阅读说明：本技术 对rna分子进行处理的方法及试剂盒和复合体 (Method for treating RNA molecule, kit and complex ) 是由 江媛 徐旭 章文蔚 席阳 汪为茂 赵霞 任悍 王欧 于 2018-06-04 设计创作，主要内容包括：本发明涉及基因测序领域,具体涉及一种对RNA分子进行处理的方法及试剂盒和复合体。所述方法包括：基于所述RNA分子,形成第一RNA-DNA复合体,所述第一RNA-DNA复合体包括：双链区,所述双链区由匹配DNA单链和匹配RNA单链形成；DNA单链区,所述DNA单链区与所述匹配DNA单链的5’末端相连；以及RNA单链区,所述RNA单链区与所述匹配RNA单链的5’末端相连；在所述匹配RNA单链的3’末端连接具有平末端的双链DNA接头,以便获得第二RNA-DNA复合体。还提供了一种对RNA处理的试剂盒。利用该方法和试剂盒,可以简化接头连接步骤,降低成本,以实现对小RNA的检测或建库测序。(the invention relates to the field of gene sequencing, in particular to a method for processing RNA molecules, a kit and a complex. The method comprises the following steps: forming a first RNA-DNA complex based on the RNA molecule, the first RNA-DNA complex comprising: a double-stranded region formed from a single matched DNA strand and a single matched RNA strand; a DNA single-stranded region linked to the 5' end of the matching DNA single strand; and an RNA single-stranded region linked to the 5' end of the matching RNA single strand; ligating a double-stranded DNA adaptor having a blunt end to the 3' -end of the single strands of the matching RNA so as to obtain a second RNA-DNA complex. A kit for RNA treatment is also provided. By using the method and the kit, the step of connecting the joints can be simplified, and the cost is reduced, so that the detection or library-building sequencing of the small RNA can be realized.)

1. A method of processing an RNA molecule comprising:

Forming a first RNA-DNA complex based on the RNA molecule, the first RNA-DNA complex comprising:

A double-stranded region formed from a single matched DNA strand and a single matched RNA strand;

A DNA single-stranded region linked to the 5' end of the matching DNA single strand; and

an RNA single-stranded region linked to the 5' end of the matching RNA single strand;

Ligating a double stranded DNA adaptor having a blunt end to the 3' end of the single strands of the matching RNA so as to obtain a second RNA-DNA complex.

2. The method of claim 1, wherein the double stranded DNA adaptor is ligated to the 3' end of the single strands of matching RNA using T4 ligase;

optionally, the 3' end of the matching DNA single strand on the double-stranded region contains a blocking group;

optionally, the blocking group comprises at least one selected from a 3' phosphate, a 3' open ring sugar such as a 3' -phosphate- α, β -unsaturated aldehyde (PA), a 3' amino modification, a 3' dideoxynucleotide, a 3' Phosphorothioate (PS) linkage or a 3' phosphate.

3. The method of claim 1 or 2, wherein the RNA molecule has a nucleic acid length of 10nt or more;

Optionally, the nucleic acid length of the double-stranded region is greater than or equal to 6 bp;

Optionally, the nucleic acid length of the RNA single-stranded region is greater than or equal to 0 nt;

optionally, the nucleic acid length of the DNA single-stranded region is greater than or equal to 1 nt;

Optionally, the RNA molecule is selected from a small RNA, or an mRNA;

optionally, the small RNA comprises SiRNA, miRNA, piRNA.

4. The method of claim 1, wherein the first RNA-DNA complex is formed by contacting the RNA molecule with a DNA probe;

Optionally, the 3' end of the DNA probe contains a blocking group;

Optionally, the blocking group comprises at least one selected from a 3' phosphate, a 3' open ring sugar such as a 3' -phosphate- α, β -unsaturated aldehyde (PA), a 3' amino modification, a 3' dideoxynucleotide, a 3' Phosphorothioate (PS) linkage or a 3' phosphate;

Optionally, the DNA probe is selected from at least one of:

A nucleic acid fragment that partially matches the RNA molecule;

a mixture of nucleic acid fragments comprising 6 to 30 arbitrary nucleotides.

5. the method of any one of claims 1 to 4, further comprising:

subjecting the second RNA-DNA complex to reverse transcription treatment to obtain a reverse transcription product;

optionally, further comprising:

subjecting the reverse transcription product to cyclization treatment so as to obtain a cyclization product;

Optionally, further comprising:

Constructing a sequencing library based on at least one of the reverse transcription product and the circularization product.

6. a kit, comprising:

A DNA probe for capturing an RNA molecule;

A double stranded DNA adaptor having blunt ends.

7. The kit according to claim 6, wherein the 3' end of the DNA probe comprises a blocking group;

Optionally, the blocking group comprises at least one selected from a 3' phosphate, a 3' open ring sugar such as a 3' -phosphate- α, β -unsaturated aldehyde (PA), a 3' amino modification, a 3' dideoxynucleotide, a 3' Phosphorothioate (PS) linkage or a 3' phosphate;

Optionally, the DNA probe is selected from at least one of:

A nucleic acid fragment that partially matches the RNA molecule;

a mixture of nucleic acid fragments comprising 6 to 30 arbitrary nucleotides;

Optionally, the DNA probe forms a first RNA-DNA complex with the RNA molecule, the first RNA-DNA complex comprising:

A double-stranded region formed from a single matched DNA strand and a single matched RNA strand;

A DNA single-stranded region linked to the 5' end of the matching DNA single strand; and

An RNA single-stranded region linked to the 5' end of the matching RNA single strand.

optionally, further comprising: t4DNA ligase.

8. the use of the kit of claim 6 or 7 in the field of sequencing of RNA molecules;

optionally, the RNA molecule has a nucleic acid length of 10nt or greater.

9. An RNA-DNA complex, comprising:

a double-stranded region formed from a single matched DNA strand and a single matched RNA strand;

A DNA single-stranded region linked to the 5' end of the matching DNA single strand;

An RNA single-stranded region linked to the 5' end of the matching RNA single strand; and

A double-stranded DNA linker region attached to the 3' end of the single strand of matching RNA, the double-stranded DNA linker having a blunt end.

10. The RNA-DNA complex of claim 9, wherein the 3' end of the matching DNA single strand on the double-stranded region comprises a blocking group;

Optionally, the blocking group comprises at least one selected from a 3' phosphate, a 3' open ring sugar such as a 3' -phosphate- α, β -unsaturated aldehyde (PA), a 3' amino modification, a 3' dideoxynucleotide, a 3' Phosphorothioate (PS) linkage or a 3' phosphate;

Optionally, the other end of the double-stranded DNA adaptor is not the same size, and the long strand of the double-stranded DNA adaptor is linked to the 3' end of the matching RNA single strand;

Optionally, the RNA molecule has a nucleic acid length of 10nt or greater;

Optionally, the nucleic acid length of the double-stranded region is greater than or equal to 6 bp;

optionally, the nucleic acid length of the RNA single-stranded region is greater than or equal to 0 nt;

Optionally, the nucleic acid length of the DNA single-stranded region is greater than or equal to 1 nt;

Optionally, the RNA molecule is selected from one of a small RNA or mRNA;

Optionally, the small RNA comprises SiRNA, miRNA, piRNA.

Technical Field

The invention relates to the field of gene sequencing, in particular to a method for processing RNA molecules, a kit and a complex.

background

small RNA (Small RNA) is an important regulatory factor of life activities, is about 10-40 nt long and widely exists in organisms. It can play an important role in the processes of regulation and control of organism gene expression, development of organism individuals, metabolism, occurrence of diseases and the like through participating in multiple ways of regulating mRNA degradation, translation inhibition, heterochromatin formation and the like.

the new generation of high-throughput sequencing can obtain the sequence information of the small RNA in sample cells in one-time sequencing, can quickly identify the expression levels and the expression differences of the known and unknown small RNAs in different tissues, different developmental stages and different disease states, provides a powerful tool for researching the effect of the small RNA on the cell process and the biological influence of the small RNA, and particularly can identify the new small RNA through sequencing. Due to the application of this technology, more and more small RNAs are discovered and functionally characterized. And the important regulation and control function in the cells also enables the polypeptide to have the potential of being used as a disease diagnosis marker or a drug target, and can be widely applied to the fields of disease diagnosis, personalized treatment, prognosis and the like.

Further improvements are then needed for sequencing means for small RNA molecules.

disclosure of Invention

The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Therefore, an object of the present invention is to provide a method for processing RNA molecules, which can detect or sequence small RNA by using high-throughput sequencing enzymes and reagents that are conventionally used for library construction, thereby simplifying the step of linker ligation and reducing the cost.

The present invention is obtained based on the following findings of the inventors:

Sequencing or analyzing technology for small RNA usually separates small RNA from total RNA, adds specific joints at two ends of the small RNA, then carries out in vitro reverse transcription to form cDNA, amplifies the cDNA, and carries out unidirectional end direct sequencing on DNA fragments by using a sequencer. The technology of sequencing or analyzing by directly connecting the small RNA with the joint and carrying out reverse transcription has the problems of some technical difficulties and higher cost, which are mainly shown in the following steps: because the template for building the library is RNA, the conventional reagent is difficult to be directly utilized for connecting the joint, and special enzyme is required for carrying out the reaction of adding the joint, so the cost is higher. And secondly the fragments of the template are too small, and the template, after addition of the linker, is more difficult to separate from the various dimers.

therefore, the invention provides the following technical scheme:

According to a first aspect of the invention, there is provided a method of treating an RNA molecule comprising: forming a first RNA-DNA complex based on the RNA molecule, the first RNA-DNA complex comprising: a double-stranded region formed from a single matched DNA strand and a single matched RNA strand; a DNA single-stranded region linked to the 5' end of the matching DNA single strand; and an RNA single-stranded region linked to the 5' end of the matching RNA single strand; ligating a double stranded DNA adaptor having a blunt end to the 3' end of the single strands of the matching RNA so as to obtain a second RNA-DNA complex. By forming a complex structure of DNA and RNA, the method can be used for capturing RNA molecules, is particularly suitable for capturing small-fragment RNA molecules, realizes the capture of RNA molecule nucleic acid information, and is beneficial to the subsequent library building and sequencing of RNA molecules.

according to the embodiment of the present invention, the above method for treating RNA molecules may further have the following technical features:

According to an embodiment of the invention, the double stranded DNA adaptor is ligated at the 3' end of the single strands of the matching RNA using T4 ligase. T4 ligase or T4DNA ligase can repair single-strand gaps on the DNA-RNA complex, and double-strand DNA joints are connected to the 3' ends of the matched RNA single strands, so that RNA molecules can be captured by using DNA probes to form a second RNA-DNA complex structure, and subsequent library building and sequencing of the RNA molecules are facilitated.

According to an embodiment of the invention, the 3' end of the single strand of matching DNA on the double-stranded region comprises a blocking group. The 3 'end of the matching single strand of DNA is linked to a blocking group that prevents the double stranded DNA linker from attaching to the 3' end of the matching single strand of DNA and prevents the DNA strand on the first RNA-DNA complex from self-attaching thereto.

According to an embodiment of the invention, the blocking group comprises at least one selected from the group consisting of a 3' phosphate, a 3' open ring sugar such as a 3' -phosphate- α, β -unsaturated aldehyde (PA), a 3' amino modification, a 3' dideoxynucleotide, a 3' Phosphorothioate (PS) linkage or a 3' phosphate.

According to an embodiment of the invention, the RNA molecule has a nucleic acid length of 10nt or more.

According to an embodiment of the invention, the nucleic acid length of said double-stranded region is equal to or greater than 6 bp.

According to an embodiment of the present invention, the RNA single-stranded region has a nucleic acid length of 0nt or more.

According to an embodiment of the present invention, the single-stranded region of DNA has a nucleic acid length of 1nt or more.

according to an embodiment of the invention, the RNA molecule is selected from one of a small RNA or an mRNA.

According to an embodiment of the invention, the small RNA comprises SiRNA, miRNA, piRNA. According to the embodiment of the present invention, the small RNA includes but is not limited to siRNA (small interfering RNA), miRNA (microRNA, small molecular RNA or microRNA), and piRNA (PIWI-interacting RNA), wherein the length of siRNA is usually between 21-25 nt, and the siRNA is processed by Dicer (enzyme in RNAase III family that has specificity to double-stranded RNA). SiRNA is primarily functional in stimulating silencing of target mRNA to which it is complementary. The length of MiRNA is usually between 21 nt and 25nt, the MiRNA is derived from non-coding RNA, the main function is to inhibit the gene expression after transcription by specifically combining with target messenger ribonucleic acid (mRNA), and the MiRNA plays an important role in the aspects of regulating and controlling the gene expression, the cell cycle, the development time sequence of organisms and the like. The piRNA is a novel non-coding small-molecule RNA, can interact with PIWI protein, is called piRNA, is usually 24-31 nt long, and can regulate and control the stability of mRNA, the synthesis of protein, the chromatin organization and the genome structure by combining with Argonaute family protein and the like.

according to an embodiment of the invention, the first RNA-DNA complex is formed by contacting the RNA molecule with a DNA probe.

According to an embodiment of the present invention, the 3' end of the DNA probe contains a blocking group.

According to an embodiment of the invention, the blocking group comprises at least one selected from the group consisting of a 3' phosphate, a 3' open ring sugar such as a 3' -phosphate- α, β -unsaturated aldehyde (PA), a 3' amino modification, a 3' dideoxynucleotide, a 3' Phosphorothioate (PS) linkage or a 3' phosphate.

According to an embodiment of the invention, the DNA probe is selected from at least one of: a nucleic acid fragment that partially matches the RNA molecule; a mixture of nucleic acid fragments comprising 6 to 30 arbitrary nucleotides. The DNA probe designed for the RNA molecule with a specific sequence is a specific target nucleic acid hybridization sequence which can be partially matched with the RNA molecule to form the first RNA-DNA complex structure. For RNA molecules with unknown sequences, such as small RNA molecules, a mixture of nucleic acid fragments containing 6-30 arbitrary nucleotides can be used as a DNA probe to capture the nucleic acid information of all RNA molecules.

According to an embodiment of the invention, the method further comprises: subjecting the second RNA-DNA complex to reverse transcription treatment to obtain a reverse transcription product.

According to an embodiment of the invention, the method further comprises: subjecting the reverse transcription product to cyclization treatment so as to obtain a cyclization product.

according to an embodiment of the invention, the method further comprises: constructing a sequencing library based on at least one of the reverse transcription product and the circularization product.

According to a second aspect of the invention, there is provided a device for processing RNA molecules, comprising:

An RNA-DNA complexing module that forms a first RNA-DNA complex based on the RNA molecule, the first RNA-DNA complex comprising:

A double-stranded region formed from a single matched DNA strand and a single matched RNA strand;

A DNA single-stranded region linked to the 5' end of the matching DNA single strand; and

An RNA single-stranded region linked to the 5' end of the matching RNA single strand;

A linker connecting module connected to the RNA-DNA complexing module, the linker connecting module connecting a double stranded DNA linker at the 3' end of the matching RNA single strands so as to obtain a second RNA-DNA complex, the double stranded DNA linker having blunt ends.

According to the embodiment of the present invention, the above apparatus for processing RNA molecules may further have the following technical features:

according to an embodiment of the invention, the adaptor ligation module ligates the double stranded DNA adaptor at the 3' end of the single strands of the matching RNA using T4 ligase.

According to an embodiment of the invention, the 3' end of the single strand of matching DNA on the double-stranded region comprises a blocking group.

according to an embodiment of the invention, the blocking group comprises at least one selected from the group consisting of a 3' phosphate, a 3' open ring sugar such as a 3' -phosphate- α, β -unsaturated aldehyde (PA), a 3' amino modification, a 3' dideoxynucleotide, a 3' Phosphorothioate (PS) linkage or a 3' phosphate.

According to an embodiment of the invention, the other end of the double stranded DNA adaptor is not the same size, and the long strand of the double stranded DNA adaptor is ligated to the 3' end of the single strand of the matching RNA.

According to an embodiment of the invention, the RNA molecule has a nucleic acid length of 10nt or more.

According to an embodiment of the invention, the nucleic acid length of said double-stranded region is equal to or greater than 6 bp.

According to an embodiment of the present invention, the RNA single-stranded region has a nucleic acid length of 0nt or more.

According to an embodiment of the present invention, the single-stranded region of DNA has a nucleic acid length of 1nt or more.

According to an embodiment of the invention, the RNA molecule is selected from one of a small RNA or an mRNA.

According to an embodiment of the invention, the small RNA comprises SiRNA, miRNA, piRNA.

According to an embodiment of the invention, the apparatus further comprises:

A reverse transcription module linked to the linker-linking module, the reverse transcription module being adapted to perform a reverse transcription process on the second RNA-DNA complex to obtain a reverse transcription product.

According to an embodiment of the invention, the apparatus further comprises:

A circularization module coupled to the reverse transcription module, the circularization module adapted to circularize the reverse transcription product to obtain a circularized product.

according to an embodiment of the invention, the apparatus further comprises:

A sequencing library construction module linked to the reverse transcription module or to the circularization processing module, the sequencing library construction module constructing a sequencing library based on at least one of the reverse transcription product and the circularization product.

The above description of the advantages and technical features of the method for processing RNA in any embodiment of the present invention is also applicable to the apparatus for processing RNA in this embodiment of the present invention, and will not be repeated herein.

According to a third aspect of the invention, there is provided a kit comprising: a DNA probe for capturing an RNA molecule; a double stranded DNA adaptor having blunt ends. The kit provided by the invention is used for capturing RNA molecules by using a DNA probe to form an RNA-DNA complex which is at least partially matched, then a double-stranded DNA joint with a flat end is connected to the 3' end of the RNA molecules on the RNA-DNA complex to form the RNA-DNA complex connected with the double-stranded DNA joint, and then the library building, the quantitative detection and the like of the RNA molecules are realized by reverse transcription PCR and/or fluorescent quantitative PCR and the like.

according to the embodiment of the invention, the kit can be further added with the following technical characteristics:

According to an embodiment of the invention, the other end of the double stranded DNA adaptor is a sticky end.

According to an embodiment of the present invention, the 3' end of the DNA probe contains a blocking group.

according to an embodiment of the invention, the blocking group comprises at least one selected from the group consisting of a 3' phosphate, a 3' open ring sugar such as a 3' -phosphate- α, β -unsaturated aldehyde (PA), a 3' amino modification, a 3' dideoxynucleotide, a 3' Phosphorothioate (PS) linkage or a 3' phosphate.

According to an embodiment of the invention, the DNA probe is selected from at least one of: a nucleic acid fragment that partially matches the RNA molecule; a mixture of nucleic acid fragments comprising 6 to 30 arbitrary nucleotides.

According to an embodiment of the invention, the kit further comprises T4DNA ligase. The T4DNA ligase is used to ligate a double stranded DNA adaptor to the 3' end of the RNA molecule on the RNA-DNA complex.

according to an embodiment of the invention, said DNA probe forms with said RNA molecule a first RNA-DNA complex comprising: a double-stranded region formed from a single matched DNA strand and a single matched RNA strand; a DNA single-stranded region linked to the 5' end of the matching DNA single strand; and an RNA single-stranded region that is linked to the 5' end of the matching RNA single strand.

according to the present invention, the kit may further comprise buffers, such as, but not limited to, hybridization buffers for hybridization of DNA probes and RNA molecules, which are commercially available or formulated by those skilled in the art, such as, for example, TA buffer available from Epicentre; a ligation buffer, which may also be referred to as a ligation mixture, may include, for example, BSA, Tris-HCl, magnesium chloride, DTT, PEG-8000, ATP, and the like, for ligating the double-stranded DNA adaptor to the 3' -end of the RNA molecule on the RNA-DNA complex.

According to the present invention, the kit may further comprise other substances such as reverse transcription primers for performing reverse transcription amplification and fluorescent quantitative PCR primers for performing fluorescent quantitation, and the like. The kit can also be used together with other commercial kits, for example, when the small RNA of whole blood is subjected to library construction and sequencing, the small RNA in a blood sample can be extracted by using the commercial small RNA kit, and the ligation product after the double-stranded DNA probe is ligated can be subjected to reverse transcription treatment by using the commercial reverse transcription kit, and the like.

According to a fourth aspect of the present invention, there is provided the use of a kit according to the third aspect of the present invention in the field of sequencing of RNA molecules. According to an embodiment of the invention, the RNA molecule has a nucleic acid length of 10nt or more.

according to a fifth aspect of the present invention, there is provided an RNA-DNA complex comprising: a double-stranded region formed from a single matched DNA strand and a single matched RNA strand;

A DNA single-stranded region linked to the 5' end of the matching DNA single strand;

An RNA single-stranded region linked to the 5' end of the matching RNA single strand; and

A double-stranded DNA linker region attached to the 3' end of the single strand of matching RNA, the double-stranded DNA linker having a blunt end.

According to an embodiment of the present invention, the RNA-DNA complex described above may be further characterized by the following features:

According to an embodiment of the present invention, in the RNA-DNA complex, the 3' -end of the single strand of matching DNA in the double-stranded region comprises a blocking group.

According to an embodiment of the present invention, in the RNA-DNA complex described above, the blocking group comprises at least one selected from a 3' phosphate, a 3' open cyclic sugar such as 3' -phospho- α, β -unsaturated aldehyde (PA), a 3' amino modification, a 3' dideoxynucleotide, a 3' Phosphorothioate (PS) linkage or a 3' phosphate.

According to an embodiment of the present invention, in the RNA-DNA complex, the other end of the double-stranded DNA linker is different in length, and the long strand of the double-stranded DNA linker is linked to the 3' -end of the single strand of the matching RNA.

According to an embodiment of the present invention, in the RNA-DNA complex, the RNA molecule has a nucleic acid length of 10nt or more.

According to an embodiment of the present invention, in the above RNA-DNA complex, the nucleic acid length of the double-stranded region is 6bp or more.

According to an embodiment of the present invention, in the above RNA-DNA complex, the RNA single-stranded region has a nucleic acid length of 0nt or more.

According to an embodiment of the present invention, in the RNA-DNA complex, the single-stranded region of DNA has a nucleic acid length of 1nt or more.

According to an embodiment of the present invention, in the RNA-DNA complex described above, the RNA molecule is selected from one of a small RNA or an mRNA.

According to an embodiment of the present invention, in the RNA-DNA complex described above, the small RNA includes SiRNA, miRNA, piRNA.

The beneficial effects obtained by the invention are as follows: by the method and the device for processing RNA, the RNA molecule can be detected or sequenced by using the enzyme and the reagent of the conventional NGS library construction, the problems of complex step and high cost of the joint connection in the prior art are mainly solved, and the method and the device are particularly suitable for library construction, sequencing or detection of small RNA.

Drawings

FIG. 1 is a schematic diagram of the structure of a first RNA-DNA complex provided according to one embodiment of the present invention.

FIG. 2 is a schematic diagram of the structure of a second RNA-DNA complex provided according to an embodiment of the present invention.

FIG. 3 is a schematic illustration of performing the ligation reaction provided according to one embodiment of the invention.

FIG. 4 is a diagram of a detection scheme for small RNAs according to an embodiment of the present invention.

FIG. 5 is a schematic diagram of the structure of a reverse transcription product provided in accordance with one embodiment of the present invention.

FIG. 6 is a diagram of a detection scheme for small RNAs according to an embodiment of the present invention.

FIG. 7 is a graph of a gel utilizing 10% denatured polyacrylamide gel for a ligation product, provided in accordance with an embodiment of the present invention.

FIG. 8 is a graph of a gel utilizing 10% denatured polyacrylamide gel for a ligation product, provided in accordance with an embodiment of the present invention.

FIG. 9 is a graph showing the results of fluorescent quantitative PCR on a reverse transcription product according to one embodiment of the present invention.

Figure 10 is a graph of cyclization results provided in accordance with one embodiment of the present invention.

FIG. 11 is a graph showing the results of performing fluorescent quantitative PCR on the circularized product according to one embodiment of the present invention.

FIG. 12 is a graph showing the results of performing fluorescent quantitative PCR on the circularized product according to one embodiment of the present invention.

FIG. 13 is a graph showing the results of fluorescent quantitative PCR on a reverse transcription product according to one embodiment of the present invention.

Fig. 14 is a schematic diagram of an apparatus for processing RNA molecules according to an embodiment of the present invention.

Fig. 15 is a schematic diagram of an apparatus for processing RNA molecules according to an embodiment of the present invention.

Fig. 16 is a schematic diagram of an apparatus for processing RNA molecules according to an embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

The invention adds the joint into RNA molecule by using conventional enzyme reaction reagent, greatly saves cost, benefits from various scientific research applications and kit packaging, and has wide market potential and prospect.

The invention utilizes the conventional NGS linker adding method to realize adding the linker on the RNA molecule, and provides a series of technical schemes based on the method, including oligonucleotide sequence composition, enzyme and reagent components, reaction conditions, method steps and the like required by the technical schemes.

the invention is realized based on the following principle: under appropriate reaction conditions, the DNA probe hybridizes with the RNA molecule to form a DNA-RNA hybrid complex, and then a blunt-end linker is ligated to the 3' end of the RNA molecule using some ligase.

Based on the basic principles and element design described above, the present invention proposes a series of embodiments, which will be described one by one hereinafter. In particular, other similar embodiments and variants thereof than those described below are also included in the scope of the claims of the present invention.

it is to be noted that in order to make the present invention easier for those skilled in the art to understand, certain terms in the present invention are explained and illustrated herein, and these explanation and illustration should not be construed as a limitation of the present invention.

Method for treating RNA molecules

the invention provides a method for processing RNA. According to an embodiment of the invention, the method comprises:

Contacting said RNA with a DNA probe to form a first RNA-DNA complex;

ligating a double-stranded DNA adaptor having a blunt end to the 3' end of the RNA molecule on the first RNA-DNA complex to obtain a second RNA-DNA complex containing a blunt-end adaptor.

When the method of the invention is used for connecting the double-stranded DNA adaptor with the flat end to the 3' end of the RNA molecule on the first RNA-DNA complex, the reagent required by the connection reaction is simple, no additional special enzyme is needed, and the cost is lower.

Herein, the first RNA-DNA complex is shown in FIG. 1. The first RNA-DNA complex comprises a double-stranded region formed by a single DNA strand and a single RNA strand that are matched with each other; a DNA single-stranded region linked to the 5' end of the matching DNA single strand; and an RNA single-stranded region that is linked to the 5' end of the matching RNA single strand. The first RNA-DNA complex comprises a DNA chain partially matched with the RNA molecule and the RNA molecule chain, and the RNA molecule is captured by forming a double-stranded region, a DNA single-stranded region and an RNA single-stranded region so as to realize subsequent library building or sequencing of the RNA molecule and the like.

according to the embodiment of the present invention, the first RNA-DNA complex may be formed without the RNA single-stranded region, i.e., the RNA single-stranded region may have a nucleic acid length of 0 nt. The RNA-DNA complex thus formed may contain only the double-stranded region and the single-stranded region of DNA.

In this context, the RNA molecule, which may also be referred to as RNA fragment, may be a sample of nucleic acid fragments of 10-500nt in length, preferably 10-100nt in length. According to the embodiment of the invention, the RNA fragment can be a small RNA fragment or other RNA fragments after being broken, such as mRNA molecules. Thus, sequencing of small RNA molecules can be achieved using the methods of the invention. It is also possible to sequence the fragmented RNA molecule and then obtain the nucleic acid information of the long RNA molecule before the fragmentation. The RNA molecule sample may be obtained or provided from any organism of interest. These organisms include plants, animals (e.g., mammals, including humans and non-human primates), pathogens (e.g., bacteria and viruses). Methods for obtaining polynucleotides (e.g., RNA, etc.) from organisms are well known in the art.

In this context, a DNA probe may be a nucleic acid fragment that can be sequence-complementary paired with an RNA molecule, or may be a nucleic acid fragment containing N. When the sequence of the RNA molecule of the target region is known, the DNA probe can be a specific nucleic acid segment which can be complementarily paired with the sequence of the RNA molecule of the target region, and the DNA probe can be contacted with the corresponding RNA molecule to react, so that the DNA probe can be used for capturing and establishing a library of the target sequence. When the sequence of the RNA molecule in the target region is unknown, for example, unknown small RNA is subjected to library construction sequencing, the DNA probe can be a random sequence segment, the bases on the random sequence segment are randomly arranged, and is selected from any one of four bases of A/T/G/C, and the DNA probe is a mixture containing the random sequence segments. The nucleic acid length of the DNA probe is preferably 6-30 bp. Each DNA probe comprises an arbitrary array of nucleic acids of these lengths. The mixture of all DNA probes is mixed with the RNA sequence with unknown sequence, and can be used for the library building and sequencing of the RNA sequence with unknown sequence.

Meanwhile, the 3 'end of the DNA probe contains a 3' blocking group, and the 3 'blocking group includes, but is not limited to, 3' phosphate, 3 'open-ring sugar such as 3' -phospho- α, β -unsaturated aldehyde (PA), 3 'amino modification, 3' dideoxynucleotide, 3 'Phosphorothioate (PS) bond or 3' phosphate, and the like. The 3' end of the DNA probe is treated by the blocking group, so that the DNA probe can be prevented from being connected with the double-stranded DNA joint in the process of connecting the double-stranded DNA joint.

According to a specific embodiment of the present invention, the first RNA-DNA complex is obtained by mixing the RNA molecule and the DNA probe, and then adding a buffer solution to perform a hybridization reaction. According to the embodiment of the invention, the hybridization reaction is carried out for 1-2 minutes at 80-95 ℃, then for 10-20 minutes at 60-70 ℃, and then for 10-20 minutes at 37 ℃. According to another embodiment of the present invention, the hybridization reaction is performed at 80-95 ℃ for 1-2 minutes, then the temperature is decreased to 37 ℃ at a rate of 0.1-0.5 second/per DEG C, and then the hybridization reaction is performed at 37 ℃ for 10-20 minutes.

After contacting the DNA probe with the RNA molecule to form the first RNA-DNA complex, a double-stranded DNA adaptor is ligated to the first RNA-DNA complex, i.e., a ligation reaction is performed to form a second RNA-DNA complex. In carrying out the ligation reaction, the ligase used in the present invention is capable of mediating ligation of DNA to the 3' end of RNA under appropriate conditions and at appropriate substrate concentrations. For example, the DNA probe and the RNA template can be ligated by using T4DNA ligase. T4DNA ligase (T4DNA ligase) is known to mediate not only ligation of blunt ends but also ligation of cohesive ends between double-stranded DNA molecules. Furthermore, T4DNA ligase can also mediate ligation between DNA-templated DNA and nicks in RNA.

According to an embodiment of the invention, the double stranded DNA adaptor has a blunt end at one end for ligation to the RNA molecule. The other end of the double-stranded DNA adaptor may be a sticky end, a blunt end, or simply a different length, and is not particularly required.

According to an embodiment of the present invention, the first RNA-DNA complex and the double-stranded DNA adaptor, together with other ligation mixture, are mixed and subjected to ligation reaction at 37 ℃. According to an embodiment of the invention, the ligation mixture comprises BSA, Tris-Cl, magnesium chloride, DTT, PEG-8000 and ATP and T4DNA ligase.

Herein, the second RNA-DNA complex is linked to a double-stranded DNA linker having a blunt end at the 3' end of the single strands of the matching RNA, as compared to the first RNA-DNA complex, as shown in fig. 2. The second RNA-DNA complex comprises: a double-stranded region formed from a single matched DNA strand and a single matched RNA strand; a DNA single-stranded region linked to the 5' end of the matching DNA single strand; an RNA single-stranded region linked to the 5' end of the matching RNA single strand; and a double-stranded DNA linker region, said double-stranded DNA linker region being linked to the 3' end of said matching RNA single strand, said double-stranded DNA linker having a blunt end.

According to the embodiment of the present invention, the second RNA-DNA complex may be formed without the RNA single-stranded region, i.e., the RNA single-stranded region may have a nucleic acid length of 0 nt. The RNA-DNA complex thus formed comprises only the double-stranded region, the DNA single-stranded region, and the double-stranded DNA linker region.

according to a specific embodiment of the invention, the invention provides a Small RNA sequencing detection protocol. And (3) performing reverse transcription treatment on the second RNA-DNA complex, namely performing thermal denaturation hybridization on the second RNA-DNA complex and a reverse transcription primer, performing reverse transcription amplification to obtain a reverse transcription product, performing direct cyclization by using an enzyme reaction to obtain a cyclization product, and performing amplification and library building.

According to an embodiment of the invention, the reverse transcription product formed is shown in FIG. 5, and comprises the RNA molecule and the DNA strand of the double-stranded DNA linker to which the RNA molecule is attached (i.e., the long strand from the double-stranded DNA linker), and a reverse transcription primer strand that is complementary-paired with the portion of the double-stranded DNA linker to which the RNA molecule is attached, the unpaired portion constituting the linker region. The linker region may be used for subsequent cyclization processes.

According to an embodiment of the present invention, a single-stranded loop containing a reverse transcription primer strand nucleic acid is formed as a cyclization product using the reverse transcription product. The circularized product contains a reverse transcription product of the RNA molecule, and the nucleic acid information of the RNA molecule, such as the length and base arrangement of the RNA molecule, can be obtained by sequencing the circularized product or sequencing after amplification.

According to an embodiment of the invention, the circularized products are pooled to obtain a library containing the nucleic acid sequences of the corresponding RNA molecules. For example, a library is constructed for circular DNA according to a BGI SEQ-500 high-throughput gene sequencer produced by Huada or a derivative product thereof, and a library construction sample can be rapidly and conveniently obtained.

According to one embodiment of the present invention, the present invention provides a small RNA sequencing assay protocol, as shown in FIG. 6. And (3) carrying out reverse transcription treatment on the second RNA-DNA complex, namely carrying out thermal denaturation hybridization on the second RNA-DNA complex and a reverse transcription primer, carrying out reverse transcription amplification to obtain a reverse transcription product, and then carrying out PCR amplification to build a library. According to an embodiment of the present invention, a portion of the DNA linker is added to the 3 'end of the RNA template using T4DNA ligase, and then a second portion of the DNA linker is added to the 3' end of the reverse transcription product using PCR.

According to another embodiment of the invention, the invention provides a Small RNA quantitative detection scheme, namely, the second RNA-DNA complex is subjected to reverse transcription treatment, namely, the second RNA-DNA complex is subjected to thermal denaturation hybridization with a reverse transcription primer, a reverse transcription product is obtained through reverse transcription amplification, and then quantitative detection is carried out by utilizing a fluorescent quantitative PCR mode.

in addition, in this paper, the expression "connection" or "connection" between nucleic acid molecules, refers to the nucleic acid molecules through 5 '-3' two phosphoric acid ester bond connection.

Herein, the RNA-DNA complex or RNA-DNA hybrid complex refers to a hybrid fragment containing both RNA and DNA molecules, and these complexes may be partially complementary-paired hybrid fragments or all complementary-paired hybrid fragments, depending on the case. The RNA-DNA complex or RNA-DNA hybrid complex may also be expressed as a DNA-RNA complex or a DNA-RNA hybrid complex.

"reverse transcription" is herein understood as conventional in the art and refers to the process of synthesizing DNA by reverse transcriptase using RNA as a template. Reverse transcription is also known as reverse transcription.

Herein, the term "complementary" or "pairing" or "matching" or "complementary pairing" is used as a general explanation in the art, and refers to a one-to-one correspondence relationship between bases of nucleic acid molecules, which is due to the fact that hydrogen bonds between bases have a fixed number and the distance between two strands of DNA remains constant, so that the base pairing follows a rule, specifically, between double-stranded DNA molecules or between some double-stranded RNA molecules, or between single-stranded DNA and single-stranded RNA molecules, adenine a and thymine T (or uracil U), and guanine G and cytosine C.

Device for processing RNA molecules

According to another aspect of the present invention, there is provided an apparatus for treating an RNA molecule, as shown in fig. 14, comprising an RNA-DNA complexing module and a linker connecting module, wherein the RNA-DNA complexing module is connected to the linker connecting module, and the RNA-DNA complexing module forms a first RNA-DNA complex based on the RNA molecule, and the first RNA-DNA complex comprises: a double-stranded region formed from a single matched DNA strand and a single matched RNA strand; a DNA single-stranded region linked to the 5' end of the matching DNA single strand; and an RNA single-stranded region that is linked to the 5' end of the matching RNA single strand. The adaptor-ligating module ligates a double-stranded DNA adaptor at the 3' end of the matching RNA single strands so as to obtain a second RNA-DNA complex, the double-stranded DNA adaptor having blunt ends.

according to another embodiment of the present invention, the apparatus is shown in fig. 15, and further comprises: a reverse transcription module and a sequencing library construction module; the reverse transcription module is connected with the joint connection module, and the reverse transcription module is suitable for performing reverse transcription treatment on the second RNA-DNA complex so as to obtain a reverse transcription product; the sequencing library construction module is connected with the reverse transcription module, and the sequencing library construction module constructs a sequencing library based on the reverse transcription product.

According to another embodiment of the present invention, the apparatus is shown in fig. 16, and further comprises: the device comprises a reverse transcription module, a cyclization treatment module and a sequencing library construction module. The reverse transcription module is connected with the joint connection module, and the reverse transcription module is suitable for performing reverse transcription treatment on the second RNA-DNA complex so as to obtain a reverse transcription product; the cyclization treatment module is connected with the reverse transcription module and is suitable for carrying out cyclization treatment on the reverse transcription product so as to obtain a cyclization product; the sequencing library construction module is connected with the cyclization treatment module, and constructs a sequencing library based on the cyclization product.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.