (modified U or T) preferably with G base pairing, without with A matches (Sochacka et al., Nucleic Acids Res.2015Mar 11；43(5):2499–2512).

The modification of the nucleobase of PNA can be the hydrogen (H) on oxygen (O) or nitrogen (N) atom and be substituted base such as carbon substitution (example Such as, such as in methyl group or other alkyl groups), to remove the hydrogen as hydrogen bond donor.Modification can be oxygen (O) or nitrogen (N) free electron of atom replaces (for example, such as in methyl group or other alkyl groups) to substituted base such as carbon, thus Remove the electronics pair as hydrogen bond receptor.Modification may include replacing O by sulphur (S) or SH, then carry out one of above-mentioned modification, The alkylation of especially S or SH.A kind of preferred method of O is replaced to mention by biosynthesis and to enzyme such as transcriptase with S or SH For the nucleotide through S or SH modification, such as s⁴U is carried out.Transcriptase can be in cell.

Modification of the invention can be step modification or by the way that more than a step, such as two steps, three steps or more step repaired Decorations.Such as.The first part of modification carries out in a kind of reaction environment such as cell, and the second modification is in another reaction environment It carries out, such as after isolating PNA in cell.Preferably, it such second or further modifies depending on the first modification, example Such as, the atom changed by the first modification is carried out.Particularly preferably multi step modification, wherein the first modification is enzyme modification, such as Modified nucleotide/nucleobase is mixed in PNA by enzyme such as RNA or archaeal dna polymerase.In this step, for enzymatic Duration only includes small modification, not damage or damage enzymatic activity in a manner of it can endure.It is small modification be for example with corresponding day Variation of the right nucleobase compared to only 1 or 2 atom (disregarding hydrogen).In further step, the modified nucleobase of incorporation It can further be modified by any means, such as be modified to modified nucleobase as described herein, such as pass through wet-chemical Method, including alkylation.Such further modification can extracellular, enzymatic or non-enzymatic.It is preferably targeted The modification introduced in one step.(first) modification in cell can be induction or enhancing modification, such as by mentioning to cell For modified nucleobase (for example, as modified nucleotide), then the cell is mixed in the PNA of biosynthesis. " enhancing " refers to the naturally-occurring beyond modification.

(the first) the modification natural process that is also likely to be cell interior, without providing modified nucleobase to cell.This Class natural process is, for example, Thiolation (Thomas et al., Eur J Biochem.1980,113 (1): 67-74 of tRNA； Emilsson et al., Nucleic Acids Res 1992,20 (17): 4499-4505；Kramer et al., J.Bacteriol.1988,170(5):2344-2351).Such naturally occurring modification can also be examined by means of the present invention It surveys, such as is modified by further (second) of the nucleobase directly or by these natural modifications, detect and these are through modifying Nucleobase base mispairing or change base pairing behavior.Certain natural modifications can be response or other environment shadows Loud result.Therefore, method of the invention can be used for detecting the influence in the such response and cell of cell.One example is Respond UV light, the s of especially close UV radiation⁴U modification, especially in tRNA (Kramer et al., ibid).s⁴U modification, especially It is that can be used for the growth rate (Emilsson et al., ibid) of measurement cell in tRNA.It can be with side according to the present invention Method detection will be used as this modification of growth indicator.Preferably, eubacteria or archeobacteria are used for such natural modifications.

In a preferred embodiment of the invention, modification step mixes PNA (modification by the nucleobase that will be modified through mercaptan First part) and the mercaptan nucleobase is alkylated (second part of modification) Lai Jinhang with alkylating agent.Thiol reactivity Alkylating agent includes iodoacetamide, maleimide, benzylic halides and bromomethyl ketone.Alkylating agent may include alkyl as described above And leaving group, such as halide, such as Br or Cl.These reagents generate stable thioether by the S- alkylated reaction of mercaptan Product.Aromatic yl reagent-ing such as NBD (2 evil -1,3- diazole of 4- nitro benzo) halide by with nucleophile to aromatic series halogenation The similar substitution of object is reacted with mercaptan or amine.Thiosulfate can also be used for reversible mercaptan modification.Thiosulfate and mercaptan The disulphide of mixing is formed with stoichiometric reaction.Mercaptan is also reacted with isothiocyanates and succinimide ester.Different sulphur Cyanate and succinimide ester can also be used for reacting with amine.

The modification of mercaptan can also include the steps that converting mercaptans to thioketones.It may then pass through addition or remove hydrogen bond Gametophyte is bonded further to modify thione group.Such asEt al. (Angew.Chem.Int.Ed.Engl.1996,35 (9): 993-995) described, being converted into thioketones may include removing hydrogen, such as on the transient metal cluster as catalyst.Conversion It is allowed for the additional option of reactive chemistry, for thioketones to carry out modification of the invention.Et al. also illustrate mercaptan Or thioketones introduces aryl, this is also that the present invention creates thio-modification (mercaptan, thioketones) in modified nucleobase of the invention Option.

The alkylation of mercaptan is also referred to as the alkylation of mercaptan (SH)-connection.The benefit of thiol alkylation is it to " soft " sulphur The selectivity of alcohol, and not Thiolation nucleobase can remain unchanged (HSAB theory- " hard and soft (Lewis) Acids and bases ", Pearson et al., JACS 1963,85 (22): 3533-3539).Iodoacetamide is easy and all sulphur Alcohol is reacted to form thioether；They slightly have reactivity than the acetbromamide that also can be used.Maleimide is selected for mercaptan Sex modification is selected, quantitative and analysis excellent reagent.In this reaction, the double bond of mercaptan across maleimide is added to generate Thioether.It can also be alkylated via above-mentioned thioketones.

Preferably, modification includes 4 upper alkylations in uridine.In the position, the natural hydrogen bonding behavior of uridine is interfered It is highly effective.Such modification can use alkylating agent, such as the alkylating agent comprising hydrogen bonding gametophyte (preferably hydrogen bond receptor), or Alkylating agent not comprising hydrogen bonding gametophyte carries out, to block the hydrogen bonding usually occurred at uridine 4.Such alkyl Change to modify as described above by 4-thiourdine with two steps and carry out.

Another preferred alkylation is at 6 of guanosine.Such alkylation increase has from standard GC to only 2 Imitate the mismatch rate of the G*A swing pair of hydrogen bond (rather than 3 in GC).In particularly preferred embodiments, draw at 6 of guanidine Entering alkylation includes that guanidine is modified into 6- Thioguanosine (s⁶G) and it is alkylated thio position.Therefore, this is by the thio bird of 6- Glycosides carries out another such alkylated preferred embodiment in two step modifications, as mentioned.It can be in 6- Thioguanosine nucleosides 6- Thioguanosine is mixed in PNA by biosynthesis in the presence of acid.

Preferred alkylating agent has formula Hal- (C)_xO_yN_z(hydrogen is not shown), wherein Hal indicates that halogen, C indicate x C atom Carbochain, branch or non-branched, x be 1 to 8, O indicate C atom y oxygen substituent group, y be 0 to 3, N expression C atom z nitrogen Substituent group, z are 0 to 3.N is preferably at least-a NH₂Or double bond=NH, O are preferably-OH or double bond=O.Hal is preferably selected from Br or I.

It is particularly preferred that PNA include one or more 4-thiourdines or 6- Thioguanosine, preferably 2,3,4,5,6,7,8, 9,10 or more 4-thiourdines or 6- Thioguanosine.Modifying one or more nucleobases may include by hydrogen bonding gametophyte (such as hydrogen bonding receptor or donor) is connected to the nucleobase modified through mercaptan.Such connection can pass through any chemical modification It completes, wherein alkylation is preferred, such as passes through the alkylating agent of halide as mentioned above.

Alkylated alternative is to pass through oxidative modification.Such modification is e.g., as disclosed in Burton, Biochem J 204 (1967): 686 and Riml et al., Angew Chem Int Ed Engl.2017；56(43):13479-13483.For example, can be with By oxidative modification nucleobase, especially Thiolation nucleobase, to change hydrogen bonding donor or receptor.As described above In the case where two-step method by Thiolation nucleobase, the sulphur of thiol group can be oxidized, such as pass through OsO₄、NaIO₃、 NaIO₄Or peroxide such as chloroperoxybenzoic acid or H₂O₂Oxidation.For example, s4U can be oxidized to C (Schofield et al., Nature Methods, doi:10.1038/nMeth.4582), base pairing/hybridization behavior is changed into C-G from U-A by this.Such as Shown in Burton (ibid), the oxidation does not need thiol intermediate, however, the thiol intermediate is preferably, especially (see below) in the case where biosynthesis modification.Such C analog is such as trifluoroethyl cytidine (such as in 2,2,2- Oxidation product in the presence of trifluoroethylamine).C analog can retain base pairing behavior and/or the pyrimidine 2- ketone ring of cytimidine.It is phonetic 4 on pyridine -2- ketone ring can be replaced by amino (as shown in C), or comprising other substituent groups such as R-NH- group, wherein R Selected from alkyl, aromatic group, alkane group, NH₂, trifluoro-ethylene, MeO etc. (referring to Schofield et al., ibid, especially Supplement Fig. 1；It is incorporated herein by reference).

In preferred embodiments, it is incited somebody to action via the biosynthesis in cell or by cellular enzymes (such as passing through in-vitro transcription) In modified nucleobase, such as the base incorporation PNA through mercaptan modification.In addition, the modified nucleobase of chemistry introducing is can Can, such as synthesized by (abiotic) chemistry PNA, such as the organic or hemizygous synthesis that becomes second nature.Biosynthesis is based on template PNA The synthesis of the PNA of (usually DNA, especially genomic DNA) and Template Dependent synthesis (transcription, reverse transcription).For such The suitable enzyme of transcription is RNA polymerase, archaeal dna polymerase, reverse transcriptase.Enzyme can be by natural and modified nucleotide (tool Have modified nucleobase) it mixes in the pna molecule of biosynthesis.Nucleotide monomer units are connected when forming PNA.Such list Body can be provided in the form of modified and from incorporation PNA.Preferably, only modify a kind of natural nucleotide type (A, G, C, T/U), that is, there is modified (non-natural) counterpart in incorporation PNA.It is also preferred that there are all natural nucleus glycoside acids Type, wherein modified nucleobase is in number less than corresponding natural (unmodified) nucleobase." corresponding " refers to naturally Nucleobase, wherein minimum atom (disregarding hydrogen) variation is necessary to restoring natural nucleobases.Such as.Modified U (or choosing From any other modified nucleotide type of A, G, C, T) outside, additionally provide A, G, C, T/U.Preferably, type is given The ratio of modified nucleotide and unmodified (natural) nucleotide be 20% or less, such as 15% or less or 10% or Less or even 5% or less (whole moles of %).Modified nucleotide will be impregnated in the corresponding natural nucleotide of replacement, but It is that will then cause atypia base pairing (the base pairing behavior of change, as detailed above) in the method for the invention, this Then will lead to can be matched with clock synchronization with modified nucleotide base with natural counterpart nucleotide base compared to it is another One complementary nucleotide.Therefore, the sequence variation of the complementary strand (such as: newly synthesized complementary strand) of hybridization will will appear.Therefore, The base pairing carried out at least one modified nucleobase can lead to matches compared to the base with the nucleobase not yet modified To the base pairing with another nucleotide, the nucleobase is identical in other respects.

Alkylated nucleobase can also be mixed in PNA by biosynthesis, such as described above but without using sulphur The alkylated nucleobase of alcohol intermediate.For example, can by alkylated nucleotide mix cell in and PNA synthesis during by The cell uses.Such method via Jao et al., Proc.Nat.Acad.Sci.USA 105 (41), 2008:15779 and Darzynkiewicz et al. Cytometry A 79A, 2011:328 description.Particularly, used according to the invention effective Modified nucleotide is 5- acetenyl-uridine (5-EU).The uridine of acetenyl label is cell permeable, and mixes new life RNA in replace its natural analog uridine.In preferred embodiments, such as by Cu (I) click chemistry (example being catalyzed Such as, such as Presolski et al., Current Protocols in Chemical Biology 3,2011:153；Or Hong etc. Described in people Angew.Chem.Int.Ed.48,2011:9879) PNA functionalized to gained acetenyl further modified, with Other official is introduced by the functionalized molecule of azide (such as NHS ester, maleimide, azido acid, azido amine) Group can be changed, to influence ketone with the hydrogen bonding capability at ortho position and ethynyl group.

In other embodiments, the functionalized molecule of such azide can be introduced into cell itself using as warp The nucleobase biosynthesis of modification is into pna molecule.Cu (I) (CuAAC) being catalyzed or the strain without Cu (I) can then be passed through Promoted type (SPAAC) click chemistry introduces functional group to detect the functionalized PNA of resulting azide, the functional group with do not repair The nucleobase (C, T/U, A, G) of decorations is compared to the hydrogen bonding capability for changing nucleobase.

Another example for modifying one or more nucleobases of PNA includes being incorporated into the nucleobase of vinyl-functional In PNA, such as 5- vinyl uridine.Vinyl groups can be modified further to change the unmodified nucleobase of other aspects Hydrogen bonding capability (referring to Rieder et al., Angew.Chem.Int.Ed.53,2014:9168).

In particularly preferred embodiments, the one or more nucleobases for modifying PNA include the cyclisation of allyl group And/or the halogenation of the nucleobase including PNA, especially iodate.Modified nucleobase is preferably allyl nucleobase, such as N⁶Allyl adenosine (" a⁶A "), it can further be modified and being related to the cyclisation of allyl group.It can be synthesized in PNA Period mixes such allyl nucleobase in PNA, especially in cell, as described in other embodiments herein.Halogenation And/or cyclisation can follow Shu et al., J.Am.Chem.Soc., 2017,139 (48): principle described in 17213-17216.It is excellent Selection of land, method include by N⁶Allyl adenosine mixes in cell, then uses such as elemental iodine (I₂) iodate is carried out, this leads to iodate Before allyl and such as nucleobase purine (in the case where modified A or G) or pyrimidine (in modified C or In the case where T/U) on nitrogen-atoms cyclisation.The modification leads to the base pairing changed, can be during sequencing or hybridization Read.Such as.a⁶A is run similar to A, and can be mixed by metabolic way in mammalian cell in newly synthesized RNA. The a under mild buffer conditions⁶The N of A⁶The iodate autonomous induction N of allyl group¹,N⁶The formation of cyclisation adenosine, and Mutation is created in the complementary DNA synthesis process of reverse transcription at its opposite sites.

In another preferred embodiment, the one or more nucleobases for modifying PNA include by 5- Broxuridine (5- BrU) nucleobase is introduced into PNA.5-BrU is one kind with mutagens existing for tautomeric forms, it means that it is with its ketone Existing with Enol forms, the mutagens and adenine or guanine base match (see Figure 37 a), this then leads to amplified reaction, As T > C conversion increases (compared with unmodified U) in PCR.Therefore, it in of the invention this embodiment generally preferably, repairs The one or more nucleobases for adoring PNA introduce tautomerism nucleobase, and the tautomeric form is in modified T/U and through modifying C in the case where can with (the A and G) base pairing of two kinds of purine bases or in modified A and G can with two kinds it is phonetic Pyridine base (T and C) base pairing.Mean with two purine/pyrimidine bases base pairings herein than unmodified A, G, base pairing behavior is more balanced (but not necessarily equal) in the case where U/T, C (they are seldom matched with Non-complementary bases). In other words, tautomerism base is compared with unmodified base (swinging behavior) with (purine is phonetic with identical base nuclear structure Pyridine) Non-complementary bases increased base pairing.The increase is at the standard conditions, especially for PCR.

It can be determined by the increased result of the mixing base of the specific location corresponding to modified nucleobase Such swing behavior.(compare Fig. 5 B, 24B, 37C) in any embodiment, swinging base stage detection is the excellent of the method for the present invention It selects to read out.

It, can be by replacing halogen further to modify 5-BrU or any with amino in other relevant embodiments Other halogenated nucleobases.For example, 5-BrU can be heated together with ammonium hydroxide to be translated into 5-aminouridine.Such ammonia The nucleobase of base modification changes base pairing in process of reverse-transcription and/or will introduce other swing behaviors.

PNA (having modified nucleobase) may include RNA or DNA or be made of RNA or DNA.Exemplary RNA is MRNA, Microrna (miRNA or miR), short hairpin RNA (shRNA), siRNA (siRNA), PIWI interaction RNA (piRNA), rRNA (rRNA), tiny RNA (tsRNA), transfer RNA (tRNA), little nucleolar RNA derived from tRNA (snoRNA), small nuclear rna (snRNA), long non-coding RNA (lncRNA) or its precursor rna molecule.DNA is such as genome DNA, cDNA, Plasmid DNA or DNA vector.PNA can be double-strand or single-stranded.

"comprising" refers to open-ended term, and can also allow for molecule includes other members, for example, other kinds of nucleosides Sour (there may be RNA or DNA, including through manually modified nucleotide, such as LNA)." Consists of " is considered as closed definition, The person of hoping for success abides by the requirement, i.e., complete RNA or complete DNA.

Preferably for every kind of nucleotide type for being selected from A, G, C, U or T, modified PNA includes than modified core The more natural nucleotides of thuja acid.Herein, PNA is related to the final PNA with all modifications according to the present invention.PNA is preferred Comprising 1,2,3,4,5,6,7,8,9,10 or more and at most 30 modified nucleotide.Preferably, seldom nucleobase It is modification, such as 20% or less in pna molecule, such as 15% or less or 10% or less or even 5% or less (complete Portion mole %) nucleobase be modification.

Pna molecule can have any length.Preferably, it has the length of at least 10nt (nucleotide).Particularly preferably Be length 10nt, 20nt, 30nt, 40nt, 50nt, 75nt, 100nt, 250nt, 500nt, 1000nt, 2500nt, 5000nt, The length of 10000nt, 25000nt, 50000nt, 100000nt or more or any range between these values.It is excellent Selecting range is the length of 10nt to 100000nt or 50nt to 50000nt.

Preferably, specific cells fraction of the PNA from nucleotide, such as total serum IgE fraction, mRNA fraction or DNA fraction, example Such as Plasmid DNA or genomic DNA.Can have common trait, such as length, nucleotide type or sequence by separation, such as The PNA of poly (A) tail or 5 ' caps in mRNA selects fraction.

Method of the invention includes the step of making PNA base pairing by complementary nucleic acid.In the base pairing, through repairing At least one of nucleobase of decorations should be base pairing (usually by the base pairing of several nucleobases of PNA).It is logical Cross the base pairing for promoting PNA and nucleic acid strand with complementary nucleic acid.This can also be during extension, such as PCR Occur, or is occurred by hybridization probe nucleic acid.Complementary nucleic acid can have any length, for example, above in relation to disclosed in PNA that A little length.

The sequence of complementary nucleic acid is identified at least at the position complementary at least one modified nucleobase.Sequence determines It can be completed by any conventional program known in the art.Such method includes completely or partially based on generation complementary strand The method of (such as passing through PCR), such as (NGS) is sequenced in the next generation, in the sequencing approach based on segment.If you want it, may be used Segment read is assembled into composite sequence.However, for purposes of the invention, this is unnecessary, as long as identifying and passing through The complementary nucleobases of the nucleobase of modification, especially with its flanking sequence (such as +/- 5nt, +/- 10nt, +/- 15nt or +/- The flanking sequence of 20nt).Determine sequence other methods include in conjunction with probe, from there through known hybridization probe sequence, The sequence of PNA is determined as complementary series.

Another option be small nucleic acids sequencing, especially in the lesser situation of complementary nucleic acid, for example, with miRNA, In the case where the nucleic acid of shRNA, siRNA complementation.The length range of small nucleic acids for example can be 10nt to 200nt, preferably 12nt To 100nt or 14nt to 50nt.The length of the length or shorter than 10nt that are longer than 200nt is also possible.The segment of complementary nucleic acid It averagely can have such length.Segment can either physically or chemically be generated by known in such as field NGS.In small nucleic acids In the case where (including the segment for example obtained during NGS), preferably adapter is connected to and can be used as the miscellaneous of primer or probe Hand over the nucleic acid of sequence.Such adapter also may include characteristic sequence, such as bar code, to identify small nucleic acids by marker.Bar shaped Code can provide for the origin of the sample of acquisition PNA or as the origin (segment origin) of the pna molecule of segment or its complementary nucleic acid Marker.Such bar code can be used for multiplexing sequencing, wherein to not homotactic many nucleic acid, such as multiple and different is mutual The multiple segments for mending nucleic acid and/or one or more complementary nucleic acids are sequenced.It is such multiple to may, for example, be 2 to 1000 cores Acid or more.The another possibility for being not necessarily required to adapter is the complementary core by making primer or probe with corresponding to PNA Acid sequence hybridization.Such primer or probe can hybridize with known array or randomer hybridization, such as by using random primer.Under Face describes random primer about kit of the invention, and any such random primer may be used to side of the invention Method.

In a preferred embodiment of the invention, the PNA of single cell is identified according to the present invention.Therefore, by the PNA of cell It separates and is separated with the PNA of other cells holding." keeping separating by PNA " refers to not by the PNA sequencing information of the cell of research In the case where being mixed with the sequencing information of other cells still can identification research cell PNA.This can be by being physically separated PNA passes through label, especially by the marker with identification cells of interest, such as passes through bar shaped code labeling PNA or complementation Nucleic acid is realized.This allows to analyze the PNA metabolism of single cell.Single cell sequencing approach (Eberwine et al. can be passed through Nat.Methods.11 (1): single cell analysis 25-27) is carried out.As the alternative of sequencing, can also be prepared in library mutually Nucleic acid or its segment are mended, preferably but not necessarily has adapter.Then independent sequencing can be carried out to library or other use are provided On the way.

Modification (such as the alkylation of mercaptan specificity) of the invention promotes quantitative " mistake " incorporation of complementary nucleotide, the complementation Nucleotide forms different hydrogen bonding patterns as described above now.Such as.It can be in modified nucleobase (such as alkylated 4- Thio uridine) during complementary nucleic acid combines, such as guanine rather than adenosine are mixed during transcription or reverse transcription.But (inverse) Transcriptase duration is usually uninfluenced, this is because can be in the case where no further obstruction by alternative base pairing Nucleotide expands together with its PNA.Preferably after the first enzymatically modifying (such as by mixing modified nucleobase) With the combination of the second modification.S establish and nontoxic is improved with as mentioned above⁴Such combination of U metabolic marker scheme.

Can by the sequencing approach of the present invention of the sequence variation caused in complementary nucleic acid due to modified nucleobase with Available high-flux sequence method such as NGS coupling.PNA/ complementary nucleic acid can be identified by available calculation method not With between individual molecules different sequence variation, if particularly it is endless fully or partially.Such as.Number can be sequenced in the next generation Track T > C conversion according to concentrating (due to caused by U modification, the U modification causes G base pairing to increase).It is such supermatic Method (combining with computerization analysis) allows the present invention to provide the quick access to intracellular rna processing dynamics, i.e. this hair Bright preferred application.Since complementary base is matched, the present invention can accurately reporter rna polymerase II dependent transcription be exported. Understand the dynamics intracellular that RNA biology occurs, processes and has enough to meet the need, influences substantially any given biology in life for illustrating The molecular basis of the gene expression pattern variation of process is most important.

Therefore, in preferred embodiments, method of the invention is determined for the modification of PNA in cell or is easy to repair The change of decorations.For example, such " change for being easy modification " refers to above-described multistage method, wherein carrying out first in cell Modification (also referred to as changes), and subsequent modification is completed in second step or further step, usually in cell after separating PNA Outer progress.

Preferably, method of the invention is used to modify RNA (as PNA), in cell, especially carried out in living cells to Few first modification/change.Due to the RNA modification that will be expressed, this allows to track rna expression variation.

The adjusting expression of hereditary information provides cell flexibility for maintaining Cell Homeostasis to be necessary to respond variation Environmental condition, and if if imbalance, cause human disease, such as cancer.The basis of these basic biological processes is strictly to adjust The molecular events of section, the molecular events are with transcript specificity pattern control rna transcription, the relative dynamics of processing and degradation.

Cell RNA pond (covering countless RNA types, including mRNA or non-coding RNA, such as Microrna) is by gene The transcription definition of selected genes seat in group, and can be qualitative by RNA sequence type analysis technology (for example, high-flux sequence) and fixed Amount assessment.However, the abundance measurement inaccuracy of steady state RNA level reflects transcriptional activity itself.In fact, rna stability is true It plays a major role in the relative abundance of fixed any given RNA molecule.Therefore, measurement transcription and RNA decay on Genome Scale The method of rate can be used for illustrating the understanding to rna expression dynamics and its potential adjustment mechanism.According to the present invention it is possible to determine RNA biology occurs and the dynamics intracellular of turnover.

RNA can be changed or be modified by the metabolism of cell itself, for example, by will through change or modified nucleosides In the RNA of acid incorporation natural process.Such change can be used for being selectively introducing modification of the invention, (individually or into one After the modification of step) change hydrogen bonding behavior.Due to metabolic effect, such method is known as " metabolism sequencing " (if then to warp If the nucleotide of modification is sequenced).Sequencing steps usually can with any base pairing step of complementary (more) nucleotide With automation, and to be processed in high-flux sequence method as mentioned above.The present invention provides be adapted to determine that RNA biology The dynamic (dynamical) high-throughput compatibility metabolic marker scheme intracellular for occurring and having enough to meet the need.Its accurate measurement rna plymerase ii dependence Gather polyadenylation transcription output, and recur global posttranscriptional gene adjustment signal, therefore solve in cell with the high time point Resolution provides the problem of rna expression dynamics (including biology occurs and turnover).

Cell can be any cell, such as bacterial cell, including eukaryon and prokaryotic cell, Gram-negative and gram Positive cell, fungal cell, alga cells, plant cell, zooblast, mammalian cell, such as rodent cells, spirit Long class zooblast, people's cell, non-human cell, archeabacterial cell, avian cell, amphibian animal cell, such as frog cell, reptile class are thin Born of the same parents, marsupial cell.

Possibly through modification time control change detection, such as by the cell RNA being not decorated expression stage with The stage of cell RNA expression with modification is compared.Preferably, more such rank in same cell or cell culture Section.Such as.It is the stage being not decorated after stage with modification, or vice versa.Therefore, excellent in of the invention one It selects in embodiment, cultivates one or more cells at least two cultivation stages, one of cultivation stage includes will be through In the RNA of the nucleotide incorporation biosynthesis of modification, which is modified by adding or removing hydrogen bonding gametophyte；Separately One cultivation stage lacks will be in the RNA of modified nucleotide incorporation biosynthesis." another cultivation stage " may also wrap It includes by the RNA of modified nucleotide incorporation biosynthesis, but with different from another cultivation stage, such as more Low concentration incorporation.Concentration different or lower from another stage should be enough to observe modified nucleotide Mix the difference (being especially different concentration) in the RNA of biosynthesis.Therefore, it is more can be defined as identification to method of the invention The method of nucleic acid (PNA) comprising PNA following steps: is expressed in cell；Modify one or more nucleobases of PNA；From thin PNA is isolated in born of the same parents；Optionally, PNA is further modified；Wherein modification addition or removing before separation or later or together The hydrogen bonding gametophyte of one or more nucleobases, to change the base-pairing abilities of one or more nucleobases；It will be mutual It mends nucleic acid and PNA carries out base pairing, including carry out base pairing at least one modified nucleobase；Identification at least with The sequence of complementary nucleic acid at the position of at least one modified nucleobase complementation.Particularly preferred metabolic marker is (i.e. by thin Born of the same parents' metabolism, such as pass through the modification of its enzyme such as RNA polymerase) event progress is mixed by 4-thiourdine.This can be used for changing Become the base pairing behavior of U.

Method particularly preferably at least two cultivation stages of cell, wherein promoting at least two cultivation stages The PNA of different level is modified, especially RNA modification.This can by cell provide various concentration modified nucleobase, It is realized to allow cell to mix the modified nucleobase of different level or concentration in PNA, especially RNA.Institute as above It states, it is preferable that modified nucleobase is the nucleobase modified through mercaptan.The level that PNA is modified in one stage can be nothing Modification.Each stage, especially those stages with PNA modification should have the preset time period for PNA modification.It is logical The incorporation compared between different phase is crossed, the turnover rate in preset time period can be calculated.In a particularly preferred implementation In scheme, turnover is calculated based on mixing modified nucleobase in PNA compared with another stage at least one stage Rate or degradation rate.Preferably, the stage is continuous cultivation stage.

It can further be compared between the cultivation stage of different cells.Such comparison allows to estimate these cells Between differential expression and PNA turnover.One of cell or one group of cell can be control, and another cell or another group thin Born of the same parents can be the candidate cell or groups of cells of research.The two cells or groups of cells may have comparing by modified core The stage of base incorporation PNA.Preferably, this is controlled by providing for cell for mixing the modified nucleobase in PNA Class mixes the stage.Preferably, same amount of modified nucleobase is provided to each cell or every group of cell, is suitable for cell generation The comparison thanked.It preferably, is the stage not being further incorporated into after the incorporation stage, for example, by stopping to cell or carefully Born of the same parents organize the nucleobase for providing and further modifying.It is also likely to be the stage of reduced incorporation after the incorporation stage or is mixed with different level Enter.It is the adaptation of cell metabolism after horizontal any variation of modified nucleobase incorporation PNA, this can through the invention Method is monitored.Such as.If being lower incorporation stage or without the incorporation stage after the incorporation stage, it is likely that monitoring is through modifying PNA degradation.If after the stage without incorporation or limited incorporation being the stage of higher limited incorporation after the incorporation stage is probable, It is possible that monitoring the accumulation of modified PNA.

Therefore, a kind of purposes of the method for the present invention is at least two different at least two cells or in cell Compare the complementary nucleic acid at least at the position complementary at least one modified nucleobase (as described above) in growth phase Sequence is identified, wherein at least two cell or growth phase have between at least two cell or the growth phase Variant expression (usually gene expression, including mRNA or modulability rna expression).Difference (gene) expression can be by thin The inhibition or stimulation of at least one of born of the same parents gene cause.Such method can be used for screening the difference table that certain in cell metabolism disturbs Up to effect.The differential expression can be unknown gene, such as in screening technique, wherein for the specific heredity in cell Effect study adjusts inhibitor or activator or any other substance with phenotypic effect.In other embodiments of this method In, target gene may be known, and have studied the further secondary influences of the gene expression to other genes.For example, Known can be known adjusting gene, such as oncogene or tumor suppressor gene.

Cell or groups of cells can be in vitro in cultures or in living organism, such as plant, bacterial cell, fungi are thin In born of the same parents, alga cells, non-human animal or people.It, can be by the way that modified nucleobase be applied in vivo in the case where cell Organism, such as being systematically applied in vascular system or be locally administered to the interested organ of organism will be through repairing The nucleobase of decorations is supplied to cell.Therefore, it is possible to monitor the metabolism of PNA in internal or specific interested organ.Then may be used With for example through biopsy or in the case where secretory PNA from humoral sample or by put to death non-human organism come from PNA is isolated in organism.Preferably, divide the analysis of variance from the PNA of the single cell of organism according to the method for the present invention, It is carried out for example, generating and/or being sequenced by single cell by label and/or library, as mentioned.Cultivation stage it is any Description is also applied for interior therapeutic, and is known as " growth phase "." growth phase " does not need cell growth or cell Proliferation, and Refer to the PNA metabolism or " growth " identified and analyzed.

The PNA and PNA for comparing different level have enough to meet the need for illustrating different conditions cell in organismal development and lysis Between cell metabolism difference be important.The conversion ratio that PNA can be measured can help to illustrate which approach be it is active with And which path is less active or inactive.It is surveyed in the Css of this aspect, turnover rate PNA, especially RNA Amount provides another measure, i.e., only measures the concentration of PNA, such as the concentration of mRNA present in cell, tissue or organ.

Preferably, PNA, the preferably RNA that the biosynthesis of two cultivation stages is collected from the cell, preferably also by it Mixing, and the base pairing that wherein complementary nucleic acid and PNA are carried out includes by transcription, is such as inverse in the case where RNA is PNA Transcription generates complementary polynucleotide chain, preferably DNA chain.

Special benefit of the invention is, there is the PNA of the creation of modification and be not decorated or less modification it is comparable PNA, or corresponding complementary nucleic acid be not required to it is to be separated.The base pairing of PNA and complementary nucleic acid can in modified PNA and not In the mixture of both PNA of modification.It is then possible to the sequence of determining PNA/ complementary nucleic acid be combined, this is because can be at this Sequence/identity of complementary nucleic acid is determined in two kinds of situations (with and without modification), and can be inferred that repair by comparing Decorations event.The sequence that such comparison is preferably computerization compares.Method of the invention is (particularly preferably below according to its Embodiment: the base pairing at least one modified nucleobase, cause compared to the nucleobase not yet modified The base pairing of base pairing and another nucleotide) further comprise determining complementary polynucleotide chain sequence and compared with chain sequence Column, wherein can by identified compared with the complementary nucleic acid being not decorated by by add or remove hydrogen bonding gametophyte into The complementary nucleic acid of change caused by capable modification.Preferably, nucleotide sequence is measured with segment, such as NGS and high pass It measures in sequence.The length of sequence (many to contain the position complementary at least one modified nucleobase) to be determined can Think 10nt to 500nt, preferably 12nt to 250nt or 15nt to 100nt.

The computer of the sequence of complementary nucleic acid at least on the position complementary at least one modified nucleobase reflects It surely may include compared with the sequence of unmodified PNA.Such relatively sequence can from sequence database, such as in EBI or NCBI is obtained, or is generated by the PNA when not introducing modification, such as pass through natural base and natural complementary base alkali Basigamy is to determining.Computer program product for such comparison or the computer-readable medium for this method may include In kit of the invention.

Invention further provides the kits for being suitable for progress the method for the present invention, and it includes the core alkali modified through mercaptan Base and the alkylated alkylating agent of nucleobase for being suitable at thiol group making to modify through mercaptan, wherein the alkylating agent includes hydrogen Key is bonded donor or receptor, and alkylating agent is any one of volume above, particularly preferred iodoacetamide preferably wherein.However, appointing What above-mentioned alkylating agent especially has the modified nucleotide of modified base suitable for the reagent of any of above modification, Such as the nucleotide modified through mercaptan, it can include in kit of the invention.

Kit preferably further includes primer, the nucleotide selected from A, G, C and T, reverse transcriptase or combinations thereof, preferably institute There are these components.Exemplary primers are random primers, are the mixtures of randomly selected primer.Such random primer mixture It can have at least 50 or at least 100, at least 500 kinds of different primers.Random primer can contain random hexamer, random five Aggressiveness, random pentamer, random eight aggressiveness etc..

Kit can further include PNA polymerase, and preferably also comprising the buffer for polymerizeing enzymatic polymerization.Polymerization Enzyme can be DNA or RNA polymerase.

Kit of the invention also may include adapter nucleic acid.Such adapter can be connect with nucleic acid to generate as above The complementary nucleic acid that the adapter combines.Adapter may include one or more bar codes as described above.Kit is also It may include ligase, such as DNA ligase.

The component of kit can be provided in suitable container, such as bottle or flask.

Kit may also include the specification or handbook for carrying out any the method for the present invention.

The present invention is further described by the following drawings and embodiment, is not necessarily limited to the aspects of the invention.

Brief description

Alkylated schematic overview of Fig. 1 for mercaptan (SH) connection of RNA metabolism sequencing.Use 4-thiourdine (s⁴U cell) is handled, the 4-thiourdine is mixed after cellular uptake in the RNA newly transcribed.It carries out at given time point After total serum IgE preparation, handled by iodoacetamide (IAA) by s present in newly-generated RNA type⁴U residue carries out carboxylic acid amides first Base, to form huge group in base pairing interface.When being combined when the library the RNA preparation method with perfect foundation, In s⁴The presence of bulky group causes to cross over alkylation s during reverse transcription (RT) at U incorporation site⁴The specificity of U and quantitative G Accidentally mix.It can be by calling T to C conversion, in high-throughput sequencing library with single nucleotide resolution rate in bioinformatics Identification contains s⁴The site of U.

The 4- thiouracil derivatization that Fig. 2 is carried out by the alkylation that mercaptan connects.(A) 4- mercaptouracil (s⁴U) with Sulfydryl reactive compounds iodoacetamide (IAA) reaction, due to nucleophilic displacement of fluorine (S_N2) it reacts, makes carboxylic acid amides methyl and s⁴In U Thiol group connection.Denote extract (4- thiouracil；s⁴U；Max ≈ 335nm) and product (carboxylic acid amides methylation 4- sulphur urine Pyrimidine；*s⁴U；λ_max≈ 297nm) maximum absorbance.(B) in the iodoacetamide (IAA) the absence and presence of prescribed concentration In the case of 4- thiouracil (s⁴U absorption spectrum).It, will in the presence of 50mM sodium phosphate buffer (pH 8.0) and 10%DMSO 1mM s⁴The U and IAA of prescribed concentration is incubated 1 hour at 37 DEG C.Data indicate at least three independent duplicate average value ± SD. (C) absorption as shown in (B) at 335nm quantifies.It indicates P value (Student t inspection).(D) exist under assigned temperature It is incubated after five minutes in the presence of 50mM sodium phosphate buffer (pH 8.0) and 10%DMSO, the absence and presence of 10mM iodacetyl 1mM 4- thiouracil (s in the case where amine (IAA)⁴U absorption spectrum).Data indicate at least three independent duplicate average values ±SD.(E) the quantifying in the absorption of 335nm as shown in (D).It indicates P value (Student t inspection).(F) in 50mM sodium phosphate After incubating specified time at 37 DEG C in the presence of buffer (pH 8.0) and 10%DMSO, the absence and presence of 10mM iodoacetamide (IAA) 1mM 4- thiouracil (s in the case where⁴U absorption spectrum).The independent duplicate average values of data expression at least three ± SD.(G) the quantifying in the absorption of 335nm as shown in (F).It indicates P value (Student t inspection).(H) in 50mM sodium phosphate buffer In the presence of liquid (pH 8.0) and the DMSO of specified amount after 50 DEG C incubate 2 minutes, the absence and presence of 10mM iodoacetamide (IAA) 1mM 4- thiouracil (s in the case where⁴U absorption spectrum).The independent duplicate average values of data expression at least three ± SD.(I) the quantifying in the absorption of 335nm as shown in (H).It indicates P value (Student t inspection).(J) specified in having for 50mM It is incubated at 50 DEG C in the presence of the sodium phosphate buffer and 10%DMSO of pH after five minutes, the absence and presence of 10mM iodacetyl 1mM 4- thiouracil (s in the case where amine (IAA)⁴U absorption spectrum).Data indicate at least three independent duplicate average values ±SD.(K) quantization of the absorption as shown in (J) at 335nm.It indicates P value (Student t inspection).(L) in 50mM sodium phosphate Under buffer (pH 8.0) and 50%DMSO presence (optimum response [rxn] condition) after 50 DEG C incubate 15 minutes, it is being not present With there are 10mM iodoacetamide (IAA) in the case where 1mM 4- thiouracil (s⁴U absorption spectrum).Data indicate at least three Independent duplicate average value ± SD.(M) the quantifying in the absorption of 335nm as shown in (J).It indicates P value (Student t inspection).

The 4-thiourdine derivatization that Fig. 3 is carried out by the alkylation that mercaptan connects.(A) 4-thiourdine (s⁴) and sulphur U Alcohol reactive compounds iodoacetamide (IAA) reaction, due to nucleophilic displacement of fluorine (S_N2) it reacts, makes carboxylic acid amides methyl and s⁴Sulphur in U Alcohol groups connection.(B) pass through analytical reagent composition s⁴U- alkylation.Standard reaction buffer (50mM NaPO4 (pH 8), 50% DMSO in), 40nmol 4-thiourdine and the iodoacetamide of prescribed concentration are incubated 15 minutes at 50 DEG C.It is terminated with 1% acetic acid Reaction.The sample of acidification is in Ulitimate U300 BioRSLC HPLC system (Dionex；Thermo Fisher Scientific on), using Kinetex F5 phenyl-pentafluoride pilum (150mm x 2.1mm；2.6μm,Phenomenex) With 100 μ l/min of flow velocity separation.After using following SRM electrospray ionisation, TSQ Quantiva mass spectrograph (Thermo is used Fisher Scientific) on-line analysis nucleosides: 4-thiourdine m/z 260 → 129, alkylated 4-thiourdine m/z 318→186.Data are explained using Trace Finder software suite (Thermo Fisher Scientific) and are tested manually Card.(C) two independent experiments in the repetition of two technologies shown in (B) quantify.It is alkylated under specified IAA concentration s⁴U score represents s⁴U and alkylation s⁴The relative normalized signal strength of the peak retention time of U.Data represent average value ± SD.

The alkylation of RNA of Fig. 4 containing 4-thiourdine does not influence reverse transcription duration.(A) in order to determine s⁴U- alkylation Influence to reverse transcriptase duration, we are had the RNA of the 76nt long of the synthesis of 5 ' and 3 ' linking subsequences using flank, contained There is 4-thiourdine (s4U) incorporation at the single location (p9) of drosophila tiny RNA dme-let-7 sequence.By following 5 '³²P mark The extension of the DNA oligonucleotides opposite and complementary with 3 ' linking subsequences in sequence of note, is existed using commercially available reverse transcriptase Reverse transcription is measured before and after being handled with iodoacetamide (IAA).(B) by polyacrylamide gel electrophoresis, phosphorus is then carried out The reaction prepared in (phosphorimaging) analysis (A) is imaged in light.It describes and uses reverse transcriptase Super-script II (SSII), what Superscript III (SSIII) or Quant-seq RT (QS) were carried out exists and there is no IAA processing In the case of include s⁴Primer extend result of the sum of U without RNA.Show the sequence for excluding the RNA component of linking subsequence；s⁴U The position of residue is indicated with red.By the way that the ddNTP specified is added to progress RNA sequencing in reverse transcription reaction.PR, 5 '³²P mark The DNA primer of note；Bg, background stop signal；* p9, the termination signal at 9；FL, full length product.(C) three shown in (B) A independent experiment is duplicate quantitative.To control and contain s using specified reverse transcriptase⁴Under the RNA of U is determined after standardization at p9 The ratio of signal (+p- IAA processing) with previous background dropping signal drops.Data represent average value ± SD.Use Student T examines for statistical analysis.

The s in single nucleotide resolution rate Quantitative measurement RNA is realized in Fig. 5 alkylation⁴U incorporation.(A) iodoacetamide (IAA) is used Processing mixes at single location (p9) or does not mix 4-thiourdine (s⁴U RNA), and carry out the reverse transcription of full length product And gel extraction, then carry out PCR amplification and high-throughput (HTP) sequencing.(B) display is existed using the reverse transcriptase of instruction Or there is no in the case where iodoacetamide (IAA) processing, compare RNA (left figure) and contain s⁴Each position of the RNA (right figure) of U Mutation rate.Bar shaped represents the duplicate average mutation rate ± SD of three independences.Sequencing in each repetitive sequence (r1-r3) is denoted to read The number of section.Show that the nucleotide identity at p9 occurs.(C) Superscript II (SSII), Superscript are used III (SSIII) or Quant-seq reverse transcriptase (QS) refer to presence or absence of iodoacetamide (IAA) processing Surely the mutation rate being mutated.For containing s⁴U's and be free of s⁴Both RNA oligonucleotides of U have the position of identical nucleotide identity Between mutation rate is averaged.Indicate P value (examined and determined by Student t).N.s., not significant (p > 0.05).

Fig. 6 .s⁴Influence of the U processing to mES cell viability and metabolism RNA label.(A) it shows relative to untreated item 4-thiourdine (s of the part in prescribed concentration⁴U the survival of 12 hours (left sides) or 24 hours (right side) mES cells is cultivated in the presence of) Power.(100 μM) of final concentration used in subsequent experimental are indicated with triangle and dotted line.(B) in s⁴U metabolic marker is with impulse form S after culture medium substitutes after up to specified time or in uridine pursuit⁴U is quantified to the incorporation of total serum IgE.It digests and goes in total serum IgE After phosphoric acid turns to single nucleosides, is analyzed by HPLC and determine s⁴U incorporation.Show relative to 24 hours pulse labeling time points and S at 260nm at the 330nm of the background correction of the absorbance standard of uridine signal strength⁴U signal intensity.It shows opposite In s⁴The column retention time (minute) of U absorption maximum value.(C) s compared with unmodified uridine is determined by HPLC⁴The substitution of U Rate.S in mES cell⁴U is metabolized the s in the total serum IgE between pulse and all time points for pursuing labelling experiment⁴U incorporation.Value represents three A duplicate average value ± SD of independence.Maximum incorporation efficiency after denoting label for 24 hours.

Fig. 7 .Quant-seq mRNA 3' end sequencing library preparation method.Quant-seq use total serum IgE as input, Therefore prior poly (A) enrichment or rRNA consumption are not needed.Library, which generates to be caused by oligomer (dT), to be started.Primer has contained Have Illumina compatibility joint sequence (with green display, top: " adapter ", then the step of: last bending). After the synthesis of one chain, RNA is removed, and originate the synthesis of the second chain with archaeal dna polymerase by causing at random.Random primer also includes Illumina compatibility joint sequence (with blue display).It does not need to purify between the first chain and the synthesis of the second chain.Insert Size has been directed to shorter read (SR50 or SR100) and has been optimized.It is the purifying step based on magnetic bead after the synthesis of second chain Suddenly.Then the library is expanded, sequence required for cluster generates (showing with red and purple) is introduced.During PCR amplification step External bar code (BC) is introduced to be multiplexed.

S after Fig. 8 metabolic marker in the mRNA of mES cell⁴U mixes event.(A) it is sequenced (top using standard mRNA Three figure), Cap analyze gene expression (CAGE；Intermediate three figures) and mRNA3' end sequencing (figure of bottom three) preparation, from The representative genome browser screenshot capture in three independent libraries mRNA that the total serum IgE of mES cell generates.Show coding Representative area in the mouse genome of gene Trim 28.(B) genomic region of the end the 3' mRNA of amplification coding Trim28 Domain, including its 3' non-translational region (UTR).It shows untreated mES cell or carries out s⁴U metabolic marker 24 hours, then into The coverage diagram in the mRNA 3' end sequencing library of the total serum IgE preparation of the mES cell of row modification and sequencing.Depict coverage diagram it Under individual reads subset.Red cylindricality in individual reads indicates T > C conversion；Black cylindricality indicates appointing in addition to T > C What is mutated.

S in Fig. 9 .mES cell⁴U is metabolized the global analysis of mutation rate in mRNA 3' end sequencing after RNA is marked.It will be in s⁴U Before metabolic marker and s⁴After U metabolic marker 24 hours, from the 3 ' end sequencing library mRNA that the total serum IgE of mES cell generates 3 ' the non-translational regions (UTR) with annotation are navigated to, and determine any given mutation rate of all expressing genes.Tukey box-shaped figure With the mutation of each UTR of percentages show.Exceptional value is not shown.Show each intermediate value observed frequency being mutated individually.Pass through Mann-Whitney, which is examined, determines that T > C converts increased statistical analysis.

Figure 10 determines the transcription output of the polyadenylation in mES cell.(A) polyadenous in mES cell is determined through the invention The experimental setup of the transcription output of nucleotide mRNA.(B) contained by what the mRNA 3' end sequencing as described in (A) detected The relative abundance of the transcript (" SLAM-seq ") and the transcript (" stable state ") containing non-T > C conversion that there is T > C to convert, with every hundred Ten thousand count (cpm) meter.For stable state, the transcript excessively presented in SLAM-seq is with (the height transcription output of red mark； N=828).The most abundant transcript (high stable state expression when with yellow mark stable state；N=825).It shows and corresponds to mES cell The transcript of specific primary miRNA cluster miR-290 and miR-182.(C) the potential transcription factor of just prediction (uses Ingenuity Pathway Analysis；Www.Ingenuity.com) and for molecular pathways (using Enrichr) compare Compared with 828 genes excessively presented from the RNA (SLAM-seq) newly transcribed and pass through routine 3 ' end sequencing of mRNA (stable state) Preceding 825 genes detected in the steady state.

The global analysis of mRNA stability in Figure 11 .mES cell.(A) polyadenylic acid in mES cell is determined through the invention Change the experimental setup of the stability of mRNA.(B) in mES cell mRNA half-life period global analysis.When relative to pulse labeling for 24 hours Between point standardization contain T > C conversion read relative fractions, the read navigates in mES cell 9430 abundant expression bases 3 ' UTR of the note of cause, and intermediate value, upper and lower part quartile using the fitting of single exponent ring-down dynamics at any time, Disclose intermediate value mRNA half-life period (~t_1/2) it is 4.0 hours.(C) half-life period of individual exemplary transcription objects calculates.It shows opposite Contain the flat of the read of T > C conversion in the duplicate 24 hour burst length point of three independences of Junb, Id1, Eif5a and Ndufa7 Equal score, and it is fitted to single exponent ring-down dynamics.Indicate being averaged for each transcript such as determined by curve matching Half-life period (t_1/2).(D) it is indicated by Tukey box-shaped figure of the mRNA 3' end sequencing to the mRNA half-life period that transcript determines, The transcript according to its correlation GO terms classification be modulability (i.e. transcriptional regulatory, signal transduction, cell cycle and development) or House keeper's (i.e. extracellular matrix, metabolic process and protein synthesis).Indicate the transcript number of each classification.Indicate P value (examined and determined by Mann-Whitney).

The alkylation of Figure 12 mercaptan connection is used for the metabolic marker of tiny RNA.(A) Drosophila S 2 cells selected from size are total The representative genome browser screenshot capture in the tiny RNA library that RNA is generated.It shows in Drosophila melanogaster genome and encodes miR- 184 representative area.The end 5' relative to every kind of tiny RNA type denotes the nucleotide of encoding thymidine pyrimidine (T, red) Position.99% read for accounting for all 5 ' isotypes is shown to miR-184-3p and -5p, and the respective number of read is with hundred Very much one (ppm) instruction.It (B) will be from s⁴Before U metabolic marker and s⁴Drosophila S 2 cells is total after U metabolic marker 24 hours The tiny RNA sequencing library that RNA is generated navigates to the miRNA with annotation, and determines and appoint to the miRNA of abundant expression (> 100ppm) What given mutation rate.Tukey box-shaped figure is with the mutation of each miRNA of percentages show.Exceptional value is not shown.It indicates per each and every one The intermediate value observed frequency not being mutated.It indicates as examined determining P value by Mann-Whitney.

The biogenous dynamics intracellular of Figure 13 Microrna.(A) pass through the RNA enzyme III enzyme Drosha and cytoplasm in core In Dicer sequence processing from containing hair clip rna plymerase ii transcript (primary Microrna, pri-miRNA) derivative it is micro- Tiny RNA leads to about 22nt Microrna duplex.Hair clip process intermediate (precursor Microrna, pre-miRNA) with RanGTP according to Property mode is relied to be output to cytoplasm from core by Ranbp21.(B) accumulation distribution figure is shown from s⁴The fruit of U processing specified time Fly ago2^koIt is enriched in the miRNA (left side) expressed or 20 kinds of miR* (right side) for 42 kinds in the tiny RNA library that the total serum IgE of S2 cell generates Value T > C mutation rate.It is examined by Kolmogorov-Smirnov and determines P value (*=p < 10 * * *^-4)。Bg^maxIndicate that maximum background is wrong Accidentally rate.(C) in stable state or pass through s⁴Display is specified at the read for containing T > C conversion of specified time point after U progress metabolic marker MiRNA (left side) or miR* (right side) average abundance.Relative to the standardized read number of total tiny RNA with hundred a ten thousandths (ppm) it shows.Instruction is higher than background maximum value (bg^max) excessive mutation rate (Mu).(D) Mirtron hair clip passes through encoding histone The montage of transcript generates.Uridine after intron lariat (lariat) Tuo Zhihou, mirtron hair clip is transcribed in cytoplasm (uriylation), the 2nt-3 ' jag of pre-mirtron before this is adjusted, to prevent from sending out by the miRNA biology of Dicer It is raw.(E) in s⁴The specified time point of U labelling experiment, specification miRNA (grey) or mirtron (red) T > C mutation rate (on) Or tiny RNA standardizes T > C read, in terms of hundred a ten thousandths (ppm).Intermediate value and quartile range are marked.Indicate P value (Mann-Whitney inspection) (*, p < 0.05；**,p<0.01；***,p<0.001).N.d., it is not detected.

The dynamics intracellular of Figure 14 Microrna load.(A) after generating, Microrna duplex is loaded into Argonaute On albumin A go1.In the process, selective retention is in Ago1 for one (miR chain) in two chains, and another chain (miR* Chain) it is discharged and degrades.Single-stranded miRNA in conjunction with Ago1 forms the silencing complex (miRISC) of mature miRNA induction. (B) in ago2^koS in S2 cell⁴The miR of 20 abundant expression and miR* pairs contains T > C during U metabolic marker is tested The intermediate value of the read of conversion accumulates (in terms of ppm).Show the intermediate value and quartile model of miR (red) and miR* (blue) It encloses.The value is obtained from independent measurement twice.The instruction of P value is as examined determining miR and miR* by Mann Whitney It is significantly separated (*, p < 0.05；****,p<0.0001).(C) aobvious to miR (red, top) and miR* (blue, bottom) in (B) The amplification for the time course shown.

The dynamics intracellular of the circumscribed molten nuclearity miRNA trimming of Figure 15.(A) the circumscribed molten nuclearity miRNA maturation mould in drosophila Type.Dicer generates one group of Microrna (such as miR-34) with longer about 24nt miRNA duplex, and the duplex exists It is loaded into Ago1 and removes and gone through 3' after miR* chain and nibble the circumscribed molten of device (Nibbler) mediation to 5' exoribonucleases Nuclearity maturation is to form the silencing complex that mature, Gene regulation miRNA is induced.(B) drosophila ago2^koIn S2 cell The equilibrium length of miR-34-5p is distributed, such as by the high-flux sequence of tiny RNA (it is left, cylindricality indicate 18 average values measured ± Standard deviation；The colony count that is averaged is indicated with every one of million (ppm)) or Northern hybrid experiment (right side) determination.(C) from into Row s⁴U metabolic marker reaches the drosophila ago2 of specified time^koMiR-34-5p distribution of lengths in the library of S2 cell preparation.Display Contain the distribution of lengths of read (label, red, top) and all reads (stable state, black, bottom) of T > C conversion.Refer to Potential read number is shown.Data show two duplicate mean+SDs of independence.(D) from progress s⁴U metabolic marker Up to the drosophila ago2 of specified time^koThe weighted average length of miR-34-5p in the library of S2 cell preparation.Data expression contains The mean+SD of the read (label, red) and all reads (stable state, black) that there is T > C to convert.It is converted containing T > C Read weighted average length the circumscribed molten nuclearity trimming (being highlighted by gray area) of reduction instruction.(E)miR-34-5p Load, such as pass through the s of Drosophila S 2 cells⁴MiR-34-5p (miR chain, red) and miR-34-3p (miR* after U metabolic marker Chain, blue) in contain T > C conversion read relative abundance determine.Show the mean+SD of two independent experiments. The discrete representation by miR and miR* is loaded, and is highlighted with gray area.

The difference stability of Figure 16 .miRNA.(A) Microrna duplex is loaded on Ago1, forms pre-miRISC, MiR* chain (blue) is degraded, and forms the silencing complex (miRISC) of mature miRNA induction.MiRNA's in miRISC Precise and stable property is still unclear.(B) in drosophila ago2^koIn s in S2 cell⁴41 are enriched between the time course of U metabolic marker The miRNA (red, left) of expression and each T location mutation rate of 20 miR* (blue, right) increase.For each tiny RNA, really Determine the intermediate value mutation rate between all T locations and standardized it relative to 24 hour time point.Show intermediate value and quartile Range.Value represents two duplicate average values of independence.Median half-life (t_1/2) and 95% confidence interval it is quasi- by single index curve It closes and determines.(C) Tukey case figure represents the half-life period of 41 miR (red) and 20 miR* (blue).Pass through Mann- Whitney, which is examined, determines P value.(D) the stable state abundance and mean half-life of the miR (red) and miR* (blue) that indicate.It is average Half-life period represents two duplicate average values of independence.Report individual half-life measurements of two independent experiments (r1 and r2).It is super Cross measurement total time half-life data be designated as > for 24 hours.(E) 41miR determined in two independent biology repeat is (red Color) and the elimination half life values of 20miR* (blue) chain compare.Show Pearson correlation coefficient (r_P) and relevant p value.(F) small The stability difference of RNA facilitates the stable state abundance of miRNA.Show the elimination half life values of the 40miR relative to its stable state abundance.Number According to representing two duplicate average values of independent biochemical.

The stability of Figure 17 .Argonaute protein identity decision tiny RNA.(A) in drosophila, miRNA is preferentially loaded into Ago1 is upper to form miRISC.In parallel, the subset of miR* is loaded into Ago2 to form siRISC.The formation of siRISC is adjoint The specific methylation for the tiny RNA that Ago2 is combined on the position 3' terminal ribose sugar 2'.If Argonaute protein degree differentiation influences Tiny RNA, then stability is unknown.(B) pie chart indicates different inscribes in the tiny RNA library from wild-type Drosophila S2 cell The relative abundance of siRNA type and miRNA.It shows the result (unoxidized, upper diagram) from standard cloning approach and comes Self enrichment has the result (oxidation, bottom graph) of the Strategies For The Cloning of the tiny RNA of the modified end 3'.For the two libraries Denote the score of miR and miR*.Show being evenly distributed for 7 data sets.Indicate average library depth.(C) thermal map is aobvious Show the relative abundance of the miR (red) and miR* (blue) in specified library (gray scale).With respect to the ratio instruction presented in library The preferential association of tiny RNA and AGO1 (green) or AGO2 (red).(D) wild type (wt) S2 cell or pass through CRISPR/Cas9 S2 cell (the ago2 of genome project abatement Ago2^ko) Western blot analysis.Actin represents load control.(E) In Wild type (wt) and ago2^koThe relative abundance of miR and miR* in Drosophila S 2 cells rich in Ago2.Indicate intermediate value and quartile Number range.P value is matched by Wilcoxon to signed rank test (Wilcoxon matched-pairs signed rank Test it) determines.(F), from wild type (wt, black) or ago2^koS in S2 cell (red)⁴U metabolic marker time course Or have the Strategies For The Cloning of the tiny RNA of modified 3 ' end from wild type S2 cell (wt is aoxidized, blue) system using enrichment The decaying kinetics of tiny RNA (right side) of the sum rich in Ago1 in standby standard library rich in Ago2 (left side).Indicate two-phase or one The intermediate value and quartile range (being provided in such as text) of phase exponential fitting.Indicate the half-life period such as determined by curve matching (t1/2).In the dynamic (dynamical) situation of two-phase, it is shown that the quickly relative contribution of gentle slow kinetics.(G) ago2ko S2 cell In 30 kinds of the most abundant miRNA (red, Ago1) or had using enrichment modification 3 ' ends tiny RNA Strategies For The Cloning it is small The half-life period of the most abundant miR and miR* (blue, Ago2) in the library RNA.Show intermediate value and quartile range.Pass through Mann-Whitney, which is examined, determines P value.

4-thiourdine metabolic marker in Figure 18 Drosophila S 2 cells.In pulse labeling experiment in Drosophila S 2 cells, S4U mixes quantifying for total serum IgE after s4U metabolic marker reaches specified time.The unmodified uridine for showing and being determined by HPLC The s compared⁴U Replacement rate, (Spitzer et al. (2014) Meth Enzymol 539,113-161.) as discussed previously.Value represents Duplicate average value ± the SD of three independences.Maximum incorporation efficiency after denoting 24 hour-symbols.

The processing of Figure 19 iodoacetamide does not influence the quality in tiny RNA library.Will before and after being handled with iodoacetamide from The tiny RNA sequencing library that the total serum IgE of Drosophila S 2 cells generates navigates to the miRNA with annotation, and analyzes abundant expression miRNA(>100ppm).(A) to every kind of miRNA in the tiny RNA library from total serum IgE handled through iodoacetamide or untreated Determine any given mutation rate.Tukey box-shaped figure is with the mutation of each miRNA of percentages show.Exceptional value is not shown.Instruction The intermediate value observed frequency being each mutated individually.(B) tiny RNA prepared from total serum IgE handled through iodoacetamide or untreated MiRNA abundance in library.Denote Pearson came relative coefficient and relevant p value.(C) from being handled through iodoacetamide or not The multiple variation of individual miRNA expression in the tiny RNA library of the total serum IgE preparation of processing.

Figure 20 s in the tiny RNA of metabolic marker⁴The frequency of U incorporation.Tukey box-shaped figure, which is shown, carries out s⁴U metabolic marker (> the 100ppm) of 71 kinds of abundant expression in the tiny RNA library of the total serum IgE preparation of the size selection of 24 hours slave Drosophila S 2 cells The score of T > C conversion read containing 1,2 or 3 T > C mutation of every kind of miRNA.Indicate the intermediate value point of T > C conversion read Number.

Figure 21 .s⁴The biology that U metabolic marker does not influence Microrna occurs or load.From 5p the or 3p arm of Microrna precursor Derivative (left side), or constitute selectivity Argonaute such as and load the individual of the given tiny RNA of defined miR or miR* chain (right side) The excessive presentation or presentation of T > C conversion on position are insufficient.As a result the Microrna for being originated from 71 abundant expression (> 100ppm) is (right It should be in 35 5p-miRNA and 36 3p-miRNA or 44 miR and 27 miR*).It is examined by Mann-Whitney, by phase To the statistically-significant difference in presentation compared with the total group of designated position.N.s., p > 0.05；N.d., since data point has It limits and can not determine.

Figure 22 precursor-miRNA hangover is offset effective miRNA biology and is occurred.With s⁴U handle specified time after from ago2^koIt is related between pre-miRNA uridine and T > C mutation rate in the SLAM-seq tiny RNA library of S2 cell preparation Property.Show the related coefficient (r of Pearson came_P) and relevant p value.

Figure 23 iodoacetamide is to 6- thioguanine (s⁶G chemical modification).Show 6- Thioguanosine and iodoacetamide Chemical reaction (A) and such as by handling 6- Thioguanosine with iodo-acetamide when by the alkylation efficiency of mass spectrometric determination (B)。

Figure 24 alkylation converts the s identified in RNA with single nucleotide resolution rate by G- to-A⁶G incorporation.(A) iodine is used Acetamide (IAA) processing is with or without 6 thioguanine (s at single location (p8)⁶G) the RNA mixed, and carry out overall length production Then the reverse transcription and gel extraction of object carry out PCR amplification and high-throughput (HTP) sequencing.(B) it indicates using specified reverse Enzyme is recorded to compare RNA (left figure) presence or absence of iodoacetamide (IAA) processing and contain s⁶The RNA's (right figure) of G The mutation rate of each position.Value represents the duplicate average mutation rate ± SD of three independences.Indicate each repetitive sequence (r1-r3) The number of middle sequencing read.Show that the nucleotide identity at p8 occurs.(C) presence or absence of iodoacetamide (IAA) In the case where processing, Superscript II (SSII), Superscript III (SSIII) or Quant-seq reverse transcription are used The mutation rate of the instruction mutation of enzyme (QS).For containing s⁶G and be free of s⁶Both RNA oligonucleotides of G, with identical nucleotide Mutation rate is averaged between the position of identity.Indicate P value (examined and determined by Student t).N.s., not significant (p > 0.05)。

Figure 25 is positioned using the time resolution of the transcription response of SLAM-seq.(A) the sample work of SLAM-seq experiment Stream, the response after positioning 15 to 60 ' of Flavopiridol (flavopiridol) processing (300nM) in K562 cell.(B) do not have Or the positioning of total and conversion (>=2T > C) read of low all transgenosis GAPDH with Flavopiridol processing.(C) consider institute There is the box-shaped figure of the expression variation of the Flavopiridol induction of read or the read with >=1 and >=2 T > C conversion.Whisker instruction The range of 5-95%.(D) the BCR/ABL effector approach and kinase inhibitor of SLAM-seq research are used in K562 cell Rough schematic view (30' pretreatment, 60's⁴U label).(E) heat for the gene that 50 most variable upper reconciliations are lowered in .SLAM-seq Figure and hierarchical cluster and their behaviors in the K562 cell handled with specified inhibitor in total mRNA level in-site.(F) make For the estimation half for adjusting the gene of >=2 times of detections is taken off in total mRNA or SLAM-seq by least one of (D) inhibitor It declines the phase.(G) as described in (D), come the principal component analysis of the SLAM-seq read of the K562 for specified inhibitor processing of using by oneself.

Figure 26 .BETi over anaphylaxis is different from by the global transcription control of BRD4.(A) AID-BRD4 knocks in equipotential base The schematic diagram of cause and Tir1 delivery vector SOP.(B) in the K562AID- for being transduceed with SOP and auxin (100 μM of IAA) being used to handle The Western blot of BOP4 in BRD4 cell.(C) in s⁴U marks the K562AID-BRD4 before 60' with auxin processing 30' thin The SLAM-seq response of born of the same parents.(D) in s4Us⁴SLAM- of the U labeled as the K562 cell for using JQ1 (200nM) processing 30' before 60' Seq response.(E) to the SLAM-seq response of JQ1 in the MV4-11 cell of K562 cell and same treatment shown in (C) Compare.R, Pearson correlation coefficient.(F) compare averagely SLAM-seq response and average CRISPR score, indicate K562, MOLM- Gene in 13 and MV4-11 cell is essential (14,15).Show all genes significantly lowered in all three cell lines (FDR≤0.1).(G) as carried out in (C), to come the s of the MOLM-13 cell for JQ1 or CDK9 inhibitor NVP-2 processing of using by oneself⁴U The SLAM-seq read of label carries out principal component analysis.(H) SLAM-seq that JQ1 and CDK9 is inhibited in specified cell line The thermal map and hierarchical cluster of Spearman rank correlation between response.

Figure 27 chromatin background determines BETi over anaphylaxis.(A) it is distinguished by the fallout predictor specified in K562 cell The ROC curve of BETi tetchiness gene and expression matching control group.(B) Vean diagram shows BETi tetchiness in K562 cell The overlapping of gene and the super enhancer target delivered.(C) the H3K27ac ChIP- of the selected genes of the classification in B is illustrated The sample tracking of seq and super enhancer annotation.(D) simplify model generate workflow, for based on away from TSS 500bp or 214 chromatin labels in 2000bp classify to BETi tetchiness gene.(E) (held-out) test set is being reserved The independent BETi over anaphylaxis model based on chromatin label of two of upper assessment such as the ROC curve in (A).(F) root According to standardized model coefficient, relative contribution of five strongest positive and negative fallout predictors to the GLM shown in (E).(G) 125 At the TSS of BETi tetchiness gene in (F) the opposite ChIP-seq density of predictive factor thermal map and hierarchical cluster.

Figure 28 .MYC is the direct activator of selectivity of the cell metabolism between each cancer types.(A) MYC-AID allele With the schematic diagram of Tir1 carrier.(B) after auxin processing specified time, the MYC Western blot in K562MYC-AID cell. (C) (pretreatment of 30' auxin, the 60 ' s of the SLAM-seq overview in K562MYC-AID cell after MYC degradation⁴U label).(D) The SLAM-seq response of the gene set of all mRNA and significant enrichment.(E) as carried out in the HCT116MYC-AID cell in (B) MYC Western blot.(F) compare the SLAM-seq response in K562MYC-AID and HCT116MYC-AID cell.(G) it distinguishes (C) MYC relies on the ROC curve of the different fallout predictors of gene (FDR≤0.1, log2FC≤- 1) and expression matching control group in. MYC/MAX ChIP, by the gene of ChIP-seq signal sequence in the 2kbp away from TSS；GLM is based on 214 chromatin overviews Elastic network(s) GLM.(H) relative contribution of the strongest positive and negative fallout predictor to GLM in (G).(I) base in 672 cancerous cell lines In the expression of the MYC target label of MYC and SLAM-seq.Highlight the sample that MYCN or MYCL level is more than MYC level. The GSEA of MYC target label in the cell line of the AML patient of (J, K) from (I) or with high or low MYC expression.(L) based on height Or low MYC expression and cancer types and the MYC- target tag expression between 5583 Patient Sample As separating.* * *, p < 0.0001 (rank sum test of Wilcoxon).

Experimental setup of Figure 29 for the SLAM-seq of position disparity gene expression.(A) targeting disturbance and its to having The schematic diagram of the main and minor effect of the mRNA level in-site of the gene of different turnover rates is handled between MYC target gene with BETi Inhibition is connect to illustrate.(B) pass through the 4-thiourdine residue in iodoacetamide subsequently mRNA.(C) with the 60' label time Contribution of the background error rate to the read with >=1 or 2 T > C conversion in SLAM-seq experiment.By from through s⁴U processing Alkylation parallel with the mRNA's of untreated cell and sequencing are to measure background signal.Box-shaped figure, which is shown, to be had from through s⁴U Height (top 10%), medium (45-55%) and low (bottom 10%) mRNA of the score estimation of read are marked in the library of processing The error rate of the gene of turnover.The range of whisker instruction 5-95%.(D) function as the T content of each read, passes through The recycling of the newly synthesized mRNA of SLAM-seq.Estimate 11.4% labeling effciency based on every T in newly synthesized mRNA.Top is straight Square figure shows the T content of 3 ' UTR reads of different read length.(E) SLAM-seq sample shown in Figure 25 E is indicated Kinase inhibitor processing K562 cell in MAPK and AKT approach inhibition verifying.

Direct JQ1 response in Figure 30 myeloid leukemia cell line.(A) box-shaped figure is summarized through SLAM-seq to figure With the global changes in gene expression of the JQ1 K562 cell measurement handled in 26D.(B, C) is for cell line MOLM-13 and MV4- 11, as shown in Figure 2 D, pass through the main response to JQ1 processing of SLAM-seq positioning.(D) to MOLM-13 and MV4- such as in (A) After 11 cells carry out JQ1 processing, the summary of global changes in gene expression.Gene expression after the JQ1 processing of (E, F) designated cell system The pairs of comparison of variation.(G) compare SLAM-seq in K562 cell shown in Figure 26, B and C to degrade to JQ1 and short-term BRD4 Response.The gene usually induced in MOLM-13, MV4-11 and K562 after JQ1 processing and after BRD4 degradation is with blue It highlights.* * * instruction is for intermediate value and 0 deviation, p < 0.0001 calculated by Wilcoxon signed rank test.

Figure 31 synergistic effect that CDK9 and BET bromine structural domain inhibits on cell and transcriptional level.(A) pass through The NVP-2 and JQ1 by prescribed dose of CellTiter-Glo luminescent cell survival amylograph measurement are to MOLM-13 and OCI- The growth inhibition of AML3 cell was up to 3 days.(B) synergistic effect of the JQ1 in (A) and NVP-2, is expressed as more than Bliss additive property. (C) in s⁴U is marked in the MOLM-13 cell of the pre-treatment 30 ' of 60', the master of JQ1, NVP-2 and combined treatment to prescribed dose It replys.(D) to the pairs of comparison of the response of JQ1 shown in (C) He centre dosage NVP-2 (6nM).R indicates Pearson came phase Relationship number.(E) as in (C), mRNA is exported after NCI-2 and JQ1 processing OCI-AML3 cell global change.Point instruction is at three S in independent duplicate SLAM-seq⁴Score of the read of U label relative to total read.(F) NVP-2 and JQ1 is used in (E) The principal component analysis of the SLAM-seq read of the OCI/AML-3 cell of processing.Percentage is indicated by the total of each Principal Component Explanation The score of body variance.

Figure 32 is derived and the fallout predictor of test JQ1 over anaphylaxis.(A) based on 90 ' JQ1 processing (30 ' pretreatments+60 ' s⁴U label) afterwards the JQ-1 tetchiness gene of SLAM-seq response and balance one group of crt gene selection workflow.It is logical It crosses iteration secondary sample (subsampling) and examines (KS) to test equal baseline by using Kolmogorov-Smirnov Expression is to select crt gene.(B) for predicting JQ1 tetchiness gene and crt gene by super enhancer (SE) ROC curve.Each SE is included into the gene closest to TSS, and is classified by super enhancer grade to gene.(C) Workflow for the classifier based on TSS of BETi over anaphylaxis in K562 cell shown in derivation graph 27E.SVM branch Vector machine is held, GLM passes through generalized linear model derived from elastic network(s) regularization, GBM gradient lift scheme.

Figure 33 predicts the characterization of the GLM of JQ1 over anaphylaxis in K562 cell.(A) all to facilitate shown in Figure 27 E GLM fallout predictor coefficient figure.Adjacent table lists each fallout predictor, the mark for the ChIP-seq tracking delivered accordingly Know symbol (being also shown in Table S1) and whether within the 500 or 2000bp away from transcription initiation site measuring signal.(B) based in (A) 5 The thermal map of the JQ1 tetchiness gene of the opposite ChIP-seq signal of a strongest positive and negative uniqueness fallout predictor and control gene and Hierarchical cluster.

To the main response of MYC degradation in the endogenous tagged MYC-AID cell line of Figure 34.(A) with life shown in Fig. 4 C The volcano figure of the SLAM-seq response of the K562MYC-AID cell of long element processing.Illustration lists the lower gene for reconciling and raising (FDR≤0.1) and de- adjusting are more than the sum of twice of gene.For clarity, ordinate value is limited to 20.(B) and refer to Determine the SLAM-seq response of the HCT116MYC-AID cell in (B) of the relevant gene of GO term.(C) volcano figure is shown such as (A) The SLAM-seq result of the middle HCT116MYC-AID cell handled with auxin.(D) different for the coding by GO term packet The gene of RNA polymerase complex subunit, the SLAM-seq response to K562MYC-AID cell middle or short term MYC degradation.

Figure 35 .MYC rely on gene expression based on chromatinic prediction.(A) ROC curve is measured and is being distinguished in Figure 28 C MYC dependent gene (FDR≤1, log2FC≤- 1) be not responding to MYC degradation gene (FDR≤0.1, -0.2≤log2FC≤ 0.2) performance of 5 classifiers in.Model such as exports in Figure 32, the test set training to 802 genes, and to 268 bases The test set of cause is assessed.(B) table of the GLM shown in ROC curve assessment (A) to another group of gene reserved to verify It is existing.(C) coefficient of all fallout predictors of the GLM shown in (A).(D) ROC, for by the 2000bp away from gene TSS MYC dependent gene is predicted in the presence at the peak MYC ChIP-seq.For each gene, based on CAGE-seq signal behavior to thin Born of the same parents' mRNA level in-site contributes strongest TSS, in Figure 32.The peak that the peak SPP is called by SPP, the peak IDR pass through irreproducible discovery rate The peak of threshold value 2%.(E) it is such as grouped into the Vean diagram of 7135 genes of MYC combination gene and MYC dependent gene above.

MYC is directly to the expression of homologue in Figure 36 human carcinoma cell line.(A) RNA-seq from 672 cancerous cell lines is general The comparison of MYC and MYCL expression in condition.(B) such as the comparison of MYC and MYCN expression in the cancerous cell line in (A).

PH dependence alkali during the RNA oligonucleotide reverse transcription that Figure 37 passes through the Broxuridine containing 5- (5BrU) of sequencing measurement Base pair frequency.(a) tautomeric form of 5BrU shows different pH dependence base pairings from adenine or guanine Characteristic.(b) experiment influenced under the background of RNA on the pH dependence that nucleotide at the site modified through 5BrU accidentally mixes is detected Schematic overview.(c) it is observed at the nucleotide position modified through 5BrU after reverse transcription under the conditions of specified pH specific The conversion ratio of conversion.Indicate the increase multiple relative to the conversion ratio of pH7 condition.The number independently measured is n=3, and Cylindricality indicates average conversion ± SD.Indicate P value (unpaired parameter Student t is examined).N.s., not significant (p > 0.05)；*,p<0.05；**,p<0.01；***,p<0.001；With * * * *, p < 0.0001.

Embodiment