CN1511957A - Antisense oligonucleotide screening method based on chip and its use - Google Patents
Antisense oligonucleotide screening method based on chip and its use Download PDFInfo
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- CN1511957A CN1511957A CNA021594155A CN02159415A CN1511957A CN 1511957 A CN1511957 A CN 1511957A CN A021594155 A CNA021594155 A CN A021594155A CN 02159415 A CN02159415 A CN 02159415A CN 1511957 A CN1511957 A CN 1511957A
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Abstract
The present invention relates to biological engineering detection technology and its use. Specially, the antisense oligonucleotide screening process includes fixing the synthesized and modified oligonucleotide of target gene onto chemically modified solid carrier to prepare gene chip, hybridizing with mRNA template of radioactive isotope or non-radioacitve molecular mark, washing off hybridizing liquid and drying in the air, RNase H cutting, adding buffering liquid to the contrast area only, washing, air drying and detection, analyzing the combining strength and RNase H cutting activity of the sequences, sorting based on the ratios between pre-cutting RNase H and post-cutting RNase H, and obtaining optimal binding target spot sequence with RNase H activating activity, optimal oligonucleotide length and modification method. Further, the optimal oligonucleotide sequence is selected, its bioactivity is determined to obtain candidate medicine as disease diagnosing probe and gene research tool.
Description
Technical field the present invention relates to a kind of biological engineering detection technology and uses thereof.Be a kind ofly to have RNase H by the chip hybridization optimization and activate active specifically in conjunction with target sequence, best oligonucleotide length and modifying method thereof.From above-mentioned optimized sequence, select best oligonucleotide sequence, can further determine its biological activity, obtain the oligonucleotide drug candidate, also can be used as the probe of medical diagnosis on disease and the instrument of gene functional research by the inside and outside activity rating of a series of bodies.
The background technology antisense nucleic acid medicament is antisense oligonucleotide (antisense oligodeoxynucleotide, ASODN) its nucleotide sequence can with said target mrna or target DNA complementation, suppress or seal this gene transcription and expression, or induce RNase H identification and cutting mRNA, make its loss of function.Antisense nucleic acid medicament is pharmacological frontier or revolution (Crooke ST.Oligonucleotide analogs might bedesigned to bind to mRNA.Anticancer drug Des, 1997,12:311-313), that is: new medicine---antisense oligonucleotide; New drug receptor---mRNA; New receptors bind mode---Watson-Crick hybridization; New drug receptor is in conjunction with afterreaction: (1) ASODN combines the effect of making it and is obstructed with the RNA polymerase.(2) ASODN inhibition mRNA precursor enters endochylema and transcribes preceding modification.(3) by certain direct stereoeffect, the rrna or the important startup factor combine with ASODN translation are obstructed.(4) ASODN activates RNase H, hydrolysis hybridization chain with the complementary hybridization of mRNA the time.ASODN dissociates from mixture then, and with another mRNA molecular hybridization, this process can be carried out repeatedly again.(5) form triple strand structure at nuclear target gene position by Hoogsteen base pairing form, stop duplicating or transcribing of target gene.
Since antisense nucleic acid can be on gene level the expression of specific inhibition specific gene, therefore be considered to a kind of highly selective that gets a good chance of and high efficiency potential disease medicine.At present, existing a kind of antisense nucleic acid (Vitravene) by FDA consultative committee (ScienceScop.Greenlightfor antisensedrug.Science, 1998,281:625), other has kind more than 20 carrying out clinical experiment.Domesticly also done a large amount of basic and applied research work in this field, but still the none medicine enters the clinical experiment stage.
Along with the enforcement of going deep into of the Human Genome Project, particularly post genome project, will have increasing disease related gene and the drug effect target is found.Antisense nucleic acid is that the medicine of new generation of target will be brought into play its bigger advantage as gene.The research of carrying out new gene function with antisense nucleic acid also receives publicity day by day, utilize antisense nucleic acid to be the trend of explosive growth as the bibliographical information of medicine and gene functional research at present, according to incompletely statistics, there were 3 antisense nucleic acid patent applications to get the Green Light in the U.S. in 2000, and calendar year 2001 has 5 to get the Green Light, 6221850), the antisense nucleic acid of target TNF-alpha (publication number: 6228642), the antisense nucleic acid (publication number: 6312900) of target AP-1 such as, the antisense nucleic acid of target JNK (publication number:.
The screening of high specific and high reactivity sequence is antisense oligonucleotide drug research and the key of succeeding in developing.For a mRNA not every target spot all be easy to reach (Branch AD, A good antisense molecule is hard to find.Trends Biochem Sci, 1998,23:45-50).It is generally acknowledged that the target spot that the design antisense drug is selected is around mRNA5 ' the end initiation codon AUG and ribosomal assembling position, antisense drug reaches easily, and is active higher.But studies show that other position of mRNA also can become the target spot of design, especially the non-translational region of 3 ' end.At present, the judgement of RNA high-affinity target spot is based on the empirical formula random screening, and this method often will could be found an ordered sequence at the synthetic a large amount of oligonucleotide sequence of a gene, relatively blindly, both uneconomical, do not know whether really to have taken the ASODN of high-affinity yet.
The very near site of distance on mRNA is found in experiment, even only differs from 2 bases, and the activity difference of its ASODN is huge.Though the composition of base may influence the formation of heteroduplex, the difference of the theoretical free energy of heteroduplex can not be explained the active greatest differences of antisense.Found afterwards that the base pairing of RNA intramolecularly forms two, tertiary structure can cause most molecule not interact with complementary nucleic acid; Some other structure as loop-stem structure, end sequence, though there is not intramolecular interaction, easily combines with other nucleic acid.This proof complementary oligonucleotide and target gene bonded major influence factors are not the compositions of base, but RNAs two, tertiary structure.There is non-specific action in many antisense oligonucleotides that studies show that, though some non-special antisense effect also has therapeutic value certainly, non-specific action is normally unpredictable, is difficult to come the appropriate design medicine with this.This non-specific action of ASODN mainly is not the reason of the chemically modified of original people's suspection, but owing to has selected the non-best target spot on the target gene.
In recent years, developed many in order to select the method for the best target spot among the mRNA better, to search out more effective antisense drug.But these methods still have a lot of problems in application, even are helpless to the screening of antisense drug at all.As predict that the folding method of RNA studied more than 20 year, and but because the restriction that the RNA structure is understood, it is still undesirable to design antisense nucleic acid with it, and difficult within recent years have big progress, and successful example is also very limited.Nearest studies have shown that the most frequently used folding software---MFOLD can not be used to seek on the mRNA can be near the site.RNase H restriction enzyme site collection of illustrative plates (Lima WF et al., Combinatorial screening and rational optimization forhybridization to folded hepatitis C virus RNA of oligonucleotides with biological antisense activity.J Biol Chem, 1997,272:626-638) with oligonucleotide chip scanning (Milner N et al., Selecting effective antisense reagents oncombinatorial oligonucleotide arrays.Nat Biotech, 1997, though 15:537-541) two empirical methods can be used for seeking best target spot on the RNA molecule and obtain to a certain degree success, but still have many weak points, miss some avtive spots as meeting, operation easier is big etc.Therefore need development new, workable, based on definite method of the best target spot of experiment, this also is the key link and the bottleneck problem of antisense nucleic acid medicament research and development.
Based on above-mentioned present Research, we have set up a kind of novel method of antisense oligonucleotide screening---based on the screening method of chip hybridization and original position RNase H cutting.The main design philosophy of this method is: the oligonucleotide by the target specific gene that will synthesize and modify is fixed on the solid phase carrier of chemically modified and is prepared into gene chip, hybridize with messenger RNA(mRNA) (mRNA) template of radio isotope or on-radiation molecule marker, hybridization finishes back flush away hybridization solution and dries, adding RNase H cuts, the check plot is only not enzyme-added with damping fluid, the certain hour after scouring, dry and carry out signal detection and scan, analyze each sequence in conjunction with said target mrna intensity and RNase H nicking activity, with before the RNase H cutting/ratio after the cutting sorts, in theory the sequence that ratio is big more its in conjunction with active and RNase H nicking activity is good more, activation is active in conjunction with target sequence thereby optimization has RNase H, best oligonucleotide length and modifying method thereof.By setting up antisense oligonucleotide triage techniques based on chip hybridization and original position RNase H cutting, we wish quickly, still less to find the best target spot of gene with omitting, for the research of gene function, the screening of medical diagnosis on disease probe and the exploitation of antisense nucleic acid medicament provide a kind of effective means.
Summary of the invention
1. the preparation of antisense oligonucleotide chip: oligonucleotide probe can be the oligonucleotide and the analogue thereof of different lengths, length 10-30 base, an optimum length 16-25 base; Also can be the antisense oligonucleotide analogue of different modifying form, comprise that thio-modification, peptide nucleic acid(PNA) (PNA), the carbon atom connecting arm that 5` is terminal modified, 3` is terminal modified, different modify, best one of modify be that the polyoxyethylene glycol connecting arm is modified.Solid phase carrier is sheet glass, the plastics of macromolecule member material, chemically modified.
2. the preparation of said target mrna and isotopic labeling: said target mrna can directly separate from tissue or cell extraction, also can obtain by in-vitro transcription.The said target mrna mark can be radio isotope (as
32P-rNTP) or non-radioactive substance (as vitamin H-rNTP, fluorescein etc.) mix during by in-vitro transcription, add radioactivity or nonradioactive labeling by enzymic catalytic reaction at its 5` end or 3` end after also can transcribing or extract mRNA.
3. chip hybridization and original position RNase H cutting: the chip in the said target mrna and 1 of mark in 2 is hybridized 10min-24hr, and Best Times is 30min-2hr, 37 ℃-65 ℃ of hybridization temperatures, and optimum temps is 40 ℃-45 ℃.After reaction finishes, the flush away hybridization solution also dries, add the damping fluid that contains RNase H in a district, the check plot is only not enzyme-added with damping fluid, hatch back flush away enzyme liquid for 37 ℃, dry, carry out the signal scanning analysis on scanner, optimization has the best oligonucleotide length of RNase nicking activity, in conjunction with target sequence and modifying method thereof.
4. antisense oligonucleotide activity rating: the best oligonucleotide sequence that screening in 3 is obtained is by further definite its biological activity of a series of inside and outsides activity rating.Adopt body outer cell proliferation inhibition activity and Western trace to verify that on cell levels the genetic expression of the antisense oligonucleotide of screening suppresses active.Further estimate the biologic activity of antisense drug candidate, obtain the oligonucleotide drug candidate in integral level.
The objective of the invention is target gene binding sequence, best oligonucleotide length and modifying method thereof by chip hybridization and RNase H cutting optimization the best.Enforcement of the present invention to quickly, still less find the best target spot of gene with omitting, and the screening of the research of gene function, medical diagnosis on disease probe and the exploitation of antisense nucleic acid medicament have important social benefit and economic benefit.
Description of drawings Fig. 1 is the chip screening process of antisense oligonucleotide
Fig. 25 ' NCR-Luciferase mRNA electrophoretogram
Scintigram behind Fig. 35 ' NCR-Luciferase mRNA and the chip hybridization
Fig. 4 is the optimization of RNaseH cutting concentration
Fig. 5 is the RNase H optimization of clipping time
Table 1 is the Antisensedigonucleotsequence sequence of target pattern gene 5 ' NCR-Luciferase
The activity checking of table 2 chip screening antisense sequences
Some illustrative of embodiment screening method of the present invention but nonrestrictive example are as follows.
Embodiment 1 chip hybridization screening oligonucleotide
1. probe is synthetic prepares with chip: according to 17 oligonucleotide probes of secondary structure design of target gene 5 ' NCR-Luciferase, length 21-23 base (sequence sees Table 1).Upward synthetic at 8909 type automatic dna synthesizers (PE company), the 3` end is amido modified, 4 polyoxyethylene glycol connecting arms are modified.Synthetic 55 ℃ of cuttings of strong aqua and the deprotection of finishing is after 15 hours; through the anti-phase purification column of Micro Pure II (Solid PhaseSciences company) purifying; ultraviolet (Beckman company) is final vacuum (Oligo Prep OP120 quantitatively; SAVANT company) drying, being diluted to concentration with sampling liquid is 15 μ mol/L-30 μ mol/L.Be distributed on the sheet glass of chemically modified with point sample instrument (Cartisian) point.
2. the acquisition of target gene mRNA and mark: with pattern gene 5 ' NCR-Luciferase is example, obtains total length target gene cDNA clone with the RT-PCR method, is cloned into the A/T carrier that contains the T7/SP6 promotor, is correct sequence through sequence verification.Introduce Hind III and BamH I restriction enzyme site by pcr amplification respectively at the 5` of target gene and 3` end, directed pSP64 poly (A) carrier that inserts is that template is carried out in-vitro transcription and prepared the said target mrna (see figure 2) with EcoR I linearization for enzyme restriction plasmid.Also can access single-gene mRNA with the single-gene specific probe.
When the said target mrna isotopic labeling can be passed through in-vitro transcription
32P-rUTP mixes, and also can transcribe or extract behind the mRNA to add at its 3` end by Poly (A) polysaccharase
32P-rATP.
3. chip hybridization and scanning: the chip of isotopic labeling said target mrna and preparation was hybridized 3 hours 42 ℃ of hybridization temperatures.The reaction back flush away hybridization solution that finishes dries, with preservative film parcel back phosphorus screen compressing tablet after 3 days on Typhoon 9410 scanners scanning analysis.After the strong and weak screening of probe bonded signal on the chip, point out preferably that candidate locus is 1,2,6,9,10,17, see Fig. 3 A and table 2.
1.RNase the optimization of H original position cutting condition: adopt the RNase H of different concns, be respectively 7.81 * 10
-3U, 1.56 * 10
-2U, 3.12 * 10
-3U, 6.25 * 10
-2U, 1.25 * 10
-1U, 2.5 * 10
-1U, 5.0 * 10
-1U, 37 ℃ of effect 20min, the result shows 1.25 * 10
-1U is the most appropriate concentration of cutting.Adopt 1.25 * 10
-1U RNase H, 37 ℃ act on 2min, 5min, 10min, 15min, 20min, 30min, 60min respectively, and the result shows that 20min cuts the time for the most appropriate.In sum, the most appropriate condition of cutting of RNase H is 1.25 * 10
-1U sees Fig. 4, Fig. 5 in 37 ℃ of cutting 20min.
2. chip hybridization, RNase H cutting and scanning: the chip of isotopic labeling said target mrna and preparation was hybridized 3 hours 42 ℃ of hybridization temperatures.The flush away hybridization solution dried after reaction finished, and added the damping fluid that contains RNase H in a district, the check plot is only not enzyme-added with damping fluid, hatch back flush away enzyme liquid for 37 ℃, dry, with preservative film parcel back phosphorus screen compressing tablet after 3 days on Typhoon 9410 scanners scanning analysis.Calculate the ratio of each point before and after enzymic digestion, in theory the sequence that ratio is big more its in conjunction with active and RNase H nicking activity is good more.Than after the screening once more, point out preferably that candidate locus is 1,2,6,7,9,17 with the signal before and after the original position RNase H cutting, see Fig. 3 B and table 2.
The candidate sequence of synthetic thio-modification, HepG
2Liver cancer cell is inoculated in 96 orifice plates, 1 * 10
4Cells/well, 37 ℃, 5%CO
2Hatch 18-20h.After the 80-90% cytogamy, under the serum-free state, adopt liposome Lipofectin (Gibco company) to carry out cell transfecting.With above-mentioned sequence respectively with pHCV-neo4 plasmid 0.1 μ g cotransfection HepG
2Liver cancer cell, effect 5h changes the DMEM substratum that contains 10% calf serum and continues to cultivate 24h.With the positive contrast of the plasmid transfection that does not add S-ASODN.Per 1 sulfo-sequence all repeats 3 holes in each experiment, and the result gets its mean value.Uciferase activity detects and adopts Luciferase Assay System (Promega company), and the by specification operation improves a little.At multiple labeling detector (VICTOR
TMWallac 1420 Multilabel Counter) go up survey 1s luminous intensity, output valve is CPS.
The result shows: after the strong and weak screening of probe bonded signal on the chip, pointing out preferably, candidate locus is 1,2,6,9,10,17, wherein 4 sequences are active good through the cytology checking, article 1, sequence is better active, article 1, sequence is invalid, and the coincidence rate of chip results of screening and cytology checking is 66.7%; Cut the signal of front and back with original position RNase H than after screening once more, pointing out preferably, candidate locus is 1,2,6,7,9,17, wherein 5 sequences are active good through the cytology checking, and 1 sequence is better active, and the coincidence rate of chip results of screening and cytology checking is 83.3%.See Table 2.
The Antisensedigonucleotsequence sequence of table 1 target pattern gene 5 ' NCR-Luciferase
The arrangement of sequence numbering base (5` → 3`)
1 GAA GAC AGT AGT TCC TCA CAG
2 CCA TGG CTA GAC GCT TTC TGC
3 GGG GTC CTG GAG GCT GCA CGA
4 TTC CGG TGT ACT CAC CGG TTC
5 CGC GGG GGC ACG CCC AAA TCT
6 GCC TTT CGC GAC CCA ACA CTA CT
7 GCA CTC GCA AGC ACC CTA TCA G
8 GTG CTC ATG GTG CAC GGT CTA CG
9 GAG GTT TAG GAT TCG TGC TCA TG
10 GTT TTT CTT TGA GGT TTA GGA TT
11 CCA，ACA，CTA，CTC，GGC，TAG，CAG，TC
12 GCC，CAA，CAC，CGG，CAT，AAA，GAA，TT
13 AGA，CCA，GTA，GAT，CCA，GAG，GAG，TT
14 AGA，GAG，GGG，AGC，GCC，ACC，AGA，AG
15 GGG，AGC，CAC，CTG，ATA，GCC，TTT，GT
16 CGT，CCA，CAA，ACA，CAA，CTC，CTC，CG
17 AGA，ATT，ACA，CGG，CGA，TCT，TTC，CG
Negative control 1 CAT GAG CAC GAA TCC TAT CCT AA
The activity checking of table 2 chip screening antisense sequences
Chip signal maximal percentage inhibition IC50 (nmol/L)
The site numbering
Before the cutting of hybridization ability/cutting back ratio (%)
1 37767 0.88 88.8 44.7
2 28804 0.91 50.2 ＞100
3 - - -48.3 ＞100
4 - - 28.1 ＞100
5 - - 30.7 ＞100
6 29406 2.70 81.9 50.2
7 20552 0.96 63.2 74.7
8 11223 0.44 87.2 41.6
9 44928 1.60 82.8 36.3
10 31185 0.54 -8.0 ＞100
11 14441 0.38 56.0 52.2
12 22415 0.51 73.2 47.3
13 - - - -
14 - - - -
15 - - - -
16 21492 0.53 72.5 66.5
17 26857 0.87 76.1 32.9
Claims (9)
1. the present invention relates to a kind of new antisense oligonucleotide screening method based on chip hybridization and original position RNase H cutting, it is characterized in that, oligonucleotide by the target specific gene that will synthesize and modify is fixed on the solid phase carrier of chemically modified and is prepared into gene chip, hybridize with messenger RNA(mRNA) (mRNA) template of radio isotope or on-radiation molecule marker, hybridization finishes back flush away hybridization solution and dries, adding RNase H cuts, the check plot is only not enzyme-added with damping fluid, the certain hour after scouring, dry and carry out signal detection and scan, analyze each sequence in conjunction with said target mrna intensity and RNase H nicking activity, with before the RNase H cutting/ratio after the cutting sorts, in theory the sequence that ratio is big more its in conjunction with active and RNase H nicking activity is good more, activation is active in conjunction with target sequence thereby optimization has RNase H, best oligonucleotide length and modifying method thereof.
2. the oligonucleotide probe in the claim 1 can be the oligonucleotide and the analogue thereof of different lengths, length 10-30 base, an optimum length 16-25 base; Also can be the antisense oligonucleotide analogue of different modifying form, comprise that thio-modification, peptide nucleic acid(PNA) (PNA), the carbon atom connecting arm that 5` is terminal modified, 3` is terminal modified, different modify, best one of modify be that the polyoxyethylene glycol connecting arm is modified.
3. the solid phase carrier in the claim 1 is sheet glass, the plastics of macromolecule member material, chemically modified.
Said target mrna mark in the claim 1 can be radio isotope (as
32P-rNTP) or non-radioactive substance (as vitamin H-rNTP, fluorescein etc.) mix during by in-vitro transcription, add radioactivity or nonradioactive labeling by enzymic catalytic reaction at its 5` end or 3` end after also can transcribing or extract mRNA.
5. the hybridization time of said target mrna and chip is 10min-24hr in the claim 1, and Best Times is 30min-2hr; 37 ℃-65 ℃ of hybridization temperatures, optimum temps are 40 ℃-45 ℃.
6. the described method of claim 1 can be used for screening the oligonucleotide binding site of transcribing group mRNA at human body or other biological body.
7. the purposes of the described method of claim 1 in the drug screenings such as oligonucleotide, RNAi/siRNA, ribozyme of antitumor, antiviral and treatment other diseases.
8. the purposes of the described method of claim 1 in medical diagnosis on disease probe screening.
9. the purposes of the described method of claim 1 in gene functional research.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101710362B (en) * | 2009-12-10 | 2011-07-20 | 浙江大学 | microRNA target position point prediction method based on support vector machine |
| CN108265053A (en) * | 2017-10-26 | 2018-07-10 | 武汉大学 | A kind of screening technique for the antisense oligonucleotides that extron shearing is inhibited to inhibit subfunction |
| CN112458080A (en) * | 2020-12-03 | 2021-03-09 | 深圳市大鳄生物科技股份有限公司 | siRNA fishing method for obtaining lncRNA LOC157273 |
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2002
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101710362B (en) * | 2009-12-10 | 2011-07-20 | 浙江大学 | microRNA target position point prediction method based on support vector machine |
| CN108265053A (en) * | 2017-10-26 | 2018-07-10 | 武汉大学 | A kind of screening technique for the antisense oligonucleotides that extron shearing is inhibited to inhibit subfunction |
| CN108265053B (en) * | 2017-10-26 | 2020-10-13 | 武汉大学 | Screening method of antisense oligonucleotide for inhibiting exon shearing inhibitor function |
| CN112458080A (en) * | 2020-12-03 | 2021-03-09 | 深圳市大鳄生物科技股份有限公司 | siRNA fishing method for obtaining lncRNA LOC157273 |
| CN112458080B (en) * | 2020-12-03 | 2023-01-31 | 深圳市华元生物技术股份有限公司 | siRNA fishing method for obtaining lncRNA LOC157273 |
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