A method for constructing a linker bridge and RNA library.

Abstract

This invention discloses a linker bridge and a method for constructing an RNA library. The linker bridge is a double-stranded nucleotide fragment composed of a first strand and a second strand. The first strand includes regions A, B, and C sequentially from 5' to 3'. Region A includes DNA bases for linking the adapter complex; region B includes DNA bases for complementary pairing with the second strand; and region C includes DNA bases for linking the target RNA. The second strand includes regions D and E sequentially from 5' to 3'. Region D includes DNA bases for complementary pairing with the target RNA; and region E includes DNA bases for complementary pairing with the first strand. This linker bridge can effectively link the adapter complex at the 5' end of the RNA.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for constructing a linker and RNA library. Background Technology

[0002] Nanopore RNA direct sequencing technology can sequence natural RNA, selectively reading full-length transcripts without reverse transcription or PCR amplification, and without sequencing bias. This technology preserves RNA base modification information, detecting single-base modifications such as m6A, m5C, and pseudouridine on individual RNA molecules; it can accurately analyze alternative splicing, alternative polyadenylation (APA), fusion genes, and identify novel transcripts; furthermore, it can provide relatively accurate estimation of poly(A) length, restoring true RNA characteristics.

[0003] Current nanopore RNA direct sequencing technology primarily performs sequencing from the 3'-5' direction. This approach relies on the helicase used in current nanopore RNA direct sequencing, which requires operation from the 3'-5' end. Furthermore, linking the adapter complex at the 3' end of the RNA is more efficient than at the 5' end. However, 3'-5' sequencing sometimes results in the loss of information from the 5' end. Summary of the Invention

[0004] To address the above problems, the present invention provides a linker bridge, wherein the linker bridge is a double-stranded nucleotide fragment composed of a first strand and a second strand.

[0005] The first strand includes regions A, B and C sequentially from 5' to 3'. Region A includes deoxyribonucleic acid bases for connecting the adapter complex. Region B includes deoxyribonucleic acid bases for complementary pairing with the second strand. Region C includes ribonucleic acid bases for connecting the RNA to be tested.

[0006] The second strand includes regions D and E sequentially from 5' to 3'. Region D includes deoxyribonucleic acid bases for complementary pairing with the RNA to be tested, and region E includes deoxyribonucleic acid bases for complementary pairing with the first strand.

[0007] Optionally, according to the aforementioned connecting bridge, region A comprises 3-5 DNA bases, region B comprises 8-20 DNA bases, region C comprises 3-6 DNA bases, region D comprises 8-15 DNA bases, and region E comprises 8-20 DNA bases. Region A may also include a spacer, such as at least one of 5SpC3, iSpC3, and iSp18.

[0008] Optionally, according to the aforementioned connecting bridge, region A comprises DNA bases and spacers sequentially from 5' to 3', wherein there are 1 to 5 spacers.

[0009] Optionally, according to the above-described connecting bridge, the first chain is as shown in SEQ ID NO.1 or SEQ ID NO.11, and the second chain is as shown in SEQ ID NO.2 or SEQ ID NO.4.

[0010] The present invention also provides a method for constructing an RNA library, comprising: ligating the above-mentioned linker bridge to the RNA to be tested to obtain a ligation product; and ligating the ligation product to the adapter complex to obtain an RNA library.

[0011] This invention also provides another method for constructing an RNA library, comprising: ligating the aforementioned linker bridge to the RNA to be tested to obtain a ligation product; reverse transcribing the ligation product to obtain the RNA / DNA hybrid strand; and ligating the RNA / DNA hybrid strand to the adapter complex to obtain an RNA library. In this method, the first strand region A of the linker bridge preferably contains a spacer, which can terminate reverse transcription, prevent the first strand cDNA from extending to the 5' end of the linker bridge, and ensure the efficiency of subsequent ligation with the adapter complex.

[0012] Optionally, in the above method for constructing the RNA library, the condition for connecting the linker bridge to the RNA to be tested is ligation at room temperature for 60 minutes.

[0013] Optionally, in the above method for constructing the RNA library, the ratio of the first strand of the linker to the second strand of the linker to the RNA substance to be tested is 3:3:1.

[0014] The present invention also provides a nanopore library preparation kit, comprising the above-described linker and adapter complex.

[0015] Optionally, the adapter complex comprises a non-RNA polynucleotide sequencing sequence and a helicase. The non-RNA polynucleotide includes a first segment for guiding the sequencing fragment into the nanopore, a second segment for binding the helicase, a third segment for controlling the speed at which the helicase moves along the sequencing fragment, and a fourth segment for connecting the linker bridge.

[0016] The application of the above-described connecting bridge, method, or kit in any of the following ways also falls within the scope of protection of this invention:

[0017] (1) Preparation of nanopore sequencing RNA library

[0018] (2) Products for preparing nanopore sequencing RNA libraries;

[0019] (3) Nanopore sequencing;

[0020] (4) Prepare nanopore sequencing products;

[0021] (5) Test for helicase;

[0022] (6) Prepare products for testing helicases.

[0023] To develop helicases for direct sequencing of nanopore 5'-3' RNA, this patent provides a linker bridge that can effectively connect adapter complexes to the 5' end of RNA, and utilizes existing DNA helicases to collect nanopore RNA 5'-3' signals. This method can then be used to test various helicases to identify suitable helicases for direct sequencing of nanopore 5'-3' RNA. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the connecting bridge result.

[0025] Figure 2 This is a denaturing polyacrylamide electrophoresis image used to determine the ligation efficiency of the linker bridge and synthetic single-stranded RNA under different ligation conditions in Example 1.

[0026] Figure 3 This is a flowchart for measuring sample connection efficiency.

[0027] Figure 4 This is an electrophoresis diagram of the amplified products used to determine the ligation efficiency of the linker bridge with single-stranded RNA of different lengths in Example 1.

[0028] Figure 5 Flowchart for library construction using direct sequencing of single-stranded RNA in the 5′–3′ direction.

[0029] Figure 6 This is the well signal for the 40nt RNA library construction sample in Example 2.

[0030] Figure 7 This is the well signal from the 500nt mRNA library construction sample in Example 2.

[0031] Figure 8 This is the well signal from the 1000nt mRNA library construction sample in Example 2. Detailed Implementation

[0032] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0034] A schematic diagram of the connecting bridge structure described in this invention is shown below. Figure 1 As shown. The linker is a double-stranded nucleotide fragment composed of a first strand and a second strand. The first strand includes regions A, B, and C sequentially from 5' to 3'. Region A includes DNA bases for linking the adapter complex, region B includes DNA bases for complementary pairing with the second strand, and region C includes DNA bases for linking the RNA to be tested. The second strand includes regions D and E sequentially from 5' to 3'. Region D includes DNA bases for complementary pairing with the RNA to be tested, and region E includes DNA bases for complementary pairing with the first strand.

[0035] Example 1: Determination of the ligation efficiency between the linker bridge and single-stranded RNA

[0036] 1. Determination of ligation efficiency of linker bridges and synthetic single-stranded RNA under different ligation conditions

[0037] Design and synthesize bridge primer sequences

[0038] Sequence 1 (i.e., the first strand of the linker), synthetic sequence, (SEQ ID NO.1): 5'phos-CACTAGATGTGTATAAGAGACAG, positions 1-17 are DNA, positions 18-23 are RNA.

[0039] Sequence 2 (i.e., the second strand of the linker), synthetic DNA sequence (SEQ ID NO.2): GTCAACCCCTGTCTCTTATACACATCTA.

[0040] Sequence 3 (i.e., the RNA to be tested), synthetic RNA sequence (SEQ ID NO.3): GGGUUGACCUUUCUCUCCCAUAUUGCAGUCGCGGCACGAU.

[0041] Sequence 1, Sequence 2, and Sequence 3 were dissolved in annealing buffer (50 mM HEPES, pH 8.0 and 100 mM KCl) to prepare 100 μM stock solutions 1, 2, and 3. Stock solutions 1, 2, and 3 were mixed with annealing buffer (specific amounts shown in Table 1) at ratios of 1:1:1, 2:2:1, and 3:3:1, respectively, and annealed using the following annealing program: the temperature was decreased from 70 °C to 22 °C at a rate of -2 °C / min, resulting in three annealing complexes with different ratios of stock solutions 1, 2, and 3. Each ratio of annealing complex was divided into three equal parts. One part served as a negative control, and the other two parts were ligated at room temperature (20 °C) for 30 min and 60 min, respectively, to obtain ligation products (ligation system details shown in Table 2). The ligation efficiency of the ligation bridge to the synthesized single-stranded RNA under different ligation conditions was detected by electrophoresis of the ligation products with denaturing polyacrylamide (10% PAGE, 7 μM).

[0042] Table 1

[0043]

[0044] Table 2

[0045] Components Dosage (μL) Annealing compound 1 NEB T4 ligase (M0202) 1.5 NEB 5×T4 Quick ligation buffer 3 <![CDATA[Nuclease-free H2O]]> 9.5 Total volume 15

[0046] Figure 2 The image shows a denaturing polyacrylamide gel electrophoresis pattern. Quantitative analysis of the remaining single-stranded RNA (SRNA) content revealed the following results: When the ratio of bridge to synthetic SRNA was 1:1:1, 2:2:1, and 3:3:1, ligated at room temperature for 30 min, the remaining SRNA content was 46.5%, 13.0%, and 8.0%, respectively. When the ratio was 1:1:1, 2:2:1, and 3:3:1, ligated at room temperature for 60 min, the remaining SRNA content was 14.0%, 1.7%, and 0.8%, respectively. These results indicate that a bridge to synthetic SRNA ratio of 3:3:1, ligated at room temperature for 60 min, yielded the optimal ligation efficiency.

[0047] 2. Determination of the ligation efficiency of the linker bridge with single-stranded RNA of different lengths

[0048] Figure 3This is a flowchart for measuring sample ligation efficiency. As shown in the figure, the ligation bridge is connected to ssRNA (single-stranded RNA) to obtain the ligation product. Reverse transcription is performed using reverse transcriptase to obtain the RNA / DNA hybrid strand (black represents DNA, blue represents RNA). Then, the RNA strand in the RNA / DNA hybrid strand is digested with RNase H. The digested product is purified. Finally, PCR amplification is performed using primers A and B. The ligation efficiency is quantified by running the PCR products on an agarose gel and using ImageJ. The amount of amplified product from primers ① and ③ in primer pair A represents the total amount of ssRNA, and the amount of amplified product from primers ② and ③ in primer pair B represents the amount of ssRNA ligated to the ligation bridge. The ligation efficiency is the percentage of the brightness of the bands of the amplified products from primer pair B and primer pair A on electrophoresis.

[0049] 1) Fabrication of connecting bridges

[0050] Design and synthesize bridge primer sequences

[0051] Sequence 4 (i.e., the second strand of the connecting bridge), synthetic DNA sequence, (SEQ ID NO.4): AGTCTCCCCTGTCTCTTATACACATCTA.

[0052] Sequence eleven (i.e., the first strand of the connecting bridge), synthetic sequence, (SEQ ID NO.11): 5'phos-CACTT / iSp18 / / iSp18 / TAGATGTGTATAAGAGACAG. Positions 1-5 and 8-21 are DNA, positions 6-7 are two iSp18 molecules, i.e., two spacers, and positions 22-27 are RNA.

[0053] Sequence 4 and sequence 11 were dissolved in annealing buffer (50 mM HEPES, pH 8.0 and 100 mM KCl) to prepare stock solutions 4 and 11 with a concentration of 100 μM. Stock solutions 1 and 4 were mixed in a 1:1 ratio to prepare a linker (I) with a final concentration of 10 μM, and stock solutions 11 and 4 were mixed in a 1:1 ratio to prepare a linker (II) with a final concentration of 10 μM. Both were annealed using an annealing program that reduced the temperature from 70 °C to 22 °C at a rate of -2 °C / min, and then stored at -20 °C for subsequent experiments.

[0054] 2) Preparation of single-stranded RNA of different lengths

[0055] Taking the preparation of 1000nt mRNA as an example, lambda DNA (NEB) was used as a template, and primers 1000nt F (SEQ ID NO. 5) and 1000nt R (SEQ ID NO. 6) were used as primers for PCR amplification to obtain a 1000nt DNA sequence containing a T7 RNA Polymerase binding site. The obtained DNA sequence was purified and used as a template for subsequent transcription reactions using HiScribe. TM Transcription was performed using the T7 High Yield RNA Synthesis Kit (NEB) to obtain lambda 1000nt mRNA. The RNA was purified using the TRIzol method. DNase I (GenScript) was used to remove the DNA template from the mRNA, and the mixture was treated at 37°C for 30 min. The RNA was then further purified using the TRIzol method to obtain 1000nt mRNA.

[0056] Using the same preparation method, 1600nt mRNA was prepared using primer 1600nt F (SEQ ID NO.7) and primer 1600nt R (SEQ ID NO.8); 2000nt mRNA was prepared using primer 2000nt F (SEQ ID NO.9) and primer 2000nt R (SEQ ID NO.10); and stored at -80℃.

[0057] 3) Connection of the bridge to single-stranded RNA of different lengths

[0058] Add the components sequentially according to Table 3 and mix well. Use the mixture to obtain the ligation product for the ligation reaction between the bridge (Ⅰ) and single-stranded RNA. Ligation program: 20℃, 1h.

[0059] Table 3

[0060]

[0061]

[0062] 4) Determination of the ligation efficiency of the linker bridge with single-stranded RNA of different lengths

[0063] Using SuperScript TM IV reverse transcriptase (Invitrogen, 18090010) was used to reverse transcribe the ligation product from step 4) to obtain the RNA / DNA hybrid strand; then the RNA strand in the RNA / DNA hybrid strand was digested with RNase H (NEB M0297s); the digested product was purified using 1.4× magnetic beads (Beckman A63882 AMPure XP); finally, the ligation efficiency was quantified by PCR amplification.

[0064] The purified 1000nt mRNA magnetic bead product was amplified using primer pair A (1000nt F paired with 1000nt R) and primer pair B (primer (SEQ ID NO. 14) paired with 1000nt R); the purified 1600nt mRNA magnetic bead product was amplified using primer pair A (1600nt F paired with 1600nt R) and primer pair B (primer (SEQ ID NO. 14) paired with 1600nt R); the purified 2000nt mRNA magnetic bead product was amplified using primer pair A (2000nt F paired with 2000nt R) and primer pair B (primer (SEQ ID NO. 14) paired with 2000nt R).

[0065] Figure 4 The electrophoresis images of the amplification products were obtained. ImageJ was used to quantify the ligation efficiency. It was found that the ligation efficiency of the ligation bridge with 1000nt, 1600nt, and 2000nt mRNA was no less than 70%, specifically 95.3%, 71.7%, and 84.3%, respectively. The ligation efficiency with 1000nt mRNA was as high as 95%.

[0066] Example 2: Direct sequencing of single-stranded RNAs of different lengths in the 5′-3′ direction

[0067] The above embodiments experimentally confirmed the feasibility of the ligation method using a linker bridge with single-stranded RNA, and the optimal ligation conditions have been optimized. This embodiment uses this method to detect the current signal from direct sequencing of single-stranded RNA of different lengths along the 5'-3' direction.

[0068] Figure 5 This is a flowchart of the library preparation process for direct sequencing of single-stranded RNA along the 5'-3' direction. As shown, there are two library preparation methods. One method involves ligating a linker bridge to ssRNA to obtain a ligation product; then ligating the ligation product to an adapter complex to obtain an RNA library. The other method involves: ligating a linker bridge to ssRNA to obtain a ligation product; reverse transcribing the ligation product to obtain an RNA / DNA hybrid strand; and then ligating the RNA / DNA hybrid strand to an adapter complex (i.e., the adapter complex in the diagram) to obtain an RNA library.

[0069] The specific steps are as follows:

[0070] 1. Connecting bridge to synthetic single-stranded RNA

[0071] The ligation of the synthesized 40nt RNA to the linker bridge was performed using the same method as in step 1 of Example 1, "1. Determination of ligation efficiency of linker bridge and synthetic single-stranded RNA under different ligation conditions". The optimal ligation conditions were: the ratio of the first strand and second strand of the linker bridge to the synthetic single-stranded RNA was 3:3:1, and the ligation was performed at room temperature for 60 min.

[0072] 500nt mRNA was prepared using primers 500nt F (SEQ ID NO.12) and 500nt R (SEQ ID NO.13) according to the method in Example 1, "2. Determination of the ligation efficiency of the linker bridge with single-stranded RNA of different lengths".

[0073] The ligation of 500nt mRNA and 1000nt mRNA (prepared in Example 1) to the linker bridge was performed using the same method as steps 2)-3) in "2. Determination of ligation efficiency between the linker bridge and single-stranded RNA of different lengths" in Example 1. Specifically, the 1000nt mRNA was ligated to linker bridge (Ⅰ); after the 500nt mRNA was ligated to linker bridge (Ⅱ), reverse transcription primers were used to form an RNA / DNA hybrid chain. Two iSp18 molecules were designed at the 5' end of linker bridge (Ⅱ) to terminate reverse transcription, prevent the first-strand cDNA from extending to the 5' end of the linker bridge, and ensure the efficiency of subsequent ligation with the adapter complex.

[0074] 2. Connector complex and purification

[0075] The linker complex was ligated and purified from the sample using a barcode library construction kit (catalog number PY-BLP101, Beijing Puyi Biotechnology Co., Ltd.), thus completing the library construction. Detailed procedures are as follows:

[0076] 1. Prepare the following reaction system in a 1.5 mL centrifuge tube:

[0077] Table 4

[0078] Components volume Connecting bridge-mRNA 1000ng Ligation buffer 25μL Connector compound 5μL DNA ligase 8μL <![CDATA[Nuclease-free H2O]]> up to 100

[0079] After gently blowing and mixing, do not shake. Place the centrifuge tube in a 20°C metal bath and incubate for 15 minutes.

[0080] 2. Add 1.2 times the volume of the resuspended RNA magnetic beads to the centrifuge tube and mix well by pipetting. Incubate at room temperature for 10 min.

[0081] 3. Place the centrifuge tube on a magnetic rack to separate the magnetic beads for about 5-8 minutes. After the solution becomes clear, carefully remove and discard the supernatant.

[0082] 4. Remove the centrifuge tube and add 200 μL of fragment recovery buffer (AWB) to resuspend the magnetic beads. Then place the centrifuge tube on a magnetic rack to separate the magnetic beads for about 3-5 minutes. After the solution becomes clear, carefully remove and discard the supernatant. Repeat this step once. When removing the supernatant for the second time, try to remove as much residual liquid as possible.

[0083] 5. Remove the centrifuge tube from the magnetic rack, add 15-30 μL of elution buffer (EB) to resuspend the magnetic beads, and incubate at 37°C for 10 min;

[0084] 6. Separate the magnetic beads on the magnetic rack for about 2 minutes. After the solution becomes clear, carefully transfer the supernatant to a new centrifuge tube, being careful not to pick up the magnetic beads.

[0085] 7. Use the Qubit fluorescence quantitative quantitation instrument to detect the library concentration, and take 200ng of library sample for sequencing.

[0086] 4. RNA sample sequencing

[0087] The library samples were sequenced using the nanopore gene sequencing platform PolyseqOne (catalog number PY-NSRO01, Beijing Puyi Biotechnology Co., Ltd.).

[0088] Figure 6 To synthesize the well signal for 40nt RNA library preparation samples, Figure 7 This is the signal from the well of a 500nt mRNA library preparation sample. Figure 8 The nanopore sequencing signal for a 1000 nt mRNA library preparation sample was collected. The results showed that for RNA of different lengths, clear step-like patterns and smooth, complete nanopore sequencing signals could be obtained in the experiment. Subsequent steps could be taken to reduce signal noise and achieve direct sequencing of RNA in the 5′–3′ direction by selecting suitable helicases and nanopore proteins. Through the collection and analysis of nanopore sequencing signals from RNA samples of different lengths, it was demonstrated that this method can achieve direct library preparation of single-stranded RNA in the 5′–3′ direction.

[0089] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for constructing an RNA library, characterized in that, include: The linker bridge is ligated to the RNA to be tested to obtain a ligation product; the ligation product is then ligated to the adapter complex to obtain an RNA library. The linker is a double-stranded nucleotide fragment composed of a first strand and a second strand. The first strand includes regions A, B, and C sequentially from 5' to 3'. Region A includes deoxyribonucleic acid (DNA) bases for linking the adapter complex. Region B includes DNA bases for complementary pairing with the second strand. Region C includes ribonucleic acid (RNA) bases for linking the RNA to be tested. The second strand includes regions D and E sequentially from 5' to 3'. Region D includes DNA bases for complementary pairing with the RNA to be tested. Region E includes DNA bases for complementary pairing with the first strand. The linker complex comprises a non-RNA polynucleotide and a helicase; The non-RNA polynucleotide includes a first segment for guiding the sequencing fragment into the nanopore, a second segment for binding helicase, a third segment for controlling the speed at which the helicase moves along the sequencing fragment, and a fourth segment for connecting the linking bridge.

2. The database construction method according to claim 1, characterized in that, Region A comprises 3-5 DNA bases, Region B comprises 8-20 DNA bases, Region C comprises 3-6 DNA bases, Region D comprises 8-15 DNA bases, and Region E comprises 8-20 DNA bases.

3. The database construction method according to claim 1, characterized in that, Region A, from 5' to 3', includes deoxyribonucleic acid bases and spacers, with 1 to 5 spacers.

4. The database construction method according to any one of claims 1-3, characterized in that, The first chain is shown as SEQ ID NO.1 or SEQ ID NO.11, and the second chain is shown as SEQ ID NO.2 or SEQ ID NO.

4.

5. The application of the library construction method according to any one of claims 1-4 in any of the following: (1) Construction of nanopore sequencing RNA library (2) Products for preparing nanopore sequencing RNA libraries; (3) Nanopore sequencing; (4) Preparation of nanopore sequencing products; (5) Test for helicase; (6) Prepare products for testing helicase.

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