Method for in-situ characterization of chemically modified DNA

文档序号：1916850 发布日期：2021-12-03 浏览：11次中文

阅读说明：本技术 一种对化学修饰的dna进行原位表征的方法 (Method for in-situ characterization of chemically modified DNA ) 是由李峰王冠唐娅楠于 2021-08-11 设计创作，主要内容包括：本发明公开了一种对化学修饰的DNA进行原位表征的方法。化学修饰是扩展天然DNA化学多样性和功能的有效途径。然而,当化学修饰的寡核苷酸用于基于DNA的反应或结构中时,预测和控制它们的动力学和热力学变得相当困难。为了应对这一挑战,本发明的方法中引入了DNA天平和砝码探针,其能够测量化学修饰的DNA在其天然环境中的临界热力学和动力学特性,例如吉布斯自由能和杂交速率常数的变化。该方法不仅可以测量稳定的化学修饰,还解决了涉及不稳定化学修饰和瞬态异构化反应的更具挑战性的问题。该方法在生物学、生物医学、材料科学、纳米光子学和纳米电子学等领域具有非常好的潜在应用价值。(The invention discloses a method for carrying out in-situ characterization on chemically modified DNA. Chemical modification is an effective way to expand the chemical diversity and function of natural DNA. However, when chemically modified oligonucleotides are used in DNA-based reactions or structures, predicting and controlling their kinetics and thermodynamics becomes quite difficult. To address this challenge, the method of the present invention incorporates a DNA balance and weight probe that is capable of measuring the critical thermodynamic and kinetic properties of chemically modified DNA in its natural environment, such as changes in gibbs free energy and hybridization rate constants. This method not only allows for the measurement of stable chemical modifications, but also solves the more challenging problems related to unstable chemical modifications and transient isomerization reactions. The method has very good potential application value in the fields of biology, biomedicine, material science, nanophotonics, nanoelectronics and the like.)

1. A method of detecting chemical modification of DNA comprising the steps of:

(1) preparing a chain C and a weight W according to the target DNA chain T;

(2) hybridizing the chain T with the chain C to obtain a chain CT;

(3) reacting the chain CT with a weight W, and detecting;

wherein the strand T is single-stranded DNA, and is divided into fragments S₁And S₂Wherein the segment S₂Comprising at least one chemical modification;

chain C is a single-stranded DNA consisting of a fragment S_r、S_m、S_fAre connected in sequence to form a compound in which S_rAnd S_mAre respectively S₁And S₂Complementary sequence of (1), S_fIs a toehold sequence;

the chain C and the chain T are respectively marked with a fluorescence reporter group and a fluorescence quenching group;

the weight W is a single-stranded DNA composed of a fragment W_fAnd W_rA connecting composition in which W_rIs S_mComplementary sequence of (1), W_fIs S_fA complementary sequence of a part of (1), and W_f-W_rAnd S_m-S_fAnd (4) complementation.

2. The method of claim 1, wherein the chemical modification is a methylation modification or a photosensitive molecular modification;

preferably, the chain T comprises at least one X structure, X beingR is a photosensitive molecular residue;

more preferably, R is an azo compound residue.

3. The method according to claim 1, wherein the length of the strand T is 15nt or more, preferably 15 to 30 nt;

S_rthe length of (a) is 1 to 14nt, preferably 4 to 10 nt;

S_fthe length of (A) is 2nt or more, preferably 2 to 20nt, more preferably 4 to 10 nt.

4. The method of claim 1, wherein S is_fComprises the nucleoside shown as SEQ ID NO. 1A sequence or consisting thereof.

5. The method of claim 1, wherein the method further comprises:

let chain T₀Hybridizing with C to obtain a control chain CT₀；

Make the control chain CT₀Reacting with a weight W, and detecting;

wherein the chain T₀Identical to the chain T sequence but containing no chemical modifications.

6. The method of claim 1, further comprising the step of computationally converting the test results into thermodynamic and kinetic parameters.

7. The method of claim 1, wherein the strand T is as set forth in SEQ ID NO 2;

the chain C comprises or consists of the nucleotide sequence shown as SEQ ID NO. 3;

the weight W is selected from: one or more of the nucleotide sequences shown by SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18 and SEQ ID NO. 19.

8. Use of the method of any one of claims 1 to 7 for the characterization and detection of chemically modified DNA.

9. Use of the method of any one of claims 1 to 7 for guidance and evaluation of the design of a DNA sequence to be chemically modified.

10. Use of the method of any one of claims 1-7 in the preparation of light-sensitive nucleic acids, light-controlled DNA materials, light-controlled DNA nanodevices, light-controlled gene expression systems, and the like.

Technical Field

The invention relates to the technical field of biology, in particular to a method for carrying out in-situ characterization on chemically modified DNA and application thereof.

Background

In addition to the biological role of storing and transmitting genetic information, DNA has also emerged in highly programmable engineering materials for the construction of various nanostructures and nanodevices. The field of research in DNA nanotechnology is revolutionary in its ability to control molecular self-assembly, thus having a significant impact not only on material science, but also on molecular computing, biosensing, diagnostics, and therapeutics. One of the biggest advantages of distinguishing DNA from any other engineered material is the predictability based on the unique Watson-Crick base-pairing rules. Important thermodynamic parameters, such as the standard gibbs free energy (Δ G °), can be accurately predicted using computer tools such as NUPACK. Such computer tools play a key role in designing nucleic acid hybridization probes and building blocks for various applications. The more advanced use of thermodynamic parameters further supports the development of analogue guidelines for the identification of super-specific hybridization probes and primers for single nucleotide variants. Key kinetic parameters, such as rate constants, also establish a good basis for the toehold-mediated strand displacement reaction, an important component of a class of dynamic DNA nanotechnologies, enabling the simulation of complex DNA reaction networks using computer tools such as Visual DSD.

To further extend the chemical diversity and functionality of DNA, a number of chemical modifications have been introduced to enable increased nuclease resistance, modulation of gene expression, or to allow spatial/temporal control of hybridization events. While the chemistry of DNA is significantly diversified, accurate prediction of hybridization kinetics and thermodynamics becomes difficult due to the changes produced by chemical modifications. Critical thermodynamic parameters such as Δ G ° and Tm can be measured using Isothermal Titration Calorimetry (ITC), Differential Scanning Calorimetry (DSC), or DNA melting analysis. However, these methods generally require cumbersome procedures and heating procedures, and thus it is difficult to measure chemically modified DNA in its natural environment. Existing tools are also limited to labile DNA modifications, such as many photoconversion molecules, including azobenzene, spiropyrans, diarylethenes, and thioindigoids, among others. Furthermore, the prior art does not provide kinetic analysis, since only the state of static equilibrium is recorded. Therefore, there is a great need for a simple, versatile and rapid method to measure the thermodynamic and kinetic properties of chemically modified DNA in its native form.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a method for detecting chemical modification of DNA, which comprises the following steps:

(1) preparing a chain C and a weight W according to the target DNA chain T;

(2) hybridizing the chain T with the chain C to obtain a chain CT;

(3) reacting the chain CT with a weight W, and detecting;

wherein the strand T is single-stranded DNA, and is divided into fragments S₁And S₂(i.e., chain T is S)₁-S₂) Wherein the segment S₂Comprising at least one chemical modification;

chain C is a single-stranded DNA consisting of a fragment S_r、S_m、S_fAre connected in sequence (i.e. chain C is S)_r-S_m-S_f) In which S is_rAnd S_mAre respectively S₁And S₂The complementary sequence of (i.e., fragment S)_r-S_mIs S₁-S₂Complementary sequence of (1), S_fIs a toehold sequence;

the chain C and the chain T are respectively marked with a fluorescence reporter group and a fluorescence quenching group;

the weight W is a single-stranded DNA composed of a fragment W_fAnd W_rConnected component (namely the weight W is W_f-W_r) Wherein W is_rIs S_mComplementary sequence of (1), W_fIs S_fA complementary sequence of a part of (1), and W_f-W_rAnd S_m-S_fAnd (4) complementation.

In some embodiments of the invention, the chemical modification is a methylation modification, for example, a covalent bond to a methyl group at the cytosine 5 carbon position of a CpG dinucleotide.

In some embodiments of the invention, the chemical modification is a photosensitive molecular modification, in particular, the insertion of photosensitive molecular residues in the form of chemical bonds on the DNA backbone; in particular, the photosensitive molecule may be selected from: azobenzene, spiropyran, diarylethene, hemithioindigo, stilbene, fulgide, etc., especially azobenzene.

Specifically, the position of the chemical modification may be a phosphate backbone, a base, or a ribosomal moiety of the chain T.

In particular, the chain T comprises at least one X structure, X beingR is a photosensitive molecular residue; more specifically, R is an azo-based compound residue, for example, and the like.

Specifically, the number of chemical modifications may be 1 or 2 or more.

Specifically, the length of the chain T may be 15nt or more (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100nt), particularly 15 to 100nt, 15 to 50nt, 15 to 40nt, 15 to 30nt, 15 to 25nt, 20 nt.

Specifically, S_rCan be 1-14nt, particularly 4-10nt (e.g., 4, 5, 6,7, 8, 9, 10 nt); in some embodiments of the invention, S_rIs 5-10nt in length.

Specifically, S_fThe length of (a) may be 2nt or more, for example 2 to 20nt, particularly 4 to 10nt (e.g. 4, 5, 6,7, 8, 9, 10 nt); in some embodiments of the invention, S_fHas a length of 10nt, wherein W_fCan be 1-9nt (e.g., 1, 2, 3, 4, 5, 6,7, 8, 9nt), particularly 3-9 nt.

In some embodiments of the invention, S_fComprises or consists of the nucleotide sequence shown as SEQ ID NO. 1.

In particular, the weight W may be one or more, in particular by adjusting the segments W individually_fAnd W_rA set of weights can be obtained, the "heavier" DNA weight being designed to be smaller than the "lighter" DNA weightContaining longer W_fAnd/or shorter W_r。

Specifically, the fluorescent reporter group may be, for example, FAM, Texas Red, ROX, TET, VIC, JOE, HEX, Cy3, Cy3.5, Cy5, Cy5.5, LC RED640, LC RED705, or the like.

Specifically, the fluorescence quenching group may be, for example, TAMRA, DABCYL, ECLIPSE, BHQ-1, BHQ-2, BHQ-3 or the like.

Specifically, the step (2) includes: mix and anneal strand C and strand T in buffer.

Specifically, the annealing step includes: the buffer containing chain C and chain T was heated to 90 ℃ for 5 minutes and then cooled 5 ℃ every 2 minutes until the temperature reached 20 ℃.

Specifically, the method further comprises a step of peeling (taring):

let chain T₀Hybridizing with C to obtain a control chain CT₀；

Make the control chain CT₀Reacting with a weight W, and detecting;

wherein the chain T₀Identical to the chain T sequence but containing no chemical modifications.

In particular, the detection may comprise the detection of a fluorescent signal.

Specifically, the reaction temperature in step (3) may be 20 to 30 ℃, for example, room temperature.

Specifically, the method further comprises a calculation step, for example, the detection result is converted into thermodynamic and kinetic parameters, such as Gibbs free energy, reaction rate constant and the like, through calculation, and further, the CT is compared with the control chain₀Comparing the thermodynamic and kinetic results to obtain the thermodynamic and kinetic changes caused by chemical modification.

In particular, the method can be used for characterizing thermodynamic and kinetic parameters of chemically modified DNA, such as Gibbs free energy changes of hybridization and photoisomerization, reaction rate constants of hybridization and photoisomerization, and the like.

In some embodiments of the invention, chain T is as set forth in SEQ ID NO 2.

In some embodiments of the invention, chain C comprises or consists of the nucleotide sequence set forth as SEQ ID NO. 3.

In some embodiments of the invention, the weight W is selected from: one or more of the nucleotide sequences shown by SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18 and SEQ ID NO. 19.

The invention also provides the use of the above method for the characterization of chemically modified DNA.

In particular, the chemical modification may be a methylation modification, for example, a covalent bond to a methyl group at the cytosine 5 carbon position of a CpG dinucleotide.

Specifically, the chemical modification may also be a photosensitive molecular modification; in particular, the photosensitive molecule may be selected from: azobenzene, spiropyran, diarylethene, hemithioindigo, stilbene, fulgide, etc., especially azobenzene.

Specifically, the position of the chemical modification may be a phosphate backbone, a base, or a ribosomal moiety of the chain T.

Specifically, the chemically modified DNA comprises at least one X structure, wherein X isR is a photosensitive molecular residue; more specifically, R is an azo-based compound residue, for example, and the like.

In particular, the characterization includes characterization of thermodynamic and kinetic parameters of the chemically modified DNA, e.g., gibbs free energy changes for hybridization and photoisomerization, reaction rate constants for hybridization and photoisomerization, and the like.

The invention also provides the use of the above method in the design guidance and evaluation of DNA sequences to be chemically modified.

Specifically, the design guidance and evaluation relates to the modification position, the number of modifications, design guidance and evaluation of nucleotides adjacent to the modification position, and the like.

The invention also provides the application of the method in the preparation of photosensitive nucleic acid, light-operated DNA materials, light-operated DNA nano devices, light-operated gene expression systems and the like.

Specifically, the photosensitive nucleic acid, the light-controlled DNA material, the light-controlled DNA nano device and the light-controlled gene expression system are prepared by the step of embedding a photosensitive molecule residue in a chemical bond form on a DNA framework.

In particular, the photosensitive molecule may be selected from: azobenzene, spiropyran, diarylethene, hemithioindigo, stilbene, fulgide, etc., especially azobenzene.

In particular, the embedded photo-sensitive molecule residue may be an azo-type compound residue, for example, and the like.

Specifically, the light-operated DNA nanodevices include, for example, light-operated DNA tweezers, light-operated DNA pedestrians, light-operated DNA nanocapsules, and the like (as described in "von seipine, zhanghong. light-operated nanodevices constructed from azobenzene-modified DNA are applied and envisioned [ J ]. chemical new materials, 2016: 34-36.").

The invention also provides the application of the method in the detection of DNA modification.

The invention also provides the use of the above method in the assessment of gene damage caused by chemical modification of DNA.

Specifically, the chemical modification is a methylation modification, for example, a covalent bond to a methyl group at the cytosine carbon position 5 of a CpG dinucleotide.

The invention also provides the application of the method in the detection of non-classical DNA structures.

In particular, the non-classical DNA structures described above are, for example, i-motif.

The invention provides a method for detecting chemical modification of DNA, which utilizes a reasonably designed DNA balance and can measure the hybridization thermodynamics and strand displacement kinetics of the chemically modified DNA by a toehold displacement principle. The inventors have successfully determined the critical thermodynamic and kinetic parameters of cyclic azobenzene (cabs), which is an optical switch modification of DNA, using a DNA balance. More importantly, Δ G ° changes for both hybridization and photoisomerization have been quantitatively described, which is not possible with classical methods such as ITC, DSC, and DNA melting analysis. The critical rate constants for strand displacement and isomerization have also been successfully determined by screening kinetic sensitive DNA weights and using DNA balance in conjunction with a mathematical model. The method does not require specialized instrumentation and can be easily extended from cabs to other chemical modifications.

Another advantage of this method is that it characterizes chemically modified DNA under targeted native conditions. In previous studies, Zhang and co-workers demonstrated that natural Characterization of Nucleic Acid Motif Thermodynamics can lead to significantly better accuracy of measured Δ G ° (Wang, C.; Bae, J.H.; Zhang, D.Y. Native charaterization of Nucleic Acid motion of Nucleic acids vitamin Non-volatile catalysis, Nat. Commin.2016, 7,10319.). Similarly, one of the most representative conditions in DNA nanotechnology (room temperature, with 12.5mM Mg)²⁺Tris-EDTA buffer) the DNA equilibrium measures the thermodynamics of hybridization of chemically modified DNA. Thus, key thermodynamic parameters identified in the present method can be readily used to guide the design and use of cAB modified DNA sequences. Notably, the thermodynamics of the photoisomerization was estimated by combining experimental measurements with the inventors' theoretical model. Assuming that the optical isomers have minimal effect on the reverse strand displacement reaction, errors may be introduced into the estimated Δ G ° values for the trans-to-cis transition. However, the inventors believe that this error is minimal because the measured Δ G ° values of isomerization reasonably reflect the overall performance of DNA equilibrium in the presence of both isomers. Setting up experiments, e.g. at constant illuminationMaking measurements to maintain the trans isomer can help improve the accuracy of the measurement.

In addition to thermodynamics, this method also offers the possibility to measure transient chemical changes (e.g. isomerization) within DNA species.

The determination of the rate constant of the chemically modified DNA strand displacement reaction can also be applied to dynamic DNA nanotechnology. Quantitative descriptions of the kinetics of the toehold-mediated strand displacement and toehold displacement reactions play a key role in guiding the design of different dynamic DNA devices and reaction networks. Although the kinetics of strand displacement are well defined for unmodified DNA sequences, chemically modified DNA is rarely explored. The inventors' work in designing DNA balances and building mathematical models provides powerful tools for characterizing the kinetic behavior of chemically modified DNA in a quantitative manner. The inventors' work also shows that cabs, which undergo drastic changes in DNA duplex stability upon photoisomerization, can be used as unique optical switches to design DNA probes with high temporal controllability (e.g., hybridization during photoisomerization and de-hybridization immediately after removal of the light source) or to design photoregulated dissipative reaction networks.

Therefore, the method provided by the invention has very good potential application value in the fields of biology, biomedicine, material science, nanophotonics, nanoelectronics and the like.

Drawings

FIG. 1 shows: (a) schematic diagram of DNA balance principle; (b) the toehold displacement is illustrative of the working principle of DNA balances; (c-d) structural and photoisomerization of cyclic azobenzenes; thermally stable cis-cabs isomerize to unstable trans-cabs by irradiation with 390nm light, and the reverse process is spontaneous upon heating at 520nm irradiation or under ambient conditions; (e) the effect of cAB modification and photoisomerization on DNA duplex stability.

FIG. 2 shows: reaction yield versus free energy of reactionThe experimental and theoretical thermodynamic diagrams were established. By plotting the theory calculated from equations S2 and S3Theoretical yield versus calculation using NUPACK softwareA graph of values (solid line) to establish a theoretical relationship graph. The reaction curves obtained from experiments using a set of DNA weight probes (dots) were fitted using a theoretical model (dotted line). Calculating sight +0.1kcal & mol according to least square error algorithm^-1And (6) correcting.

FIG. 3 shows: study of thermodynamic characterization of cis-cabs modification using a DNA balance; (a) schematic representation of the toehold displacement reaction and DNA weight probe set for measuring the thermodynamic change induced by cis-cAB modification; (b) then, drawing a graph of the reaction yield of each weight probe relative to the Gibbs free energy of each weight probe through experiments, and establishing a thermodynamic curve; further comparison with an "empty" DNA balance allows for direct reporting of changes in Δ G °; (c-f) experiments to determine the thermodynamic curves for a single cAB modification or multiple cAB modifications (f, g) at different sites (c-e), and for each modificationThe value is obtained.

FIG. 4 shows: mapping the reaction yield in equilibrium to the standard free energy of a group of weight probes, and then fitting by using a theoretical model; the scheme is suitable for cis-CAB (CT)_cis) And trans CAB (CT)_trans) Modified DNA, the number of modifications varies from 1(a), 2(b) to 3 (c). Observation of the thermodynamic diagrams of cis-and trans-cabs shows that it is not possible to directly use thermodynamic measurements to determine thermodynamic parameters due to spontaneous relaxation from the trans to cis isomer.

FIG. 5 shows: kinetic study of unmodified DNA balance with/without irradiation; (a) a schematic of the toehold displacement reaction without DNA modification served as a control to exclude possible interruption of the light irradiation; (b-j) kinetic curves of DNA balances with different DNA weight probes. The results show that light irradiation has no effect on the toehold displacement reaction.

FIG. 6 shows: the effect of the position of cis-cabs modification on kinetics; kinetic Curve equilibrium CT of DNA_cis-1NAnd CT_cis-1F. The slight difference between the two single cabs modification sites indicates that the modification sites have no significant effect on DNA balance kinetics.

FIG. 7 shows: kinetic profiles of DNA duplexes containing varying amounts of cabs modification. The presence of cis-cabs modification accelerates the kinetics of the toehold displacement when using the same DNA weight probe compared to unmodified DNA duplexes. Increasing the amount of modification further increases the rate of toehold displacement.

FIG. 8 shows: using a DNA balance to research the kinetic characteristics of cis-trans isomeric modification; (a) a schematic of the reaction pathway involving the toehold displacement reaction with isomerization-coupled cis-or trans-cabs; (b-j) the toehold displacement reaction of the DNA balance was monitored in real time with different weight probes. Comparison with light with or without illumination reveals a weight probe that is sensitive to isomerization reactions in DNA.

FIG. 9 shows: containing two cAB-modifications (CT)_cis-2) The kinetic profile of the DNA double strand in the presence or absence of light irradiation.

FIG. 10 shows: containing three cAB-modifications (CT)_cis-3) The kinetics of the DNA duplex of (1) in the presence or absence of light irradiation.

FIG. 11 shows: determining the rate constant of reaction of each element by a combination of experimental measurements and simulations using a DNA balance; (a) simplified reaction pathway of toehold displacement coupled with isomerization, apparent isomerization being considered a zero order reaction (k)₁) (ii) a The DNA equilibrium reaction is simplified to an apparent bimolecular reaction (k)_2app，k_3app) (ii) a (b) Determining k in sequence₁、k_3appAnd k_2appThe workflow of (1); (c-e) experimental validation of each step in the workflow using a single cAB modification of W (7, 7) as isomerization-sensitive weight probe; (d-f) experiments determined the rate constants for DNA duplexes with one (f), two (g) and three (h) cAB modifications.

FIG. 12 shows: the simulation results are used to evaluate the potential of the DNA balance to probe the dynamics of transient reactions or events in DNA; (a) the simulated kinetic curve of the toehold displacement reaction combined with the transient chemical change obtains the rate constants of the displacement reaction of the front and rear chains of the transient reaction by utilizing the experimental data of the work; (b) the upper and lower thresholds were set to 5% away from the independent strand displacement reaction before and after the transient chemical event.

Detailed Description

Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

In the present invention, the "azo-based compound" refers to a compound in which a phenyl group is bonded to both ends of an azo group.

In the present invention, nt means a nucleotide base.

In the present invention, "DNA modification" means that some change occurs in the structure of DNA through a series of chemical processes after it is synthesized.

The disclosures of the various publications, patents, and published patent specifications cited herein are hereby incorporated by reference in their entirety.

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The experimental method comprises the following steps:

synthesis of cyclic azobenzene (cAB) modified oligonucleotides: as previously reported, cAB modified oligonucleotides were generated by solid phase synthesis based on phosphoramidite chemistry and purified by PAGE.

DNA sequence: all non-cAB-modified DNA oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA) and purified by high performance liquid chromatography. The sequences and modifications are listed in the following table.

TABLE 1 DNA sequences

Buffer conditions: the DNA oligonucleotides were dissolved in 1 XTris-EDTA (TE) buffer (10mM Tris-HCl, pH 8.0, 1M EDTA, purchased from Sigma as 100 Xstock) and stored at-20 ℃. Unless otherwise stated, MgCl containing 12.5mM is used₂And 0.1% (v/v) TWEEN 20 in 1 XTE buffer (Sigma) as reaction buffer. TWEEN 20 was used to prevent potential loss of DNA oligonucleotides during dilution and pipetting steps.

Preparation of a DNA balance: chains C and T were first mixed and diluted from stock concentration 50. mu.M to 5. mu.M and 7.5. mu.M using reaction buffer, respectively. The DNA balance solution was then annealed in a BioRad T100 thermal cycler. The annealing program was set as follows: heat to 90 ℃ and hold for 5 minutes, then cool 5 ℃ every 2 minutes until the temperature reaches 20 ℃. The prepared DNA balance solutions, in particular those carrying the cabs modification, were covered with tin foil and stored protected from light.

Protocol for measuring cAB modification using DNA balance: the DNA balance and weight were diluted to 200nM and desired concentration, respectively, using the reaction buffer at room temperature. First, 90. mu.L of the diluted DNA weight solution was transferred to a 96-well plate. The DNA balance solution, stored in advance protected from light, was then irradiated by a 395nm LED for 30 minutes in a laboratory-made irradiation chamber. Once irradiation was complete, 10. mu.L of unirradiated and irradiated DNA balance (CT) was used_ciscABAnd CT_transcAB) The solution was added simultaneously to two parallel wells containing the same DNA weight design. Each DNA balance experiment was repeated twice. The plates were then read in a SpectraMax i3 fluorescent plate reader for 2 hours at room temperature with excitation/emission set at 485/535 nm.

Establishing a mathematical model: free energy information of DNA strands and complexes was estimated using the freeware NUPACK. For the parameter settings in NUPACK, the temperature was set at 25 ℃ and Mg was set²⁺Was set to a concentration of 10 mM. The other parameters are default settings. The thermodynamic mathematical model and the standard yield- Δ G ° relationship were solved and plotted by the numerical method of MATLAB (2019a, MathWorks). By usingCode was fitted and real-time kinetic analysis was performed in MATLAB.

Example 1: principle of design

The inventors propose a DNA balance that enables accurate measurement of thermodynamic and kinetic parameters to challenge DNA at constant room temperature (as shown in figure 1). Similar to a real world scale, the DNA scale measures thermodynamic and kinetic parameters of chemically modified DNA by comparing it to a set of DNA weights made of unmodified DNA with predetermined thermodynamic and kinetic parameters. The photoswitchable modification, cyclic azobenzene (cabs), was chosen as a test platform to demonstrate the feasibility of the inventors' strategy (fig. 1 c). Optical switch molecules such as azobenzene are a common means of building photoresponsive/controllable materials or systems by photoisomerization. Diazobenzenes and derivatives have been well studied and incorporated into DNA to construct photoresponsive constitutive dynamic networks, hydrogels, origami nanostructures, and drug delivery capsules. Since trans (E) -to-cis (Z) conversion of azobenzenes is generally limited by UV light, efforts have been made to modulate the light into the visible region and isomerize at room temperature for in vivo applications. Introduction of a cyclic structure pulls the absorption band into the visible region and produces an abnormal cis (Z) -to-trans (E) photoisomerization. The unstable trans (E) configuration can be converted back to the cis (Z) isomer by ambient heat and green illumination (520nm, FIG. 1 d). In this study, the inventors studied thermodynamic and kinetic changes caused by cAB modification in DNA with a DNA balance. Determination of the free energy Displacement of hybridization caused by Stable cis (Z) -cAB modification Using the static equilibrium State of a DNA balanceDetermination of the rate constant of the Strand Displacement reaction by real-time kinetic analysis and estimation of the contribution of the unstable Trans (E) -cAB

As shown in FIG. 1a, the DNA balance is designed as duplex DNA (CT) in which one strand is labeled with a fluorescent group (C) and the other strand is labeled with a quenching group (T). It also comprisesThe sticky ends, called DNA toehold, allow them to react rapidly with a set of single stranded DNA (ssDNA) weights (W) by the principle of toehold displacement (as shown in FIG. 1 b). The "weight" W (f, r) of each probe is defined by the length of the forward and reverse toolholes (f, r). "heavier" DNA weights are designed to contain longer forward-toolholes and/or shorter reverse-toolholes than "lighter" DNA weights, thus resulting in higher reaction yields when reacted with a DNA balance. The standard free energy, yield and rate constant of the toehold displacement reaction at the DNA day time for each DNA weight can be quantified on a computer by theoretical modeling (as shown in example 2) or by monitoring the fluorescence signal in real time. To measure chemical modification, a "peeling" step is first performed, in which an unmodified DNA balance (CT) is set up by reaction with a set of DNA weights with different f and r_N) Kinetic and thermodynamic diagrams (equation 1). The modification was then placed on a DNA balance by chemically binding it to the quencher-labeled DNA strand. Re-interrogation of the vector with chemical modification (CT) using the same set of DNA weights_cis) To obtain the thermodynamic and kinetic changes (equations 2-4) caused by chemical modification.

Unmodified DNA balance (eq.1)

Chemically modified DNA balance (eq.2)

The present study uses cyclic azobenzene (cabs), a representative class of optical switch DNA modifications in which direct probing of thermodynamics and kinetics using conventional techniques is challenging, as an experimental platform to validate the DNA balance strategy of the inventors. For this purpose, cabs were chemically bound to the phosphate backbone of the quencher-labeled chain T by solid phase synthesis based on phosphoramidite chemistry (as shown in fig. 1 c). The thermostable cis-cAB isomer can be photo-converted to trans-cAB upon 390nm illumination and reverse isomerization occurs spontaneously at ambient temperature or 520nm illumination (as shown in FIG. 1 d). The introduction of cis-cAB destabilizes the DNA duplex, while trans-cAB stabilizes the duplex by a more favorable planar conformation (as shown in FIG. 1 e).

Mathematically, cis-cAB modified duplex CT_cis Can be deduced as(equation 4), andcan be easily calculated using software tools such as NUPACK. Therefore, it is determinedAll that needs to be measured is(equation 3). In principle, the amount of the solvent to be used,it can be measured using a single DNA weight, which is almost impossible and unreliable for chemical modifications with unknown thermodynamics. In contrast, DNA weight sets were designed to cover a wider range of free energies of reaction. Thus, the inventors were able to easily establish a standard thermodynamic curve (as a standard thermodynamic curve) before and after placing chemical modifications on a DNA balanceA function of). The MATLAB code standard may then be usedAccurately determiningAs a shift of the standard curve.

For kinetic analysis, the rate constant of the cis-cAB-modified DNA strand displacement reaction can be directly measured by monitoring the fluorescence signal in real time. However, for strand displacement reactions involving trans-cabs, direct experimental measurements are not possible. This is because trans-cabs spontaneously relax to cis-cabs and thus lead to more complex kinetic behavior. Using a DNA balance, the inventors demonstrated that by combining experimental measurements and mathematical models, it was possible to address this challenge and accurately determine the rate constants for strand displacement and the transition from the trans to the cis isomer. Once critical kinetic parameters were obtained, the inventors were able to further derive the standard gibbs free energy of the transition between trans-and cis-cabs.

Example 2: theoretical framework

1.1 basis of thermodynamic simulation

The principle of the DNA balance is a series of toehold displacement reactions by which a thermodynamic diagram of chemical modifications can be established. Theoretically, the reaction yield (η) can be modeled using a bimolecular reaction:

wherein K_eq≠1； (eq.S2)

OrWherein K_eq＝1；(eq.S3)

Wherein W is a DNA weight and C is a partially complementary strand of the target T; equilibrium constantReaction yield eta＝[W_(f，r)C]/[CT_cAB]₀(ii) a Experimental parameters γ: is ═ T_cAB]/[CT_cAB]₀And τ: is ═ W_(f，r)]/[CT_cAB]₀(subscript 0 denotes initial state). Free energy of each DNA substanceCalculations can be performed using NUPACK software. When the experimental conditions of tau and gamma are constant, the only variable that changes in the DNA balance is the free energy of reaction corresponding to the DNA weightTheThe yield dependence is reflected in a sigmoidal calibration curve.

To obtain dimensionless reaction yield values from fluorescence data, the inventors normalized the fluorescence values by:

wherein F_CT、F_P.C.And F_BGRepresenting the equilibrium fluorescence signal generated from the DNA balance, the background level of the CT duplex, and the maximum signal level generated by C alone, respectively.

1.2 mathematical model of dynamics

For containing thermolabile trans-cAB modification (CT)_trans) The DNA balance of (a), which participates in two parallel reactions: direct DNA counterweight and isomerization to thermally stable CT_cis. Mathematical models of the exact kinetics of the reaction process are shown in equations S5 through S7.

Positive/negative rate constant k_E-f/k_E-r

Positive/negative rate constant k_Z-f/k_Z-r

CT_trans→CT_cis(ii) a Isomerization rate constant k_iso

T_trans→T_cis(ii) a Isomerization rate constant k_iso

In the same way as above, the first and second,

the boundary conditions are as follows: [ CT ]]_total＝[CT_trans]+[CT_trans]+[CW] (eq.S7)

The collective fluorescence signal generated by CW is:

clearly, fitting kinetic rate constants only from fluorescence data is challenging, as all reactions are highly coupled. Thus, the inventors have simplified the exact model to a mathematically solvable model (equations S9-S11):

CT_trans+W→CW+T_trans(ii) a Apparent bimolecular rate constant k_E-app

CT_cis+W→CW+T_cis(ii) a Apparent bimolecular rate constant k_Z-app

CT_trans→CT_cis(ii) a Constant intramolecular isomerization rate k_iso-app

The simplified mathematical model is therefore:

in particular, the inventors consider the generally accepted first-order isomerization reaction to be zero-order for two reasons: first, the incorporation of cycloazobenzene into the DNA strand, so that the isomerization is intramolecular; secondly, the kinetic sensitive linear region observed in the fluorescence curve indicates the presence of a zero-order rate-limiting step in the reaction.

The real-time fluorescence data was first normalized to a dimensionless yield. To reduce experimental variation from batch to batch, the inventors applied a normalization method as shown below:

wherein F_e.q.And K_e.q.Respectively representing the fluorescence level in equilibrium and the equilibrium constant of the corresponding DNA weight. DNA duplex CT due to Trans-cAB modification_transWill relax into CT during weighing_cisTherefore, CT_transAnd CT_cisBoth will achieve the same reaction yield. Thus, in the presence of a given DNA weight probe, the same K is applied to both_e.q.The value is obtained.

Example 3: design of DNA weight library and corresponding free energy of reaction

In total 16 DNA weights were designed for DNA balances, with forward toolhold lengths ranging from f-4 nt to f-9 nt and reverse toolhold lengths ranging from r-5 nt to r-10 nt. Each DNA weight is represented by the numerical values of f and r, and for example, the DNA weights with f equal to 9nt and r equal to 4nt are represented as W (9, 4). Predicting inverse per weight probe using NUPACKFree energy should be used. Combination of f and r, and prediction of each WAre listed in the following table.

TABLE 2.16 summary of DNA weight probes and predicted free energies of reactions

Example 4: measurement of the thermodynamic Properties of cis (Z) cAB-modified DNA

By predicting the experimentally determined yield for each DNA weightPlotting and establishing a standard thermodynamic curve for the unmodified DNA balance (as shown in figure 2). The experimentally determined calibration curve was found to be highly consistent with the computer prediction, only-0.1 kcal-mol^-1The small offset of (a) indicates that the thermodynamic curve can be accurately established experimentally using the DNA balance of the inventors.

The cis-cAB modification was then placed on a DNA balance for thermodynamic measurements. Three single cAB modifications were chemically incorporated into the DNA balance, but at different positions: near forward toe hold (TC)_cis-1NFIG. 3c), intermediate (TC)_cis-1MFIG. 3d) or in reverse grip (TC)_cis-1FFig. 3 e). The inventors also changed the number of modifications from 1 to 2 (FIG. 3f) and 3 (FIG. 3 g). Each dot in fig. 3c-3g represents the experimental measurement of a particular DNA weight. By fitting the experimental results of a set of DNA weights to a theoretical model using the least squares method, the inventors were able to establish thermodynamic calibration curves (dashed lines in fig. 3c-3 g) for cis-cAB modified DNA.

For all cis-cAB modifications, observedThis is consistent with previous reports on destabilization of DNA duplexes by cis-cabs. Quantitatively, a single cis-cAB modification results in +1.6 kcal.mol^-1And the position of the modification has little effect on the value of the energy shift. Similarly, differences in adjacent nucleotides also had little effect on the thermodynamic shift caused by a single cis-cAB (FIGS. 3c-3 e). As expected, rapidly increasing the number of cis-cAB modifications expanded(FIGS. 3f-3 g). It was observed that the modification ratio with two cis-cABs was two single modifications (+ 3.2 kcal. mol.)^-1) By carrying outMuch larger (+6.3 kcal. mol.)^-1) Indicating that multiple modifications may act synergistically to destabilize the DNA duplex.

After achieving thermodynamic characterization of cis-cabs, the next goal of the inventors was to measure the thermodynamics of trans-cabs and the transition between the two isomers. However, the inventors found that the thermodynamic parameters of trans-cabs cannot be directly measured due to spontaneous conversion from the trans-isomer to the cis-isomer (as shown in fig. 4). Since the DNA balance is governed by kinetics in this case, the inventors next measured critical kinetic parameters of the cabs modified DNA. Then, once the kinetic behavior of cabs is fully characterized, thermodynamic parameters can be derived.

Example 5: measurement of the kinetics of cAB-modified DNA

Kinetic characterization of cis-cAB modified DNA in the toehold-mediated strand displacement reaction was simple (FIGS. 5-7). The kinetic diagram in fig. 7 demonstrates that the binding of a single cis-cabs modification accelerates the kinetics of strand displacement with several DNA weights including W (7, 8), W (7, 7), W (9, 8) and W (8, 7). This is expected because cis-cAB destabilizes the CT duplex. For the other 'heavier' and 'lighter' weights, no observable kinetic differences were found (fig. 8b-8f, 8i-8j) as the overall reaction became much faster or slower. In summary, these observations indicate that some DNA weights are kinetically more sensitive than others in chemical modifications that are more suitable for probing the kinetics of chemically modified DNA.

The next goal of the inventors was to screen the DNA weights that were kinetically more sensitive to DNA balance carrying the trans-cAB modification. For this purpose, the cis-cAB-modified DNA duplex CT is used_cisIrradiation at 390nm for 30 min to ensure complete conversion to obtain a trans-cAB modification (CT)_trans). Immediately after irradiation, the DNA balance carrying the trans-cabs was interrogated with a set of DNA weights to initiate the toehold displacement reaction (fig. 8 a). At the same time, spontaneous relaxation from the trans-isomer to the cis-isomer occurs in parallel, which converts CT to_transConversion back to CT_cis(FIG. 8 a). By comparing the kinetic curves with and without light irradiation (fig. 8b-8j), the inventors found that most DNA weights were either too "light" (fig. 8b-8f) or too "heavy" (fig. 8i-8j) to reflect the isomerization-induced kinetic differences. In contrast, W (7, 7) and W (9, 8) showed observable kinetic changes in the presence of trans-cabs (fig. 8g and 8 h). As expected, trans-cabs stabilize the CT duplex and thus significantly slow the rate of strand displacement. However, due to spontaneous relaxation, a linear kinetic domain is observed before equilibrium is reached. Interestingly, the inventors also found that the most kinetically sensitive DNA weights W (7, 7) resulted in final reaction yields approaching 50% (fig. 8 g). For CT with two and three cAB modifications_transSimilar observations were also made (fig. 9, 10).

Due to the complexity of the reaction network (fig. 11a), kinetic parameters of trans-cabs modified DNA could not be directly measured. The inventors therefore propose to address this challenge by combining computer simulations with experimental validation (fig. 11 b). Specifically, the inventors first created a mathematical model (details in 1.2 in example 2) that quantitatively describes the dynamic behavior of a dynamic sensitive weight. The inventors' model showed that the linear region observed in FIG. 8g is from CT_transTo CT_cisAs a result of the spontaneous isomerization ofConstant of rate of change (k)₁) The value of (c) can be obtained from the slope of the linear region (fig. 11 c). Observation Rate constants (k) of cis-cAB-modified DNA balances₃) It can be determined experimentally without irradiation with light using a DNA balance (FIG. 11 d). Once k is measured₁And k₃Can be determined using the workflow outlined in FIG. 11b₂. Although different kinetic curves can be established on a computer using the inventor's mathematical model, only when the correct k is reached₂The predicted kinetic curve can only be superimposed with the experimental measurements (fig. 11 e).

For DNA balances with Single cAB modification (CT)_CAB-1Fig. 11f), DNA weights W (7, 7) are most kinetically sensitive to trans-cabs. By fitting a mathematical model to the experimental data using the workflow outlined in FIG. 11b, the inventors determined the apparent rate constant k₁、k₂And k₃Are respectively 2.17 multiplied by 10^-12M·s^-1、3.73×10²M·s^-1And 1.25X 10⁵M·s^-1. The trans isomer was observed to slow strand displacement by over 300-fold, indicating that trans cabs can effectively enhance adjacent base pairs and inhibit branch migration processes.

When increasing the number of cabs modifications, lighter weights (where W (4, 9) was used for 2 modifications and W (5, 10) for 3 modifications) were determined to be sensitive to photoisomerization kinetics. When increasing the number of cAB modifications, a slight increase in isomerization rate was observed, with k for both modifications₁The value was 6.40X 10^-12M·s^-1Three modifications, k₁The value was 1.03X 10^-11M·s^-1. Rate constant k compared to the rate constant of a single modification₂And k₃Remaining of the same order of magnitude. In summary, kinetic analysis quantitatively revealed an approximately 300-fold difference in the ability of trans and cis cabs to hybridize on stable/unstable DNA, and a single modification was best suited to produce significant kinetic differences when used for strand displacement.

After the kinetic parameters were determined, the final objective was to estimate the thermodynamic shift of the heat labile trans-cAB modification relative to the unmodified oneSince the main difference between trans and cis isomers is in DNA duplexes rather than single-stranded DNA, the inventors concluded that the rate of reverse strand displacement between T and CW was for T in a DNA balance_transAnd T_cisAre close. Thus, the equilibrium constant ratio K₂/K₃Is equal to k₂/k₃And the free energy change of trans-cis isomerization can be calculated using equation 5

Using the kinetic parameters determined in FIGS. 11f-11h, the inventors estimated that for one, two, and three cAB modifications, trans-to-cis isomerization results in a change in free energyRespectively-5.04, -4.55 and-4.06 kcal.mol^-1. The reaction free energy of trans-cAB modified DNA equilibrium can then be derived from equations 6 and 7:

table 3 summarizes the unmodified DNA duplexes with different numbers of cis-or trans-isomers studied in this studyAnd ca modifies Δ G ° of the duplex. For other cAB-modified duplexes,. DELTA.G.can be prepared by first using DNA analysis software such asNUPACK determines Δ G ° for the unmodified duplex and then estimates by calculating Δ G ° for the cAB modified duplex using equation 4 (cis) or 7 (trans), values are listed in table 3.

TABLE 3 summary of Standard free Gibbs energy values for DNA duplexes (CTs) with varying amounts of cis/trans cAB modification

The kinetic parameters obtained in this study provide the inventors with a method to roughly assess the upper and lower boundaries of a DNA balance to measure transient kinetic events in DNA. The simulation results in FIG. 12 show that the inventors' method can accurately measure (experimental error less than 5%) a rate constant of 1.08X 10^-11M·s^-1And 7.50X 10^-14M·s^-1The isomerization reaction of (2). A wide (over 2 orders of magnitude) kinetic window will allow the determination of multiple types of transient reactions and/or events in DNA.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and the like that are within the spirit and principle of the present invention are included in the present invention.

The foregoing embodiments and methods described in this disclosure may vary based on the abilities, experience, and preferences of those skilled in the art.

The mere order in which the steps of a method are listed in the present invention does not constitute any limitation on the order of the steps of the method.

Sequence listing

<110> Sichuan university

<120> a method for in situ characterization of chemically modified DNA

<130> 1

<160> 19

<170> SIPOSequenceListing 1.0

<210> 1

<211> 10

<212> DNA