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CN-122012509-A - Aptamer, tRNA, reagent, kit and tRNA detection method for recognizing tRNA

CN122012509ACN 122012509 ACN122012509 ACN 122012509ACN-122012509-A

Abstract

The invention provides nucleic acid aptamers, tRNA's, reagents, kits and tRNA detection methods that specifically recognize tRNA's. The tRNA is mature tRNA, the aptamer comprises a 3' -end protruding part and a stem loop structure forming part, wherein the 3' -end protruding part is complementary with a CCA sequence, an identification base and an aminoacyl tRNA synthetase receptor domain at the 3' -end of the mature tRNA, and the aptamer is single-stranded DNA. Compared with the existing four-leaf grass qRT-PCR method, the nucleic acid aptamer does not need to carry out connection reaction with tRNA, simplifies the reverse transcription reaction steps of tRNA, and has the advantages of high reaction efficiency, high detection sensitivity and capability of realizing accurate, rapid and low-cost detection of tRNA.

Inventors

  • LI YINGSHAN
  • FU XIAN
  • HUANG MEIPING
  • ZHANG SHANGJIE
  • SHEN YUE

Assignees

  • 常州新一产生命科技有限公司
  • 华大工程生物学长荡湖研究所
  • 深圳华大生命科学研究院

Dates

Publication Date
20260512
Application Date
20241111

Claims (11)

  1. 1. A nucleic acid aptamer that specifically recognizes a tRNA, wherein the tRNA is a mature tRNA, the aptamer comprising: A 3' end protruding portion; a stem-loop structure forming portion; Wherein the 3 '-terminal overhang is adapted to complementarily pair with the CCA sequence, the recognition site base, and the aminoacyl tRNA synthetase receptor domain of the 3' -terminal of the mature tRNA; the nucleic acid aptamer is single-stranded DNA.
  2. 2. The nucleic acid aptamer of claim 1, wherein the 3' terminal overhang has a length of 11nt or greater; optionally, the nucleic acid aptamer is an oligonucleotide aptamer; Optionally, the length of the oligonucleotide aptamer is 50-60 nt; optionally, the tRNA comprises a tRNA corresponding to a natural amino acid, a tRNA corresponding to a nonstandard amino acid; optionally, the tRNA comprises at least one of the 20 naturally occurring amino acid corresponding tRNAs; optionally, one tRNA is suitable for transporting one or more nonstandard amino acids; Optionally, the tRNA includes, but is not limited to, at least one of the nonstandard amino acids pyrrolysine, selenocysteine, nα -Boc-L-lysine, 4-methoxy-L-phenylalanine; Optionally, the nucleic acid aptamer has a nucleotide sequence as set forth in any one of SEQ ID NO:2、SEQ ID NO:6、SEQ ID NO:10、SEQ ID NO:14、SEQ ID NO:18、SEQ ID NO:22、SEQ ID NO:26、SEQ ID NO:30、SEQ ID NO:34、SEQ ID NO:38、SEQ ID NO:42、SEQ ID NO:46、SEQ ID NO:50、SEQ ID NO:54、SEQ ID NO:58、SEQ ID NO:62、SEQ ID NO:66、SEQ ID NO:70、SEQ ID NO:74、SEQ ID NO:78、SEQ ID NO:81 or a nucleotide sequence having at least 90% identity thereto.
  3. 3. A tRNA, characterized in that the CCA sequence, the recognition site base and the aminoacyl tRNA synthetase receptor domain at the 3 'end of the tRNA are complementarily paired with the 3' overhang of the aptamer of any one of claims 1 to 2.
  4. 4. A reagent comprising the nucleic acid aptamer according to any one of claims 1 to 2, wherein the reagent is used for specifically recognizing tRNA.
  5. 5. A kit comprising the nucleic acid aptamer according to any one of claims 1 to 2 or the reagent according to claim 4, wherein the kit is used for detecting tRNA; optionally, reverse transcription reagents and/or PCR reagents are included.
  6. 6. A method for tRNA detection comprising: Contacting the nucleic acid aptamer according to any one of claims 1-2, the reagent according to claim 4 or the reagent in the kit according to claim 5 with a sample of RNA to be tested to obtain a reverse transcription template, wherein when the sample to be tested comprises a tRNA, the self secondary structure of the tRNA in the reverse transcription template is eliminated; Performing reverse transcription reaction based on the reverse transcription template to obtain a cDNA template; Performing PCR or qPCR reaction based on the cDNA template to obtain a PCR or qPCR reaction product; And determining the type or the content of tRNA in the RNA sample to be detected based on the PCR or qPCR reaction product.
  7. 7. The method of claim 6, wherein the contacting comprises: mixing the nucleic acid aptamer or a reagent containing the nucleic acid aptamer with an RNA sample to be detected and a dNTP Mix, and then carrying out high-temperature denaturation and low Wen Fuxing; Optionally, the high-temperature denaturation is carried out at 50-75 ℃, preferably 65 ℃, the high-temperature denaturation is carried out for 4-15 min, preferably 5min, the low-temperature Wen Fuxing is 0 ℃, and the low-temperature renaturation is carried out for not less than 2min.
  8. 8. The method according to claim 6, wherein the temperature of the reverse transcription reaction is pulsed, the pulse variation range of the temperature is 15-55 ℃, and the maintenance time of each pulse variation is 50-105 s; Optionally, each pulse variation comprises at least two pulse temperatures, wherein the first pulse temperature is 15-17 ℃, preferably 16 ℃, and the second pulse temperature is 45-55 ℃, preferably 50 ℃; optionally, the first pulse temperature is maintained for 25-35 s, preferably 30s, and the second pulse temperature is maintained for 25-35 s, preferably 30s.
  9. 9. The method of claim 8, wherein each of the pulse variations further comprises a third pulse temperature, wherein the first pulse temperature is lower than the second pulse temperature, the second pulse temperature is higher than the third pulse temperature, and the third pulse temperature is higher than the first pulse temperature; further, the third pulse temperature is 40-50 ℃, preferably 45 ℃; further, the maintaining time of the third pulse temperature is 25-35 s, preferably 30s; optionally, in the reverse transcription reaction, a temperature which can be tolerated by the reverse transcriptase employed is 50 ℃ or higher.
  10. 10. The method of claim 8, wherein the reverse transcription reaction is performed according to the following procedure: 40 cycles of 16 ℃ 30s,50 ℃ 30s,45 ℃ 30s; 42°C1h; 80°C10min; stored at 4 ℃.
  11. 11. The method of claim 6, wherein the amplification system of the PCR or qPCR reaction comprises a primer pair specific for both ends of tRNA, the cDNA template; further, the amplification system of the PCR reaction is as follows: Component (A) Addition amount of 2X GreenTaq mix 10μL TRNA forward primer (50. Mu. Mol/L) 1μL TRNA reverse primer (50. Mu. Mol/L) 1μL The cDNA template 0.5μL H 2 O 7.5μL Further, the amplification system of the qPCR reaction is: Component (A) Addition amount of SYBR Green qPCR Mix(2X,High ROX,UDG ) 10μL TRNA forward primer (50. Mu. Mol/L) 1μL TRNA reverse primer (50. Mu. Mol/L) 1μL The cDNA template 1μL H 2 O 7μL Further, the tRNA forward primer has a nucleotide sequence as set forth in at least one of SEQ ID NO:3、SEQ ID NO:7、SEQ ID NO:11、SEQ ID NO:15、SEQ ID NO:19、SEQ ID NO:23、SEQ ID NO:27、SEQ ID NO:31、SEQ ID NO:35、SEQ ID NO:39、SEQ ID NO:43、SEQ ID NO:47、SEQ ID NO:51、SEQ ID NO:55、SEQ ID NO:59、SEQ ID NO:63、SEQ ID NO:67、SEQ ID NO:71、SEQ ID NO:75、SEQ ID NO:79、SEQ ID NO:82, and the tRNA reverse primer has a nucleotide sequence as set forth in at least one of SEQ ID NO:4、SEQ ID NO:8、SEQ ID NO:12、SEQ ID NO:16、SEQ ID NO:20、SEQ ID NO:24、SEQ ID NO:28、SEQ ID NO:32、SEQ ID NO:36、SEQ ID NO:40、SEQ ID NO:44、SEQ ID NO:48、SEQ ID NO:52、SEQ ID NO:56、SEQ ID NO:60、SEQ ID NO:64、SEQ ID NO:68、SEQ ID NO:72、SEQ ID NO:76、SEQ ID NO:80、SEQ ID NO:83; further, the PCR reaction was performed as follows: 94°C3min; 94 ℃ 15s,60 ℃ 15s,72 ℃ 15s; 72°C5min; Preserving at 4 ℃; further, the PCR reaction was performed as follows: 50°C3min; 95°C2min; 40 cycles of 95℃15s,58℃15s,68℃15s.

Description

Aptamer, tRNA, reagent, kit and tRNA detection method for recognizing tRNA Technical Field The invention relates to the technical field of tRNA detection, in particular to a nucleic acid aptamer, tRNA, reagent, kit and tRNA detection method for specifically recognizing tRNA. Background A transfer RNA (tRNA) is a non-coding RNA with a highly secondary structure in a cell, typically between 70 and 90 bases in length. All organisms, including eukaryotes, bacteria, archaea, and some viruses, contain tRNA's. tRNA is one of the core elements in the gene expression process and is responsible for encoding the genetic information in messenger RNA (mRNA) into amino acids in proteins. Taking the eukaryotic model species Saccharomyces cerevisiae (Saccharomyces cerevisiae) as an example, the Saccharomyces cerevisiae nuclear genome and mitochondrial genome together encode 20 amino acids, together with 286 tRNA genes. In addition to classical amino acid coding functions, tRNAs can be processed into short tRNA fragments that perform other important molecular biological functions. In humans, abnormal expression of tRNA is associated with some diseases such as cancers and neurodegenerative diseases. Therefore, qualitative and quantitative analysis of intracellular tRNA expression becomes important. The most classical method for detecting tRNA is Northernblot experiment, the basic principle is that RNA in cell to be detected is first gel electrophoresis, separated according to molecular weight, then RNA is transferred to nylon membrane or nitrocellulose membrane and immobilized, finally hybridization detection is carried out by using probe marked with isotope or other marker. However, this method has several obvious disadvantages, including complex and time-consuming experimental procedures, including gel electrophoresis, transfer of membrane, incubation of probe, washing of membrane, signal detection, etc., which often take two days or more, involving radioisotope manipulation, high requirements for safe handling by the experimenter, and low sensitivity in detecting tRNA's, and inability to distinguish between different tRNA's with very high sequence similarity. These disadvantages limit the application of Northern blot methods. The basic principle of the current clover qRT-PCR method is that an RNA sample to be detected is subjected to deacylation reaction to remove amino acid at the 3' -end of tRNA, then an oligonucleotide aptamer with a stem-loop structure is designed, the oligonucleotide is a complex of DNA (5 ' -end to penultimate nucleotide) and RNA (two nucleotides at the 3' -end), the 3' -end of the aptamer is protruded with four bases (5 ' -TGGN-3 '), and the aptamer can be complementarily paired with 4 bases (5 ' -NCCA-3 ') protruded from the 3' -end of mature tRNA. Following annealing, the oligonucleotide aptamer may be ligated to tRNA using T4 RNA ligase (Rnl 2) to form a clover-like structure, since the 5 'end of the aptamer contains a phosphate group and the 3' end contains a hydroxyl group. Finally, fluorescent quantitative PCR was performed by a primer complementary to the D-and T-loops of the tRNA. However, this method has several significant disadvantages (1) the structure of the hybrid oligonucleotide aptamer is complex, the hybrid oligonucleotide aptamer is a chimeric body composed of DNA and RNA, the synthesis difficulty is high, and the cost is high, (2) the reverse transcription process needs to be carried out by taking DNA as a template, and the reverse transcription reaction by taking DNA as a template has low efficiency, so that tRNA detection efficiency is low, and (3) the hybrid oligonucleotide aptamer needs to carry out the ligation reaction with tRNA by T4 RNA ligase (Rnl 2) during the reaction, and the cost of the T4 RNA ligase (Rnl 2) is high, so that the detection cost is high. These disadvantages limit the application of this clover qRT-PCR based method in tRNA detection. Thus, there is a need to further develop accurate, simple, rapid, low cost tRNA detection methods. Disclosure of Invention The present invention aims to solve at least one of the technical problems existing in the prior art to at least some extent. Compared with the existing four-leaf grass qRT-PCR method, the nucleic acid aptamer does not need to carry out connection reaction with tRNA, simplifies the reverse transcription reaction step of tRNA, takes RNA as a template for all reverse transcription reactions, has high reaction efficiency and high detection sensitivity, and can realize accurate, rapid and low-cost detection of tRNA. The present invention has been made based on the findings and knowledge of the inventors of the following problems: the presence of many self-complementary paired sequences within the tRNA sequence results in the ability of the tRNA to form complex secondary structures, which is also one of the difficulties in reverse transcription of tRNA into cDNA. To solve this problem, the inventors designed