KR20240002713A - A method for preparing library for rna sequencing - Google Patents

Abstract

The present invention relates to a method for manufacturing an RNA library comprising a step of ligating a first adaptor nucleic acid molecule, and a second adaptor nucleic acid molecule. Furthermore, guide nucleic acid molecules can be added to increase the ligation efficiency of the adapter nucleic acid molecules, thereby increasing the efficiency of RNA library construction.

Description

Library production method for RNA sequencing {A METHOD FOR PREPARING LIBRARY FOR RNA SEQUENCING}

The present invention relates to a method for producing an RNA library including the step of ligation of a first adapter nucleic acid molecule and a second adapter nucleic acid molecule. Furthermore, the efficiency of RNA library production can be increased by adding a guide nucleic acid molecule strand to increase the ligation efficiency of the adapter nucleic acid molecule.

A genome or genome refers to all the genetic information possessed by an organism. For sequencing or sequence analysis of an individual's genome, various technologies such as DNA chips, Next Generation Sequencing (NGS), and Next Next Generation Sequencing (NNGS) are being developed. NGS is widely used for research and diagnostic purposes. NGS varies depending on the type of equipment, but broadly speaking, it can be divided into three steps: sample collection, library creation, and nucleic acid sequence analysis. After nucleic acid sequence analysis, various genetic information, such as the presence or absence of genetic mutations, can be confirmed based on the sequence analysis data produced.

In particular, as the quality of life improves, interest in early diagnosis of diseases is growing, and because molecular diagnostic technology directly detects the genetic information (DNA/RNA) of disease-causing pathogens, it replaces existing antigen/antibody reactions. It is receiving a lot of attention as a technology that can solve the shortcomings of immunodiagnosis technology that detects indirect factors of disease based on immunotherapy.

As disease-related genetic information can be explored and secured to build a library, which can be used to discover candidate substances for new drug development, there is an increasing demand for the development of technology that allows faster and more accurate library construction.

Korean Patent Publication No. 10-2021-0141915

The purpose of the present invention is to provide a method for constructing an RNA library containing a first adapter nucleic acid molecule and a second adapter nucleic acid molecule.

Another object of the present invention is to provide a composition for constructing an RNA library comprising a first adapter nucleic acid molecule, a second adapter nucleic acid molecule, and a guide nucleic acid molecule strand.

Another object of the present invention is to provide an RNA library produced by the above method.

One aspect of the present invention for achieving the above object includes the steps of a) ligating a first adapter nucleic acid molecule to the 3' end of the target RNA; b) synthesizing cDNA through complementary sequence polymerization of the molecules ligated in step a); c) removing RNA by treating it with an RNA degrading enzyme; d) ligating a second adapter nucleic acid molecule to the 3' end of the cDNA; and e) PCR amplifying the nucleic acid molecule ligated in step d).

Specifically, the target RNA may be small RNA. The small RNA may refer to RNA consisting of about 50 nucleotides or less in addition to miRNA and siRNA.

In the present invention, the “first adapter nucleic acid molecule” is a nucleic acid molecule that is ligated to a target RNA, and the phosphate group at the 5' end of the first adapter nucleic acid molecule recognizes and binds to the 3' end of the target RNA. This can then be followed by a complementary sequence polymerization reaction using a molecule in which the first adapter nucleic acid molecule is ligated to the 3' end of the target RNA as a template.

In the present invention, the 'complementary strand polymerization' refers to polymerizing a sequence complementary to a molecule ligated to the 3' end of the target RNA as a template. One of these complementary sequence polymerization methods may include reverse transcription, but is not limited thereto, and may be included without limitation as long as a sequence that is complementary to the template molecule can be polymerized.

Specifically, the first adapter nucleic acid molecule may be in the form of DNA, but is not limited thereto.

Additionally, specifically, the first adapter nucleic acid molecule may have the 5' end adenylated, but is not limited thereto.

Additionally, specifically, the first adapter nucleic acid molecule may have its 3' end modified to prevent other adapter molecules from ligating to the 3' end, for example, may be amine modified, but is limited thereto. That is not the case.

In the present invention, the “second adapter nucleic acid molecule” is a nucleic acid molecule ligated to the 3’ end of cDNA synthesized through complementary sequence polymerization using a molecule in which the first adapter nucleic acid molecule is ligated to the 3’ end of the target RNA as a template. am. The phosphate group at the 5' end of the second adapter nucleic acid molecule recognizes and binds to the 3' end of the cDNA.

Specifically, the second adapter nucleic acid molecule may be in the form of DNA, but is not limited thereto.

Additionally, specifically, the second adapter nucleic acid molecule may be one in which the 5' end is adenylated, but is not limited thereto.

Additionally, specifically, the second adapter nucleic acid molecule may have its 3' end modified to prevent other adapter molecules from ligating to the 3' end, for example, may be amine modified, but is limited thereto. That is not the case.

Specifically, the complementary sequence polymerization reaction in step b) may be performed using DNA polymerase, but is not limited thereto, and any reverse transcriptase or polymerase used in a typical reverse transcription reaction may be applied.

Additionally, specifically, an adenine (A) base may be included at the end of the cDNA through the complementary sequence polymerization reaction.

Also specifically, the step of increasing the ligation efficiency of the second adapter nucleic acid molecule to the 3' end of the cDNA by treating the guide nucleic acid molecule strand before the PCR amplification reaction simultaneously with step d) or after step d). You can add

In the present invention, the “guide strand” is a strand in which part of the guide strand is complementary to the cDNA, and the other part is complementary to the second adapter nucleic acid molecule.

Accordingly, the guide nucleic acid molecule strand includes sequences complementary to the 3' end of the cDNA and the 5' end of the second adapter nucleic acid molecule. More specifically, the guide nucleic acid molecule strand may include a thymine (T) base, and the thymine may bind complementary to adenine at the end of cDNA.

More specifically, the guide nucleic acid molecule strand may bind complementary to the 3' end of the cDNA and the 5' end of the second adapter nucleic acid molecule, and through this binding, the guide nucleic acid molecule strand may bind to the 3' end of the cDNA and the first adapter nucleic acid molecule. 2 Adapter serves as a support for ligation of the 5' end of the nucleic acid molecule. The sequence of the guide nucleic acid molecule strand of the present invention can be applied without limitation as long as it is a sequence capable of complementary binding to the 3' end of cDNA and the 5' end of the second adapter nucleic acid molecule.

The guide nucleic acid molecule strand may include about 10 to 20 bases, and more specifically may include about 10 bases, but is not limited thereto. The length of the guide nucleic acid molecule strand of the present invention can be changed as necessary as long as it can stably ligate the 3' end of the cDNA and the 5' end of the second adapter nucleic acid molecule produced during the RNA library production process of the present invention. can do.

In one embodiment of the present invention, it was confirmed that the ligation efficiency of the second adapter nucleic acid molecule to the 3' end of cDNA was increased when the guide nucleic acid molecule strand was bound (FIG. 5), which further increased the efficiency of RNA library production. You can.

Another aspect of the invention includes a) a first adapter nucleic acid molecule ligated to the 3' end of a target RNA; b) a second adapter nucleic acid molecule bound to the 3' end of the cDNA synthesized through complementary sequence polymerization of the ligated molecule; and c) relates to a composition for constructing an RNA library, comprising the guide nucleic acid molecule strand.

The first adapter nucleic acid molecule, the second adapter nucleic acid molecule and the guide nucleic acid molecule strand are as described above.

The composition for producing an RNA library may further include a buffer solution, PCR primers, etc., and the additional components may be changed and applied as needed.

Another aspect of the present invention relates to an RNA library produced by the above production method. RNA related to disease, condition change, etc. of an individual can be designed in advance in the RNA library constructed as above, and this library can be used for the exploration and development of genetic markers and therapeutic candidates for disease treatment or condition improvement.

The RNA library production method of the present invention includes a ligation step of a first adapter nucleic acid molecule and a second adapter nucleic acid molecule, thereby increasing production efficiency compared to a conventional library production method, thereby reducing time and maintaining accuracy. Furthermore, the efficiency of RNA library production can be increased by adding a guide nucleic acid molecule to increase the ligation efficiency of the adapter nucleic acid molecule.

The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention.

Figure 1 shows a schematic diagram of RNA library production of the present invention. The X at the 3' end in the schematic means that the amine region has been modified and additional bonds are blocked.
Figure 2 shows the results of confirming the library produced by the method of the present invention through electrophoresis.
Figure 3 shows the results of confirming the library produced by the method of the present invention through qPCR (NTC: negative control, Library: library produced by the present invention).
Figure 4 shows a schematic diagram showing the binding of the guide nucleic acid molecule strand of the present invention in ligation of the second adapter nucleic acid molecule to the 3' end of cDNA. The X at the 3' end in the schematic means that the amine region has been modified and additional bonds are blocked.
Figure 5 shows the results of confirming the library production efficiency according to guide nucleic acid molecule strand binding (Figure 5A: electrophoresis results of GS-0 and GS-1, Figure 5B: electrophoresis results of GS-0 and GS-1 qPCR comparison results).

Hereinafter, the present invention will be described in detail by examples. However, the following examples only illustrate the present invention, and the present invention is not limited by the following examples.

Example 1. RNA library production

1-1. RNA extraction

Small RNA was extracted from blood using the XENOPURE blood small RNA purification kit (Xenohelix) according to the product instructions.

1-2. Binding of small RNA and first adapter nucleic acid molecule

The small RNA extracted from blood and the first adapter nucleic acid molecule with adenylation at the 5' end were combined using the T4 RNA Ligase 2, truncated KQ kit (NEB). 3 μl of extracted small RNA, 1 μl of the first adapter nucleic acid molecule with adenylated 5’ end (1 pmol/μl), 2 μl of 10X T4 RNA ligase reaction buffer (NEB), and 10 μl of 50% PEG8000 (NEB) ), 1 ㎕ of T4 RNA ligase 2, truncated KQ (NEB), 1 ㎕ of RNase inhibitor (Takara), and 2 ㎕ of RNase/DNase free water were added and mixed, and then incubated at 28°C for 60 minutes using a PCR device. Reaction was carried out at ℃ for 20 minutes and at 4℃ for 5 minutes.

1-3. Complementary strand polymerization

A small RNA cDNA to which the first adapter nucleic acid molecule was bound was synthesized using a DNA polymerase kit (Xenohelix). Small RNA (20 μl) to which the first adapter nucleic acid molecule is bound, 4 μl of 10X PCR buffer (Xenohelix), 4 μl of 10 mM dNTP (Xenohelix), 1 μl of DNA polymerase (Xenohelix), and 1 μl of RNase inhibitor. (Takara), add 1 ㎕ of index sequence-containing primer (1 pmol/㎕), 9 ㎕ of RNase/DNase free water, mix, and react at 95 ℃ for 30 seconds and 60 ℃ for 10 minutes using a PCR device. I ordered it.

1-4. RNA degrading enzyme (RNase) treatment

RNA was degraded using the Thermostable RNase H kit (Enzynomics). RNA/DNA chimera product (40 ㎕) synthesized by complementary sequence polymerization reaction, 10 ㎕ of 10X RNase H buffer (Enzynomics), 1 ㎕ of Thermostable RNase H (Enzynomics), and 49 ㎕ of RNase/DNase free water. was added and mixed, and then reacted at 50°C for 20 minutes using a PCR device. After the reaction was completed, cleanup was performed using a PCR purification kit (Xenohelix) according to the product manual.

1-5. Second adapter nucleic acid molecule binding

A second adapter nucleic acid molecule whose 5' end was adenylated was ligated using the Thermostable 5´App DNA/RNA Ligase kit (NEB). After treatment with RNase H, the cleaned product (37 ㎕) was added with 5 ㎕ of 10x NE Buffer 1 (NEB), 5 ㎕ of 50 mM MnCl ₂ (NEB), 2 ㎕ of Thermostable 5' App DNA/RNA Ligase (NEB), Add 1 ㎕ of a second adapter nucleic acid molecule (1 pmol/㎕) whose 5' end is adenylated and 1 ㎕ of a guide nucleic acid molecule strand (1 pmol/㎕), mix, and then use the PCR device. and reacted at 65°C for 60 minutes and at 90°C for 3 minutes. After the reaction was completed, cleanup was performed using a PCR purification kit (Xenohelix) according to the product manual.

1-6. amplification

Amplification was performed using the HotStart Taq DNA Polymerase kit (Xenohelix). After binding the second adapter nucleic acid molecule, the cleaned up product (37 ㎕) was mixed with 8 ㎕ of 10 Primer (10 pmol/㎕), 1 ㎕ of reverse primer (10 pmol/㎕), and 24 ㎕ of RNase/DNase free water were added and mixed, and then using a PCR device, 1 cycle at 95°C for 15 minutes, 95°C for 15 seconds, The reaction was performed under the following conditions: 60°C for 30 seconds, 22 cycles of 72°C for 15 seconds, 1 cycle of 72°C for 2 minutes, and 1 cycle of 4°C for 5 minutes. After the reaction was completed, cleanup was performed using a PCR purification kit (Xenohelix) according to the product manual.

1-7. Checking the completed library

The completion of the RNA library produced through the above process was confirmed through electrophoresis. Specifically, the RNA library prepared on a 6% polyacrylamide gel (19:1) was electrophoresed and the completed library was confirmed using Gel Doc.

As a result, as shown in Figure 2, the length of the completed library was about 140 to 150 bp, and a band near about 150 bp was observed in the electrophoresis gel, confirming that the miRNA library was successfully created. Furthermore, the dimer bands (120-130 bp) of the first adapter and the second adapter did not appear, confirming that there was no non-specific reaction due to the adapter nucleic acid molecule used in the present invention.

Additionally, qPCR was performed using primers made from the sequences of both ends of the library. Specifically, 9 μl of TB Green Premix Ex Taq II (Takara), 2 μl of primer mix (0.5 pmol/μl each), and 12 μl of RNase/μl were added to the library (2 μl) diluted 1/500. DNase free water was added and mixed, and reaction was performed using a real-time PCR device under the following conditions: 1 cycle of 95°C for 3 minutes, 40 cycles of 95°C for 10 seconds, and 64°C for 30 seconds.

As a result, as shown in Figure 3, it was confirmed that the library was successfully created with a Cq value of about 15. In addition, as a result of qPCR using primers made from both ends of the library and the miR92a-3p sequence, the Cq values were approximately 32 and 33, confirming that the first and second adapter nucleic acid molecules were successfully combined with the miRNA and the library was completed. did.

Example 2. Confirmation of library production efficiency according to guide nucleic acid molecule guide strand binding

In ligation of the second adapter nucleic acid molecule to the 3' end of the cDNA synthesized during the library construction process, the efficiency of adding a guide nucleic acid molecule strand was confirmed.

The guide nucleic acid molecule strand is i) a complementary base sequence to the 5' end of the second adapter nucleic acid molecule, ii) thymine (T), a base complementary to the A tail of cDNA generated using polymerase. , iii) It was manufactured so that A, C, G, and T all contain base N that can be combined. The sequence of the exemplary guide nucleic acid molecule strand in the present invention is SEQ ID NO: 4 listed in Table 3 below. In addition, a schematic diagram of how a guide nucleic acid molecule strand binds in ligation of a second adapter nucleic acid molecule to the 3' end of cDNA is shown in FIG. 4.

An RNA library was produced according to the production method of Example 1, except that a library was produced that did not include the guide nucleic acid molecule strand added in 1-5 (GS-0), and a library containing the guide nucleic acid molecule strand added in 1-5 was produced. Library production (GS-1) was performed to confirm the libraries of GS-0 and GS-1, respectively.

As a result, as shown in Figure 5A, bands around about 150 bp were observed for both GS-0 and GS-1, and the dimer bands (120-130 bp) of the first and second adapters did not appear. It was confirmed that the miRNA library was successfully created.

Additionally, qPCR was performed in the same manner as in 1-7 above to confirm the completed library. qPCR was performed using primers made from both ends of the library and the miR92a-3p sequence.

As a result, as shown in Figure 5B, it was confirmed that the Cq value of GS-1 containing the guide nucleic acid molecule strand was decreased compared to GS-0 not containing the guide nucleic acid molecule strand.

As described above, in the library production method of the present invention, it was confirmed that adding a guide nucleic acid molecule strand can increase the ligation efficiency of the second adapter nucleic acid molecule to the 3' end of cDNA and increase RNA library production efficiency. did.

Example 3. Confirmation of library produced using synthetic miRNA 92a-3p

As an example, a library was produced according to the library production method of the present invention using synthetic miRNA 92a-3p, and then the constructed library was confirmed by sequencing.

As a result of analysis after trimming the read length to 16-30 bp according to the miRNA with a length of 21-23 nt, 3,122,512 of the total 3,534,990 reads were mapped to human miRNA data, as shown in Table 1 below. ) was done.

Furthermore, as shown in Table 2 below, the detected miRNAs were has-miR-92a-3p and has-miR-92b-3p, and most of the reads were has-miR-92a-3p, confirming that the library was successfully created. did.

Each adapter nucleic acid molecule, guide nucleic acid molecule strand, and primer sequence used in Examples 1 to 3 are summarized in Table 3 below. The primer sequence of the present invention can be synthesized using the nucleic acid sequence of an adapter nucleic acid molecule regardless of the type of target nucleic acid molecule to be amplified.

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive. For example, each component described as unitary may be implemented in a distributed manner, and similarly, components described as distributed may also be implemented in a combined form.

The scope of the present invention is indicated by the claims described below, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be construed as being included in the scope of the present invention.

Claims (15)

a) ligating a first adapter nucleic acid molecule to the 3' end of the target RNA;
b) synthesizing cDNA through complementary sequence polymerization of the molecules ligated in step a);
c) removing RNA by treating it with an RNA degrading enzyme;
d) ligating a second adapter nucleic acid molecule to the 3' end of the cDNA; and
e) A method of producing an RNA library, comprising the step of PCR amplifying the nucleic acid molecule ligated in step d). According to paragraph 1,
A method of producing an RNA library, wherein the target RNA is small RNA. The method of claim 2, wherein the small RNA is miRNA. According to paragraph 1,
A method of producing an RNA library, wherein the first adapter nucleic acid molecule is in the form of DNA. According to paragraph 1,
A method of producing an RNA library, wherein the first adapter nucleic acid molecule is adenylated at the 5' end. According to paragraph 1,
A method of producing an RNA library, wherein the second adapter nucleic acid molecule is in the form of DNA. According to paragraph 1,
A method of producing an RNA library, wherein the second adapter nucleic acid molecule has adenylated 5' end. According to paragraph 1,
A method of producing an RNA library, wherein the complementary sequence polymerization reaction in step b) uses DNA polymerase. According to paragraph 1,
A method of producing an RNA library, wherein the 3' end of the cDNA contains an adenine (A) base. According to paragraph 1,
RNA, adding the step of increasing the ligation efficiency of the second adapter nucleic acid molecule to the 3' end of the cDNA by treating the guide nucleic acid molecule strand before the PCR amplification reaction simultaneously with step d) or after step d). How to create a library. According to clause 10,
A method of producing an RNA library, wherein the guide nucleic acid molecule strand includes sequences complementary to the 3' end of the cDNA and the 5' end of the second adapter nucleic acid molecule. According to clause 10,
A method of producing an RNA library, wherein the guide nucleic acid molecule strand includes a thymine (T) base. According to clause 10,
The guide nucleic acid molecule strand is complementary to the 3' end of the cDNA and the 5' end of the second adapter nucleic acid molecule. a) a first adapter nucleic acid molecule ligated to the 3' end of the target RNA;
b) a second adapter nucleic acid molecule bound to the 3' end of the cDNA synthesized through complementary sequence polymerization of the ligated molecule; and
c) A composition for producing an RNA library, comprising the guide nucleic acid molecule strand. An RNA library produced by the method of claim 1.