CN107488660A - The palindrome and complementary palindrome tiny RNA and its application - Google Patents

The palindrome and complementary palindrome tiny RNA and its application Download PDF

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CN107488660A
CN107488660A CN201710815083.3A CN201710815083A CN107488660A CN 107488660 A CN107488660 A CN 107488660A CN 201710815083 A CN201710815083 A CN 201710815083A CN 107488660 A CN107488660 A CN 107488660A
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palindrome
tiny rna
sequence
complementary
rna
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高山
陈泽
刘光远
姚雪
殷红
罗建勋
王青松
王芳
任巧云
罗金
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Nankai University
Lanzhou Veterinary Research Institute of CAAS
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Nankai University
Lanzhou Veterinary Research Institute of CAAS
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    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Abstract

The invention belongs to biological technical field.Especially, the present invention relates to the palindrome or complementary palindrome tiny RNA, and the method that siRNA is prepared according to both new tiny RNAs is proposed.By the siRNA being prepared be used for cell RNA interference experiments be study the biological question such as tiny RNA function and microorganism infection mechanism a kind of effective means.

Description

The palindrome and complementary palindrome tiny RNA and its application
Technical field
The invention belongs to biological technical field, specifically proposes and defines the palindrome and complementary palindrome tiny RNA, and proposes The method that siRNA (small interfering RNA, siRNA) is prepared according to both new tiny RNAs.The palindrome with mutually Refill literary tiny RNA to be found and defined first in the world by Nankai University's high mountain etc., prepared according to both new tiny RNAs small dry Disturb RNA be used for cell RNA interference experiments be study the biological question such as tiny RNA function and microorganism infection mechanism effective hand Section.
Background technology
Tiny RNA (small RNA, sRNA) is general name of the length in below 200nt RNA molecule, and it is most ripe that it includes people Multiple species such as the Microrna (micro RNA, miRNA) known.2016, Nankai University's high mountain etc. was excavated by big data Find first in the world and define palindrome tiny RNA (palindrome small RNA, psRNA) and complementary palindrome tiny RNA (complemented palindrome small RNA, cpsRNA).Palindrome tiny RNA is characterized in the nucleic acid sequence of positive and negative reading Arrange just the same (for example, as shown in Figure 1A)；The nucleotide sequence of complementary palindrome tiny RNA forward read and the nucleic acid sequence of reverse read Row are in the relation of base pair complementarity (for example, as shown in Figure 1B).Further investigations have shown that both tiny RNAs are widely present, And there is very important biological function：Some palindrome tiny RNAs are relevant with gene expression regulation；And some complementary palindrome are small RNA works in microorganism (for example, virus, bacterium, fungi etc.) infects.The present invention propose first in the world the palindrome with Simultaneously their feature is described in detail in the definition of complementary palindrome tiny RNA.With reference to real case, the present invention is also demonstrated according to two kinds SiRNA prepared by tiny RNA can cause the cell effect of highly significant, therefore, available for research tiny RNA function and micro- life The biological questions such as thing infection mechanism.
The content of the invention
An object of the present invention is to provide two kinds of new tiny RNAs.
A kind of palindrome tiny RNA (psRNA), it is characterised in that：The nucleotide sequence read along the tiny RNA positive (5 ' → 3 ') The nucleotide sequence read with reverse (3 ' → 5 ') is identical (for example, as shown in Figure 1A), and its length range is 15 to 200 Individual nucleotides.
The nucleotide sequence of the palindrome tiny RNA screens acquisition by the following method, the described method comprises the following steps：
(1) tiny RNA high-flux sequence (small RNA sequencing, sRNA-seq) is carried out to biological sample, from survey Ordinal number finds palindrome tiny RNA in；
(2) each palindrome tiny RNA that will be found in step (1), the Blast instruments (https of NCBI websites is passed through：// Blast.ncbi.nlm.nih.gov/Blast.cgi) with NCBI nucleic acid databases (Nucleotide collection, nt/ Nr) it is compared, the parameter of comparison is that Seed Sequences length (Word size) is 7, and matching score (Match scores) is 1, Mispairing score (Mismatch scores) is -1, and initial vacancy point penalty (Gap costs for existence) is 2, and extension is empty Position point penalty (Gap costs for extension) is 1；
(3) for each bar palindrome tiny RNA found in step (1), if existed extremely in the comparison result that step (2) obtains A few sequence and the sequence identity of palindrome tiny RNA described in step (1) are at least more than 90%, and the palindrome tiny RNA determines The palindrome tiny RNA obtained for screening；
Or it the described method comprises the following steps：Palindrome nucleic acid fragment is found from the genome sequence of animal or microorganism, The palindrome nucleic acid fragment is to be read along the nucleotide sequence that the nucleic acid fragment positive (5 ' → 3 ') is read and reverse (3 ' → 5 ') Nucleotide sequence it is identical；Pair reverse transcription is carried out with the RNA of the genomic source species identical biological sample, work as reverse transcription When existing in product with the palindrome nucleic acid fragment sequence identical cDNA, by the T in sequence corresponding to the palindrome nucleic acid fragment Replace with U, you can obtain the palindrome tiny RNA that screening obtains.
Preferably, the nucleotide sequence of the palindrome tiny RNA such as SEQ ID NO：Shown in 5.
A kind of complementary palindrome tiny RNA (cpsRNA), it is characterised in that：The core read along the tiny RNA positive (5 ' → 3 ') Base pair complementarity (for example, as shown in Figure 1B) in the nucleotide sequence that base in acid sequence is read with reverse (3 → 5 '), and And its length range is 15 to 200 nucleotides.
The nucleotide sequence of the complementary palindrome tiny RNA screens acquisition by the following method, the described method comprises the following steps：
(1) tiny RNA high-flux sequence (small RNA sequencing, sRNA-seq) is carried out to biological sample, from survey Ordinal number finds complementary palindrome tiny RNA in；
(2) each complementary palindrome tiny RNA that will be found in step (1), passes through the Blast instruments of NCBI websites (https：//blast.ncbi.nlm.nih.gov/Blast.cgi) and NCBI nucleic acid databases (Nucleotide Collection, nt/nr) it is compared, the parameter of comparison is that Seed Sequences length (Word size) is 7, matching score (Match scores) is 1, and mispairing score (Mismatch scores) is -1, initial vacancy point penalty (Gap costs for Existence it is) 2, extension gap penalty (Gap costs for extension) is 1；
(3) for each bar complementation palindrome tiny RNA found in step (1), if deposited in the comparison result that step (2) obtains It is at least more than 90% in the sequence identity of at least one sequence and the complementary palindrome tiny RNA, the complementary palindrome tiny RNA is true It is set to the complementary palindrome tiny RNA that screening obtains；
Or it the described method comprises the following steps：Complementary palindrome nucleic acid piece is found from the genome sequence of animal or microorganism Section, the complementary palindrome nucleic acid fragment is along the base in the nucleotide sequence of the nucleic acid fragment positive (5 ' → 3 ') reading and instead Base pair complementarity in the nucleotide sequence read to (3 ' → 5 ')；Pair with the genomic source species identical biological sample RNA carries out reverse transcription, when existing in reverse transcription product with the complementary palindrome nucleic acid fragment sequence identical cDNA, this is mutual The T refilled in sequence corresponding to literary nucleic acid fragment replaces with U, you can obtains the complementary palindrome tiny RNA that screening obtains.
Preferably, the nucleotide sequence such as SEQ ID NO of the complementary palindrome tiny RNA：1st, shown in 3 or 4.
An object of the present invention, which also resides in, provides a kind of method for preparing siRNA, and methods described includes, and synthesis is small RNA interfering, the sequence of the positive-sense strand of the siRNA is the palindrome or complementary palindrome tiny RNA identical sequence with the present invention, Or the sequence obtained at the palindrome of the present invention or 5 ' ends of complementary palindrome tiny RNA and/or 3 ' end additions or 1-5 nucleotides of deletion Row；Or the nucleic acid molecules of composite coding ShorthairpinRNA (short hairpin RNA, shRNA), expression vector is inserted into, is turned Enter in host cell and express, obtain siRNA, the double stranded section of the shRNA refills comprising the palindrome with the present invention or mutually The sequence identical sequence of literary tiny RNA, or the present invention the palindrome or complementary palindrome tiny RNA 5 ' end and/or 3 ' end addition or Delete the sequence that 1-5 nucleotides obtains.
Present invention also offers a kind of siRNA, its just chain nucleic acid sequence such as SEQ ID NO：2nd, shown in 3,4 or 6.
In certain preferred embodiments, the length of the palindrome tiny RNA or complementary palindrome tiny RNA is 15-150nt, excellent Selection of land, be 15-100nt, it is highly preferred that be 15-50nt, for example, 15nt, 16nt, 17nt, 18nt, 19nt, 20nt, 21nt, 22nt、23nt、24nt、25nt、26nt、27nt、28nt、29nt、30nt、31nt、32nt、33nt、34nt、35nt、36nt、 37nt、38nt、39nt、40nt、41nt、42nt、43nt、44nt、45nt、46nt、47nt、48nt、49nt、50nt。
In certain preferred embodiments, the animal includes people.
In certain preferred embodiments, the biological sample is from the blood of animal (such as people), saliva, urine Liquid, excrement, various tissues or cell line；It is highly preferred that it is blood or cell line.
In certain preferred embodiments, the microorganism includes virus, bacterium or fungi, it is preferable that the virus is SARS virus；Preferably, the bacterium is brucella；Preferably, the fungi is aspergillus fumigatus.
In certain preferred embodiments, the sequenator of the tiny RNA high-flux sequence based on Illumina companies.
In certain preferred embodiments, the sequencing data is from the document or database published；It is highly preferred that From NCBI SRA (Sequence Read Archive) database.
In certain preferred embodiments, the homogeneity be at least more than 91%, at least more than 92%, at least 93% with Above, at least more than 94%, at least more than 95%, at least more than 96%, at least more than 97%, at least more than 98%, at least 99% More than, or 100%.
An object of the present invention is that the palindrome as described above or complementary palindrome tiny RNA are viral, thin for detecting in preparation Application in the reagent of bacterium and/or fungal infection, wherein the palindrome or complementary palindrome tiny RNA from virus, bacterium and/or Fungi.Preferably, the reagent is included according to the palindrome or the sequences Design of complementary palindrome tiny RNA and the primer synthesized or spy Pin.Preferably, the nucleotide sequence such as SEQ ID NO of the complementary palindrome tiny RNA：Shown in 1 and the virus is SARS virus；It is excellent Selection of land, the nucleotide sequence such as SEQ ID NO of the complementary palindrome tiny RNA：Shown in 3 and the bacterium is brucella；Preferably, The nucleotide sequence such as SEQ ID NO of the complementary palindrome tiny RNA：Shown in 4 and the fungi is aspergillus fumigatus.
An object of the present invention is that the palindrome as described above or complementary palindrome tiny RNA are used for external evoked cell in preparation Application in apoptosis or the external reagent for promoting cell to breed.Preferably, the reagent includes siRNA or includes coding The carrier of shRNA gene, wherein the sequence of the positive-sense strand of the siRNA is and the palindrome as described above or the complementary palindrome The sequence identical sequence of tiny RNA, or in 5 ' ends of the palindrome as described above or complementary palindrome tiny RNA and/or 3 ' end additions or Delete the sequence that 1-5 nucleotides obtains；The double stranded section of the hairpin structure of the shRNA include with the palindrome as described above or The sequence identical sequence of complementary palindrome tiny RNA, or in the palindrome as described above or 5 ' ends of complementary palindrome tiny RNA and/or 3 ' The sequence that 1-5 nucleotides obtains is deleted in end addition.
An object of the present invention, which also resides in, provides a kind of vitro induction of apoptosis or the external side for promoting cell propagation Method, methods described include transfecting the cell with siRNA or the table of the gene comprising coding shRNA are transferred to the cell Up to carrier, wherein the sequence of the positive-sense strand of the siRNA is the 5 ' ends in the palindrome as described above or complementary palindrome tiny RNA And/or 3 ' end addition or delete the obtained sequence of 1-5 nucleotides；The double stranded section of the hairpin structure of the shRNA is included in The sequence that 1-5 nucleotides obtains is deleted at the 5 ' ends and/or 3 ' end additions of the palindrome as described above or complementary palindrome tiny RNA.
In certain preferred embodiments, " in 5 ' ends of the palindrome or complementary palindrome tiny RNA and/or 3 ' end additions or Delete 1-5 nucleotides " include adding or deleting 1,2,3,4 or 5 nucleotides.
In certain preferred embodiments, the carrier includes plasmid or viral vector.
In certain preferred embodiments, the cell includes zooblast, such as people's cell.
The beneficial effect of invention
The invention provides palindrome tiny RNA or complementary palindrome tiny RNA, according to the palindrome tiny RNA or complementary palindrome tiny RNA The siRNA of preparation being capable of inducing cell apoptosis or promotion cell propagation.Compared to traditional siRNA preparation method, It is simpler, accurate, quick, effective according to the method that the palindrome of the present invention or complementary palindrome tiny RNA prepare siRNA.
In the present invention, unless otherwise stated, Science and Technology noun used herein has art technology The implication that personnel are generally understood that.Also, involved laboratory operation step is to be widely used in corresponding field herein Conventional steps.
In order that the object of the invention, technical scheme and advantage are more clearly understood, below in conjunction with the accompanying drawings and embodiment, to this Invention is further elaborated.It should be appreciated that the following example is merely to illustrate the present invention, and it should not be regarded as limiting this hair Bright scope.Unreceipted actual conditions person in embodiment, the condition suggested according to normal condition or manufacturer are carried out.Agents useful for same Or the unreceipted production firm person of instrument, being can be by the conventional products of acquisition purchased in market.
Brief description of the drawings
Fig. 1 is the feature schematic diagram of the palindrome or complementary palindrome tiny RNA, and wherein A is the feature schematic diagram of palindrome tiny RNA, and B is The feature schematic diagram of complementary palindrome tiny RNA.
Fig. 2 is that the siRNA designed according to complementary palindrome tiny RNA SARS-CoV-cpsR-22 induces PC-9 cells to wither The testing result died.
Embodiment
Embodiment 1
(1) by being preced with to serious acute respiratory syndrome (Severe Acute Respiratory Syndrome, SARS) The mouse of shape virus infection carries out tiny RNA high-flux sequence and obtains a kind of complementary palindrome tiny RNA of 22-nt length, and its sequence is UCUUUAACAAGCUUGUUAAAGA(SEQ ID NO：1), the sequence is positioned at sars coronavirus reference gene group DQ497008 (GenBank database accession number) 25962bp to 25983bp, is named as SARS-CoV- cpsR-22。
(2) according to three fragments of (1) described SARS-CoV-cpsR-22 full length sequence, three kinds of tiny RNAs are designed：Respectively It is 18-nt tiny RNAs, its sequence is UUAACAAGCUUGUUAAAG；19-nt tiny RNAs, its sequence are UUAACAAGCUUGUUAAAGA(SEQ ID NO：2)；20-nt tiny RNAs, its sequence are UUUAACAAGCUUGUUAAAGA.In order to The advantage of complementary palindrome is embodied, specially upsets 19-nt tiny RNA complementation palindromes, design control tiny RNA, its sequence For AGUACUAGGAAUACUUAUA.Four kinds of double-stranded DNAs are respectively synthesized according to the sequence of these four tiny RNAs.
(3) four kinds of double-stranded DNAs described in (2) are connected into carrier pSIREN-RetroQ (Clontech, USA) and be transferred to big Enterobacteria expands, and prepares four kinds of plasmids that can express shRNA respectively, is named as plasmid 18, plasmid 19, plasmid 20 and plasmid Control, 18-nt tiny RNAs described in corresponding (2), 19-nt tiny RNAs, 20-nt tiny RNAs and control tiny RNA.
(4) there is the pathogenic effect of equal authenticity to verify SARS-CoV-cpsR-22 that this experiment detects with SARS virus Fruit, this experiment are specially tested from the lung cancer cell line PC-9 sensitive to SARS virus.All experimental cells are divided into 12 Sample (each sample about 2 × 105Individual cell), four groups are further divided into, four kinds of different plasmids are transfected respectively, equivalent to every kind of plasmid Carry out repeating to test three times.First group of transfection comes from the plasmid Control of (3)；Second group of transfection comes from the plasmid 18 of (3)； 3rd group of transfection comes from the plasmid 19 of (3)；4th group of transfection comes from the plasmid 20 of (3).All samples are according to (5-6) stream Cheng Jinhang is tested.
(5) the previous day is tested with PBS cell, sucks PBS washing lotions；0.25% pancreatin digestion is added into blake bottle Attached cell；After adding culture medium to terminate digestion, the cell for repeating experiment three times enough is collected with 15 milliliters of centrifuge tubes；4 DEG C of centrifugations (1000rpm) removes supernatant in 10 minutes；Add Gbico RPMI-1640 culture mediums (the Thermo Fisher containing 10% hyclone Scientific, USA) dilution, cell count, determine volume, it is 2 milliliters (about 2 × 10 to ensure each sample volume5Individual cell), It is seeded to 1 hole of 6 orifice plates.Transfected (after about 12 hours) within second day, each sample is proceeded as follows：Use first Opti-MEM culture mediums (Thermo Fisher Scientific, USA) dilute the plasmid of 2 micrograms to 250 microlitres, use Opti- The Lipofectamine 2000 (Thermo Fisher Scientific, USA) to 250 that MEM culture mediums dilute 5 microlitres is micro- Rise, be each stored at room temperature 5 minutes after soft mixing；Plasmid mixed liquor is mixed with Lipofectamine mixed liquors again, room temperature is incubated Educate 20 minutes；After sucking cell conditioned medium culture medium, add 1.5 milliliters of Opti-MEM culture mediums, so with PBS cell twice Plasmid and Lipofectamine mixed liquors (0.5 milliliter) are drop by drop added cell solution afterwards, all around slightly shaken It is even；37 DEG C of incubator cultures change the Gbico RPMI-1640 culture mediums containing 10% hyclone after 6 hours, continue culture 42 hours, Renew and collect cell after being further cultured for 24 hours after culture medium.
(6) according to cell apoptosis detection kit FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen, USA) explanation each sample is proceeded as follows：With PBS cell, PBS washing lotions are sucked；To each 0.25% trypsin solution digestion attached cell is added in hole；Add the GbicoRPMI-1640 culture mediums containing 10% hyclone After stopping digestion, all cells are collected with 5 milliliters of centrifuge tubes；4 DEG C of centrifugations (1000rpm) remove supernatant in 10 minutes；With the PBS of precooling Clean cell twice, be resuspended in buffer solution (1 × Binding Buffer), regulation concentration to 1 × 106Cells/ml；Order takes one Individual new centrifuge tubes, 100 microlitres of cell suspensions are added, add 5 microlitres of FITC-Annexin V solution and 5 microlitres of PI solution, Lucifuge is incubated at room temperature 15 minutes after soft mixing；400 microlitres of buffer solution (1 × Binding Buffer) is added, in 1 hour Detected with flow cytometer (BD Biosciences, USA).
Test result indicates that complementary palindrome tiny RNA SARS-CoV-cpsR-22 18-nt fragments (Fig. 2 B), 19-nt fragments (Fig. 2 C) and 20-nt fragments (Fig. 2 D), compared with compareing (Fig. 2A), 1.37 (6.2/ are improved by PC-9 apoptosis rates 4.54), 7.94 (36.04/4.54) and 3.99 (18.12/4.54) times.Particularly 19-nt fragments directly cause serious cell Apoptosis, its action effect are suitable with viral infection.Therefore, from cellular level demonstrate this complementary palindrome tiny RNA have it is important Biological function.
Embodiment 2
(1) tiny RNA high-flux sequence is carried out by the sheep for infecting brucella (Brucella Suis 019) to obtain A kind of complementary palindrome tiny RNA of 20-nt length, its sequence are GAUUGGAACAUGUUCCAAUC (SEQ ID NO：3), the sequence It is positioned at the reference gene group CP013963's (GenBank database accession number) of No. 1 chromosome of brucella 531380bp to 531399bp.
(2) in order to embody the advantage of complementary palindrome, specially by the complementary palindrome knot of (1) the complementary palindrome tiny RNA Structure is upset, and design control tiny RNA, its sequence is GAUGAUCUGACAUUGCAAUC.According to (1) described complementary palindrome tiny RNA with The sequence of control tiny RNA is respectively synthesized two kinds of double-stranded DNAs.
(3) two kinds of double-stranded DNAs described in (2) are connected into carrier pSIREN-RetroQ (Clontech, USA) and be transferred to big Enterobacteria expands, and prepares two kinds of plasmids that can express shRNA respectively, is named as plasmid 20 and plasmid Control, corresponding (1) Described in compare tiny RNA described in complementary palindrome tiny RNA and (2).
(4) this experiment carries out cell experiment from common breast cancer cell line MCF-7.All experimental cells are divided into 6 Sample (each sample about 2 × 105Individual cell), two groups are further divided into, two kinds of different plasmids are transfected respectively, equivalent to every kind of plasmid Carry out repeating to test three times.First group of cell transfecting comes from the plasmid Control of (3)；Second group of cell transfecting comes from (3) the plasmid 20.All samples are tested according to (5-6) described flow.
(5) it is described with reference to step (5) in embodiment 1, carry out cell transfecting and culture with the plasmid of (4).
(6) described with reference to step (6) in embodiment 1, the cell collected to step (5) detects.
Test result indicates that complementary palindrome tiny RNA (SEQ ID NO described in (1)：3) it is, compared with the control, that MCF-7 is thin Born of the same parents' apoptosis rate improves 47%.Therefore, demonstrating this complementary palindrome tiny RNA from cellular level has important biology work( Energy.
Embodiment 3
(1) tiny RNA high pass measurement is carried out by infecting the human cell line of aspergillus fumigatus (Aspergillus fumigatus) Sequence obtains a kind of complementary palindrome tiny RNA of 20-nt length, and its sequence is UGGCUCAGAAUUCUGAGCCA (SEQ ID NO：4), The sequence is positioned at reference gene group NC_007194 (the RefSeq databases accession of No. 1 chromosome of aspergillus fumigatus Number 4463736bp to 4463755bp).
(2) in order to embody the advantage of complementary palindrome, specially by the complementary palindrome knot of (1) the complementary palindrome tiny RNA Structure is upset, and design control tiny RNA, its sequence is UGAUCAAGCUUGCUGAGCCA.According to (1) described complementary palindrome tiny RNA with The sequence of control tiny RNA is respectively synthesized two kinds of double-stranded DNAs.
(3) two kinds of double-stranded DNAs described in (2) are connected into carrier pSIREN-RetroQ (Clontech, USA) and be transferred to big Enterobacteria expands, and prepares two kinds of plasmids that can express shRNA respectively, is named as plasmid 20 and plasmid Control, corresponding (1) Described in compare tiny RNA described in complementary palindrome tiny RNA and (2).
(4) this experiment carries out cell experiment from common breast cancer cell line MCF-7.All experimental cells are divided into 6 Sample (each sample about 2 × 105Individual cell), two groups are further divided into, two kinds of different plasmids are transfected respectively, equivalent to every kind of plasmid Carry out repeating to test three times.First group of cell transfecting comes from the plasmid Control of (3)；Second group of cell transfecting comes from (3) the plasmid 20.All samples are tested according to (5-6) described flow.
(5) cell transfecting and culture are carried out with the plasmid of (4) with reference to step (5) identical operation in embodiment 1.
(6) detected with reference to the cell that the operation of step (6) identical is collected to step (5) in embodiment 1.
Test result indicates that complementary palindrome tiny RNA (SEQ ID NO described in (1)：4) it is, compared with the control, that MCF-7 is thin Born of the same parents' apoptosis rate improves 53%.Therefore, demonstrating this complementary palindrome tiny RNA from cellular level has important biology work( Energy.
Embodiment 4
(1) complementary palindrome tiny RNA (SEQ ID NO described in embodiment 1：1), complementary palindrome tiny RNA described in embodiment 2 (SEQ ID NO：3) and embodiment 3 described in complementary palindrome tiny RNA (SEQ ID NO：4) respectively by from by SARS diseases The tiny RNA of poison, the animal sample of brucella and aspergillus fumigatus infection or cell line carries out high-flux sequence acquisition.
(2) the Blast instruments (https of NCBI websites is passed through：//blast.ncbi.nlm.nih.gov/Blast.cgi), By SEQ ID NO：1st, 3 and 4 it is compared respectively with NCBI nucleic acid databases (Nucleotide collection, nt/nr), The parameter of comparison is that Seed Sequences length (Word size) is 7, and matching score (Match scores) is 1, mispairing score (Mismatch scores) is -1, and initial vacancy point penalty (Gap costs for existence) is 2, extension gap penalty (Gap costs for extension) is 1.Only retain and complementary palindrome tiny RNA (SEQ in (1) in obtained comparison result ID NO：1), complementary palindrome tiny RNA (SEQ ID NO：Or complementary palindrome tiny RNA (SEQ ID NO 3)：4) at most there are 2 mispairing Sequence.
(3) UCSC databases (https is passed through：//genome.ucsc.edu/) download mouse genome (version number mm10) Full sequence, Blast softwares locally are being used, by complementary palindrome tiny RNA (SEQ ID NO in (1)：1) enter with mouse genome Row compares, and the parameter of comparison is that Seed Sequences length (Word size) is 7, and matching score (Match scores) is 1, mispairing Score (Mismatch scores) is -1, and initial vacancy point penalty (Gap costs for existence) is 2, and extension room is penalized It is 1 to divide (Gap costs for extension).Only retain and complementary palindrome tiny RNA (SEQ in (1) in obtained comparison result ID NO：1) at most there is the sequence of 3 mispairing.
(4) UCSC databases (https is passed through：//genome.ucsc.edu/) download ovine genome (version number OviAri3) full sequence, Blast softwares locally are being used, by complementary palindrome tiny RNA (SEQ ID NO in (1)：And sheep 3) Genome is compared, and the parameter of comparison is that Seed Sequences length (Word size) is 7, matching score (Match scores) For 1, mispairing score (Mismatch scores) is -1, and initial vacancy point penalty (Gap costs for existence) is 2, is prolonged Gap penalty (Gap costs for extension) is stretched for 1.Only retain and the complementary palindrome in (1) in obtained comparison result Tiny RNA (SEQ ID NO：3) at most there is the sequence of 3 mispairing.
(5) UCSC databases (https is passed through：//genome.ucsc.edu/) download human genome (version number hg38) entirely Portion's sequence, Blast softwares locally are being used, by complementary palindrome tiny RNA (SEQ ID NO in (1)：4) compared with human genome Right, the parameter of comparison is that Seed Sequences length (Word size) is 7, and matching score (Match scores) is 1 mispairing score (Mismatch scores) is -1, and initial vacancy point penalty (Gap costs for existence) is 2, extension gap penalty (Gap costs for extension) is 1.Only retain and complementary palindrome tiny RNA (SEQ in (1) in obtained comparison result ID NO：4) at most there is the sequence of 3 mispairing.
All experimentss result：
For SEQ ID NO：Complementary palindrome tiny RNA sequence shown in 1, the result that (2) return show that it is positioned at SARS Coronavirus reference gene group DQ497008 (GenBank database accession number) 25962bp is arrived 25983bp, without discovery and SEQ ID NO in the genome of other source of species：1 identical or homogeneity more than 90% sequence Row.And the result that (3) return has been reaffirmed in host mouse genome not with being isolated from the mutual of mouse samples in embodiment 1 Refill literary tiny RNA (SEQ ID NO：1) identical or homogeneity more than 90% sequence；Thus prove, SEQ ID NO：Shown in 1 Complementary palindrome tiny RNA uniquely comes from SARS virus, and it can be as the detection of nucleic acids mark of SARS virus.
For SEQ ID NO：Complementary palindrome tiny RNA sequence shown in 3, the result that (2) return show that it is positioned at Bu Lu The reference gene group CP013963 (GenBank database accession number) of No. 1 chromosome of Salmonella 531380bp To 531399bp, without discovery and SEQ ID NO in the genome of other source of species：3 identical or homogeneity more than 90% Sequence.And the result that (4) return has been reaffirmed in host's ovine genome not with being isolated from sheep sample in embodiment 2 Complementary palindrome tiny RNA (SEQ ID NO：3) same or analogous sequence；Thus prove, SEQ ID NO：Mutually refilling shown in 3 Literary tiny RNA uniquely comes from brucella, and it can be as the detection of nucleic acids mark of brucella.
For SEQ ID NO：Complementary palindrome tiny RNA sequence shown in 4, the result that (2) return show that it is positioned at cigarette song The reference gene group NC_007194 (RefSeq database accession number) of No. 1 chromosome of mould 4463736bp To 4463755bp, without discovery and SEQ ID NO in the genome of other source of species：4 identical or homogeneity more than 90% Sequence.And the result that (5) return has been reaffirmed in host's human genome not with being isolated from the mutual of human sample in embodiment 3 Refill literary tiny RNA (SEQ ID NO：4) identical or homogeneity more than 90% sequence；Thus prove, SEQ ID NO：Shown in 4 Complementary palindrome tiny RNA uniquely comes from aspergillus fumigatus, and it can be as the detection of nucleic acids mark of aspergillus fumigatus.
Embodiment 5
(1) a kind of palindrome tiny RNA, its sequence are obtained by carrying out tiny RNA high-flux sequence to human mitochondrial enriched product Row are GACACCCCCCACAG (SEQ ID NO：5), the sequence is positioned at human mitochondrial reference gene group NC_012920 564bp to the 577bp of (GenBank database accession number).
(2) a kind of full length sequence and sequence spreading of palindrome tiny RNA according to (1), design two kinds of tiny RNAs：Point It is not 14-nt tiny RNAs, its sequence is GACACCCCCCACAG；20-nt tiny RNAs, its sequence are AAAGACACCCCCCACAGAAA (SEQ ID NO：6).In order to embody the advantage of palindrome, specially palindrome is upset, design control tiny RNA, its sequence For AGAACACCACCACCACAGAA.According to three kinds of double-stranded DNAs of sequent synthesis of these three tiny RNAs.
(3) three kinds of double-stranded DNAs described in (2) are connected into carrier pSIREN-RetroQ (Clontech, USA) and be transferred to big Enterobacteria expands, and prepares three kinds of plasmids that can express shRNA respectively, is named as plasmid 14, plasmid 20 and plasmid Control, 14-nt tiny RNAs described in corresponding (1), 20-nt tiny RNAs and control tiny RNA.
(4) this experiment specially carries out cell experiment from the high liver cancer cell lines HepG2 of Mitochondria content.All experiments are thin Born of the same parents are divided into 9 samples (each sample about 2 × 105Individual cell), three groups are further divided into, transfects three kinds of different plasmids respectively, quite Carry out repeating to test three times in every kind of plasmid.First group of cell transfecting comes from the plasmid Control of (3)；It is second group thin The plasmid 14 of the dysuria with lower abdominal colic dye from (3)；3rd group of cell transfecting comes from the plasmid 20 of (3).After transfection, each sample is used 6 holes of 96 orifice plates carry out the cytoactive detection of 6 repetitions.All samples are tested according to (5-6) described flow.
(5) the previous day is tested with PBS cell, sucks PBS washing lotions；0.25% pancreatin digestion is added into blake bottle Attached cell；After adding culture medium to terminate digestion, the cell for repeating experiment three times enough is collected with 15 milliliters of centrifuge tubes；4 DEG C of centrifugations (1000rpm) removes supernatant in 10 minutes；Add Gbico RPMI-1640 culture mediums (the Thermo Fisher containing 10% hyclone Scientific, USA) dilution, cell count, determine volume, it is 2 milliliters (about 2 × 10 to ensure each sample volume5Individual cell), It is seeded to 1 hole of 6 orifice plates.Transfected (after about 12 hours) within second day, each sample is proceeded as follows：Use first Opti-MEM culture mediums (Thermo Fisher Scientific, USA) dilute the plasmid of 2 micrograms to 250 microlitres, use Opti- The Lipofectamine 2000 (Thermo Fisher Scientific, USA) to 250 that MEM culture mediums dilute 5 microlitres is micro- Rise, be each stored at room temperature 5 minutes after soft mixing；Plasmid mixed liquor is mixed with Lipofectamine mixed liquors again, room temperature is incubated Educate 20 minutes；After sucking cell conditioned medium culture medium, add 1.5 milliliters of Opti-MEM culture mediums, so with PBS cell twice Plasmid and Lipofectamine mixed liquors (0.5 milliliter) are drop by drop added cell solution afterwards, all around slightly shaken It is even；37 DEG C of incubator cultures change the Gbico RPMI-1640 culture mediums containing 10% hyclone after 4 hours, continue culture 24 hours After collect cell.
(6) according to cytoactive detection kit Vybrant MTT Cell Proliferation Assay Kit The explanation of (Thermo Fisher Scientific, USA) proceeds as follows to each sample：With PBS cell, suck PBS washing lotions；0.25% trypsin solution digestion attached cell is added into each hole；After adding culture medium to terminate digestion, with 5 milliliters Centrifuge tube collects all cells；4 DEG C of centrifugations (1000rpm) remove supernatant in 10 minutes；It is resuspended in 2 milliliters and contains 10% hyclone Gbico RPMI-1640 culture mediums, regulation concentration to 1 × 104Cells/ml；96 new orifice plates are separately taken, the sample after each transfection 6 holes of corresponding 96 orifice plates of product, 200 microlitres of cell suspensions (about 2000 cells) are added per hole, are cultivated 3 days；After cultivating 3 days Cell, abandon supernatant, per hole add 200 microlitres of fresh cultures after, add 20 microlitres with PBS configuration MTT solution (5 milli Grams per milliliter, pH=7.4), continue to be incubated 4 hours at 37 DEG C；Culture is terminated, abandons supernatant, 150 microlitres of DMSO, vibration 10 are added per hole Minute；490 nano wave lengths are selected, each hole absorbance (OD) value is determined on enzyme linked immunological monitor.
(7) three samples for each group, each sample detection obtain a cell viability, cell viability=(experiment Group OD values-zeroing hole OD values)/(control wells OD values-zeroing hole OD values) * 100；The indexs such as experimental group OD values are by 6 holes of the sample OD measured values in 3 immediate numerical value average to obtain.
Test result indicates that according to stating palindrome tiny RNA (SEQ ID NO in institute (1)：5) the SEQID NO of design：Shown in 6 SiRNA, compared with the control, HepG2 cell viabilities are improved 20%.Therefore, this palindrome is demonstrated from cellular level Tiny RNA has important biological function.

Claims (10)

  1. A kind of 1. palindrome tiny RNA (psRNA), it is characterised in that：Along the nucleotide sequence that the tiny RNA positive (5 ' → 3 ') is read with The nucleotide sequence that reversely (3 ' → 5 ') are read is identical, and its length range is 15 to 200 nucleotides；
    The nucleotide sequence of the palindrome tiny RNA screens acquisition by the following method, the described method comprises the following steps：
    (1) tiny RNA high-flux sequence (small RNA sequencing, sRNA-seq) is carried out to biological sample, from sequencing number Palindrome tiny RNA is found in；
    (2) each palindrome tiny RNA that will be found in step (1), the Blast instruments (https of NCBI websites is passed through：// Blast.ncbi.nlm.nih.gov/Blast.cgi) with NCBI nucleic acid databases (Nucleotide collection, nt/ Nr) it is compared, the parameter of comparison is that Seed Sequences length (Word size) is 7, and matching score (Match scores) is 1, Mispairing score (Mismatch scores) is -1, and initial vacancy point penalty (Gap costs for existence) is 2, and extension is empty Position point penalty (Gap costs for extension) is 1；
    (3) for each bar palindrome tiny RNA found in step (1), if having at least one in the comparison result that step (2) obtains Bar sequence and the sequence identity of palindrome tiny RNA described in step (1) are at least more than 90%, and the palindrome tiny RNA is defined as sieving Select the palindrome tiny RNA obtained；
    Or it the described method comprises the following steps：Palindrome nucleic acid fragment is found from the genome sequence of animal or microorganism, it is described Palindrome nucleic acid fragment is the core that the nucleotide sequence read along the nucleic acid fragment positive (5 ' → 3 ') is read with reverse (3 ' → 5 ') Acid sequence is identical；Pair reverse transcription is carried out with the RNA of the genomic source species identical biological sample, work as reverse transcription product When middle presence is with the palindrome nucleic acid fragment sequence identical cDNA, the T in sequence corresponding to the palindrome nucleic acid fragment is replaced For U, you can obtain the palindrome tiny RNA that screening obtains；
    Preferably, the nucleotide sequence of the palindrome tiny RNA such as SEQ ID NO：Shown in 5.
  2. A kind of 2. complementary palindrome tiny RNA (cpsRNA), it is characterised in that：The nucleic acid read along the tiny RNA positive (5 ' → 3 ') Base pair complementarity in the nucleotide sequence that base in sequence is read with reverse (3 → 5 '), and its length range arrives for 15 200 nucleotides；
    The nucleotide sequence of the complementary palindrome tiny RNA screens acquisition by the following method, the described method comprises the following steps：
    (1) tiny RNA high-flux sequence (small RNA sequencing, sRNA-seq) is carried out to biological sample, from sequencing number Complementary palindrome tiny RNA is found in；
    (2) each complementary palindrome tiny RNA that will be found in step (1), passes through the Blast instruments (https of NCBI websites：// Blast.ncbi.nlm.nih.gov/Blast.cgi) with NCBI nucleic acid databases (Nucleotide collection, nt/ Nr) it is compared, the parameter of comparison is that Seed Sequences length (Word size) is 7, and matching score (Match scores) is 1, Mispairing score (Mismatch scores) is -1, and initial vacancy point penalty (Gap costs for existence) is 2, and extension is empty Position point penalty (Gap costs for extension) is 1；
    (3) for each bar complementation palindrome tiny RNA found in step (1), if existed extremely in the comparison result that step (2) obtains A few sequence and the sequence identity of the complementary palindrome tiny RNA are at least more than 90%, and the complementary palindrome tiny RNA is defined as Screen the complementary palindrome tiny RNA obtained；
    Or it the described method comprises the following steps：Complementary palindrome nucleic acid fragment is found from the genome sequence of animal or microorganism, The complementary palindrome nucleic acid fragment is along the base in the nucleotide sequence of the nucleic acid fragment positive (5 ' → 3 ') reading and reversely (3 ' → 5 ') base pair complementarity in the nucleotide sequence read；Pair with the genomic source species identical biological sample RNA carries out reverse transcription, when existing in reverse transcription product with the complementary palindrome nucleic acid fragment sequence identical cDNA, this is mutual The T refilled in sequence corresponding to literary nucleic acid fragment replaces with U, you can obtains the complementary palindrome tiny RNA that screening obtains；
    Preferably, the nucleotide sequence such as SEQ ID NO of the complementary palindrome tiny RNA：1st, shown in 3 or 4.
  3. 3. according to the tiny RNA described in any one of claim 1 or 2, wherein, the biological sample be from animal blood, Saliva, urine, excrement, various tissues or cell line, preferably blood or cell line；The animal is preferably people.
  4. 4. according to the tiny RNA described in any one of claim 1 or 2, wherein, the microorganism includes virus, bacterium or fungi.
  5. 5. a kind of method for preparing siRNA, methods described include, siRNA, the justice of the siRNA are synthesized The sequence of chain be with complementary palindrome tiny RNA identical sequence described in palindrome tiny RNA described in claim 1 or claim 2, or 1- is deleted in 5 ' ends of complementary palindrome tiny RNA described in palindrome tiny RNA described in claim 1 or claim 2 and/or 3 ' end additions The sequence that 5 nucleotides obtains；Or the nucleic acid molecules of composite coding ShorthairpinRNA (short hairpin RNA, shRNA), will It inserts expression vector, is transferred in host cell and expresses, and obtains siRNA, the double stranded section of the shRNA includes and right It is required that the sequence identical sequence of complementary palindrome tiny RNA described in 1 palindrome tiny RNA or claim 2, or in claim 1 1-5 nucleotides is deleted in 5 ' ends of complementary palindrome tiny RNA described in the palindrome tiny RNA or claim 2 and/or 3 ' end additions Obtained sequence.
  6. 6. complementary palindrome tiny RNA described in palindrome tiny RNA described in claim 1 or claim 2 is viral, thin for detecting in preparation Application in the reagent of bacterium and/or fungal infection, wherein the palindrome tiny RNA or complementary palindrome tiny RNA are from virus, bacterium And/or fungi；Preferably, the reagent includes mutually refilling described in palindrome tiny RNA or claim 2 according to claim 1 The sequences Design of literary tiny RNA and the primer or probe synthesized；Preferably, the nucleotide sequence such as SEQ of the complementary palindrome tiny RNA ID NO：Shown in 1 and the virus is SARS virus；Preferably, the nucleotide sequence such as SEQ ID of the complementary palindrome tiny RNA NO：Shown in 3 and the bacterium is brucella；Preferably, the nucleotide sequence such as SEQ ID NO of the complementary palindrome tiny RNA：4 Shown and described fungi is aspergillus fumigatus.
  7. 7. complementary palindrome tiny RNA described in palindrome tiny RNA described in claim 1 or claim 2 is being prepared for external evoked thin Application in born of the same parents' apoptosis or the external reagent for promoting cell to breed；Preferably, the reagent includes siRNA or includes coding The carrier of shRNA gene, wherein the sequence of the positive-sense strand of the siRNA be with palindrome tiny RNA described in claim 1 or Complementary palindrome tiny RNA identical sequence described in claim 2, or in palindrome tiny RNA described in claim 1 or claim 2 institute The sequence that 1-5 nucleotides obtains is added or deleted at the 5 ' ends and/or 3 ' ends for stating complementary palindrome tiny RNA；The hair fastener of the shRNA The double stranded section of structure includes and complementary palindrome tiny RNA identical described in palindrome tiny RNA described in claim 1 or claim 2 Sequence, or add at 5 ' ends of complementary palindrome tiny RNA described in palindrome tiny RNA described in claim 1 or claim 2 and/or 3 ' ends Add or delete the sequence that 1-5 nucleotides obtains；Preferably, the carrier includes plasmid or viral vector；Preferably, it is described thin Born of the same parents include zooblast, such as people's cell.
  8. 8. a kind of vitro induction of apoptosis or the external method for promoting cell propagation, methods described include being turned with siRNA Contaminate the cell or the expression vector of the gene comprising coding shRNA is transferred to the cell, wherein the siRNA is just The sequence of adopted chain be with complementary palindrome tiny RNA identical sequence described in palindrome tiny RNA described in claim 1 or claim 2, or In 5 ' ends of complementary palindrome tiny RNA described in palindrome tiny RNA described in claim 1 or claim 2 and/or 3 ' end additions or delete The sequence that 1-5 nucleotides obtains；The double stranded section of the hairpin structure of the shRNA includes small with the palindrome described in claim 1 Complementary palindrome tiny RNA identical sequence described in RNA or claim 2, or will in palindrome tiny RNA described in claim 1 or right Ask the 5 ' of the 2 complementary palindrome tiny RNAs to hold and/or the sequence that 1-5 nucleotides obtains is added or deleted at 3 ' ends；Preferably, institute Stating carrier includes plasmid or viral vector；Preferably, the cell includes zooblast, such as people's cell.
  9. 9. a kind of siRNA, its just chain nucleic acid sequence such as SEQ ID NO：2nd, shown in 3,4 or 6.
  10. 10. siRNA inducing cell apoptosis or the external application promoted in cell propagation in vitro described in claim 9.
CN201710815083.3A 2017-09-07 2017-09-07 The palindrome and complementary palindrome tiny RNA and its application Pending CN107488660A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109355365A (en) * 2018-11-25 2019-02-19 中国农业科学院兰州兽医研究所 A kind of method of tiny RNA high-flux sequence detection microorganism
CN110875084A (en) * 2018-08-13 2020-03-10 深圳华大基因科技服务有限公司 Nucleic acid sequence comparison method

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110875084A (en) * 2018-08-13 2020-03-10 深圳华大基因科技服务有限公司 Nucleic acid sequence comparison method
CN110875084B (en) * 2018-08-13 2022-06-21 深圳华大基因科技服务有限公司 Nucleic acid sequence comparison method
CN109355365A (en) * 2018-11-25 2019-02-19 中国农业科学院兰州兽医研究所 A kind of method of tiny RNA high-flux sequence detection microorganism

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