CN101634047A - Method for screening external-direction sequence libraries - Google Patents
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Abstract
The invention discloses a method for screening external-direction sequence libraries, which comprises the following steps: (a) carrying out PCR amplification on external-direction sequence expression cassettes by using external-direction sequence clones as templates; (b) carrying out transcription preparation on external-direction sequences by using PCR products as templates; and (c) and mixing transcription products with target RNAs and Rnase P to react so as to select functional external-direction sequences in vitro. New target external-direction sequences can be obtained by using the technique.
Description
Technical field
The present invention relates to a kind of biological technical field, more specifically, the present invention relates to a kind of method for screening external-direction sequence libraries, thus the convenient outer guide sequence EGS that can be used as the gene therapy meaning that from the library, filters out.
Background technology
Outer sequence (the External Guided Sequence that guides, below all being called for short EGS) technology at first finds in the later stage eighties, its major function is a body interior orientation deactivation functional gene, and EGS is the microRNA functional molecular, thereby should belong to generalized RNA perturbation technique (RNAi).But different with present popular RNAi technology is that EGS adopts shorter RNA molecule (10mer), gets final product specific action in target gene, thereby causes the target gene targeted inactivation.At present the international monopoly at EGS exists, but US Patent specification US6, and 057,153, US6,783,981 and US5,877,162 grades mainly are the designs at single EGS molecule.The EGS library (being called for short LEGS) that does not still have at present the practical screening that can be used for high-throughput EGS, and the generation in this library will be of value to the gene new drug development at special disease such as AIDS, hepatitis and cancer etc.
Summary of the invention
The invention provides a kind of method for screening external-direction sequence libraries, comprising: a) guiding sequence clone in addition is template, externally guides sequence S expression cassette to carry out pcr amplification; B) be template with the PCR product, externally guide sequence to transcribe preparation; C) transcription product and target RNA and RNase P hybrid reaction, guide sequence outward with the in-vitro screening function.
Description of drawings
Fig. 1 is an EGS library schema, wherein:
Fig. 1-1: " 3/4 EGS " design framework derives from the precursor sequence of intestinal bacteria tyrosine transfer RNA;
Fig. 1-2: external-direction sequence libraries, guide the recognition sequence structural domain by based composition at random outward; Directly be exposed to nuclease for fear of " CCA " sequence, " UUU " sequence is connected its 3 ' end;
Fig. 1-3: the complex body that external-direction sequence libraries and potential target RNA form, arrow has been indicated the cleavage site of nuclease P.
Fig. 2 is primers F LEGSp and RLEGSp synoptic diagram; The oligonucleotide FLEGSp and the RLEGSp of incomplete randomization are made up of two portions, a part is used to increase the pET28a-D carrier (except not comprising the fragment between T7 promotor and the T7 terminator, can be equal to the pET28a carrier), another part external-direction sequence libraries expression cassette that is used to increase;
Fig. 3 is an EGS library construction synoptic diagram, wherein:
The schema that Fig. 3-1:pET28a-LEGS makes up;
The PCR product of Fig. 3-2:pET28a-LEGS is identified figure, and arrow has indicated size to be the DNA band of 5-kb;
Fig. 4 identifies synoptic diagram for the EGS library, wherein:
Fig. 4-1:pET28a-EGS clone enzyme is cut the evaluation mode chart, the RV1 swimming lane is that the enzyme after the pET28a-EGS clone that contains a HincII restriction enzyme site is cut by the HincII enzyme is cut synoptic diagram, the RV2 swimming lane is that the enzyme after the pET28a-EGS clone that contains two or three HincII restriction enzyme sites is cut by the HincII enzyme is cut synoptic diagram, the V swimming lane is that the enzyme after pET28a is cut by the HincII enzyme is cut synoptic diagram, and arrow has indicated size to be the DNA band of 5-kb;
Fig. 4-2:pET28a-EGS clone enzyme is cut evaluation figure, 1 to No. 42 swimming lane is the restriction enzyme mapping after the pET28a-EGS clone who contains a HincII restriction enzyme site is cut by the HincII enzyme, the V swimming lane is the restriction enzyme mapping after pET28a is cut by the HincII enzyme, and arrow has indicated size to be the DNA band of 5-kb;
Fig. 4-3:pET28a-EGS clone enzyme is cut evaluation figure, No. 1 swimming lane is the restriction enzyme mapping after being cut by the HincII enzyme for the pET28a-EGS clone who contains two or three HincII restriction enzyme sites, the V swimming lane is the restriction enzyme mapping after pET28a is cut by the HincII enzyme, and arrow has indicated size to be the DNA band of 5-kb;
Fig. 4-4: the The result of multiple comparisons of part EGS expression cassette sequence.
Fig. 5 prepares the EGS synoptic diagram for in-vitro transcription, wherein
Fig. 5-1:EGS preparation flow figure;
The in-vitro transcription template of Fig. 5-2:EGS is identified figure;
Fig. 5-3: the EGS product of transcribing preparation;
Fig. 6 is an EGS library functional screening intention, wherein:
Fig. 6-1: the functional EGS synoptic diagram of in-vitro screening;
Fig. 6-2: checking synoptic diagram in the functional EGS body;
Fig. 7 is an EGS-c-myc Function Identification synoptic diagram, wherein:
Fig. 7-1: be the EGS-c-myc structural representation;
Fig. 7-2: be EGS-D-c-myc structural representation (negative control of EGS-c-myc), garden shape labeling indicia base mutation;
Fig. 7-3: the synoptic diagram of the product that is produced by the external cutting of RNaseP for c-myc mRNA, swimming lane M is RNA Marker, arrow has indicated size to be the RNA band of 2-kb, asterisk has indicated size to be the RNA band of 0.5-kb, the product that swimming lane 1 is produced by the external cutting of RNaseP for c-myc mRNA;
Fig. 7-4 is a c-myc expression level detected result, wherein:
Fig. 7-4-1 is the fluorescence real-time quantitative PCR detected result,
Fig. 7-4-2: be Western blota detected result;
Fig. 7-5 is the apoptosis detected result, wherein:
Fig. 7-5-1: be the apoptosis rate that EGS-D-c-myc caused;
The apoptosis rate that Fig. 7-5-2:EGS-c-myc caused.
Embodiment
1, the design in EGS library
1.1 select the design framework of " 3/4 EGS " (Fig. 1-1) as the EGS library.The primary structure (nucleotide sequence) of " 3/4 EGS " and secondary structure derive from the tyrosine transfer RNA (tRNA of intestinal bacteria (Escherichia coli)
Tyr), it has been widely adopted and a large amount of experiment shows of quilt.
1.2 the target pairing region of " 3/4 EGS " is by based composition at random, the theoretical library (Fig. 1-2) of Here it is EGS.
1.3 " 3/4 EGS " the end of " CCA " sequence of 3 ' end add " UUU " sequence and directly be exposed to nuclease (RNAase) (Fig. 1-2) to avoid " CCA " sequence.
Because of this library has comprised the functional EGS (Fig. 1-3) of any target RNA of target, so this library also can be called as the EGS random library
2, the construction strategy in EGS library
By replacing the zone between T7 terminator and the T7 promotor, the expression cassette in EGS library is cloned into the pET28a carrier and makes up pET28a-LEGS.PET28a-LEGS is the recombinant vectors that contains EGS library expression cassette, and wherein EGS library expression cassette transcribes the control that is subjected to T7 terminator and T7 promotor.PET28a-LEGS is converted in the DH5 α competent cell with screening pET28a-EGS-clone---contain the recombinant vectors of a certain specific EGS expression cassette, wherein EGS library expression cassette transcribes the control (Fig. 3-1) that is subjected to T7 terminator and T7 promotor.
This tactful PCR-based technology directly is cloned into destination carrier to EGS library expression cassette, and this can be avoided in the EGS library some expression cassette to be omitted.
3, the construction process in EGS library
3.1 utilize Vector NTI software design primer: FLEGSp SEQ ID NO:1 and RLEGSp SEQ ID NO:2 (Fig. 2), second primer is RLEGSp.
This is made up of two portions primer, the part pET28a-D carrier (except not comprising the fragment between T7 promotor and the T7 terminator, can be equal to the pET28a carrier) that is used to increase, another part external-direction sequence libraries expression cassette that is used to increase.This can directly be cloned into destination carrier to EGS library expression cassette by round pcr.
3.2 utilize synthetic FLEGSp of 9700 DNA synthesizer (Applied biosystem) and FLEGSp, and their 5 ' end carried out phosphorylation modification.
3.3 utilize Phusion
TMHigh-Fidelity PCR Kit (NEB) carries out pcr amplification (Fig. 3-2) according to following reaction system and response procedures at 9700 Thermal cycler (Applied biosystem).By pcr amplification the expression cassette in the theoretical library of EGS is cloned into pET28a carrier (MERCK company) and prepares pET28a-LEGSL.
Fig. 3-2 result shows that PCR product size is consistent with theoretical prediction.
Reaction system:
Response procedures
3.4 utilize E.Z.N.A.Gel Extraction Kit (Omega) purified pcr product (Fig. 3).Measure the concentration of the PCR product of purifying with ultraviolet spectrophotometer DU530 (Beckman).
3.5 utilize T4 DNA Ligase (NEB) that the PCR product of purifying is carried out the intramolecularly connection.DNA concentration is rare more, and easy more generation intramolecularly connects, with reference to following calculating formula: 51.1/ (DNA concentration: g/l) ÷ (molecular weight: dalton) 0.5＞2, the concentration of calculating PCR product in the ligation system is 1ng/ μ l.According to calculation result, to get 1 μ gpET28a-LEGSL and connect, reaction volume is 1ml, the ligation condition is 15 ℃, 16h.
Carry out the ethanol sedimentation purifying 3.6 connect product, the connection product of purifying (is less than or equal to 1: 10) and the abundant mixing of competent cell (DH5 α: purchase the company in Invitrogen), ice bath 30min according to a certain percentage; 42 ℃, 45S, ice bath 2-3min; Add 10 volume LB substratum, 37 ℃, 225r/min, shaking culture 1h; Get in a small amount above-mentioned culture (the residue culture is placed 4 ℃ of preservations) and carry out gradient dilution and separate application in the LB flat board that contains 30 μ g/ml Kanamycin, 37 ℃, the Mammals back-off is cultivated about 14h.Observe the clone's number that calculates above-mentioned culture plate.According to calculation result, remaining several LB flat boards (containing 30 μ g/ml Kanamycin) that culture dilutes (making each flat board grow about 300 clones) and coats.
The theoretical size of pET28a-LEGSL is 5143bp, and the PCR product is carried out the agarose gel electrophoresis analysis---the fragment length of pET28a-LEGSL all meets theoretical size (Fig. 3-2); PET28a-LEGSL to purifying carries out the intramolecularly connection; Connect product and be converted into competent cell DH5 α respectively, converted product carries out colony screening on the LB flat board that contains 30 μ g/ml.Its transformation efficiency is 3.72 * 10
7/ μ g.
3.7 utilize Vector NTI software that the sequence of pET28a-LEGS is carried out the restriction enzyme site analysis---selectional restriction restriction endonuclease HincII carries out enzyme respectively to above-mentioned plasmid and cuts evaluation (Fig. 4-2).
3.8. utilize Vector NTI software design sequencing primer, use
Terminator v3.1 Cycle SequenceingKit (ABI) and 3730 DNA Analyzer (ABI) carry out two-way sequencing analysis (Fig. 4), and sequencing result is seen Fig. 4-3.
The pET28a carrier has two HincII restriction enzyme sites, and one of them is in the T7prometer and the zone between the T7 terminator that are replaced by EGS library expression cassette, but the EGS expression cassette is introduced 0,1 or 2 HincII restriction enzyme site with different probability.In theory, general 98%pET28a-EGS-clone has only 1 HincII restriction enzyme site, and its HincII restriction enzyme mapping is (Fig. 4-1, laneRV1), general 2%pET28a-EGS-clone has 2 or 3 HincII restriction enzyme sites, their HincII restriction enzyme mapping be (Fig. 4-1, laneRV2).For whether the cloning efficiency of checking the EGS library meets Design Theory, picking pET28a-EGS-clone carries out restriction enzyme (HincII) analysis at random.The HincII restriction enzyme mapping of general 96%pET28a-EGS-clone is consistent with theoretical prediction, wherein the HincII restriction enzyme mapping of 94%pET28a-EGS-clone (Fig. 4-2) and Fig. 4-1, the laneRV1 unanimity, HincII restriction enzyme mapping (Fig. 4-3 of 6%pET28a-EGS-clone wherein, lane1) with Fig. 4-1, the laneRV2 unanimity.Above-mentioned restriction analysis shows that EGS library clone efficient satisfies the Design Theory requirement substantially.For whether the composition of the EGS expression cassette of checking the EGS library meets Design Theory, picking pET28a-EGS-clone checks order and compare of analysis at random.General 94%pET28a-EGS-clone has the EGS expression cassette that sequence is played total correctness.Sequence to these EGS expression cassettes compare (Fig. 4-4) show that the composition of EGS expression cassette satisfies Design Theory.
EGS library application example:
One, the foundation of EGS library screening program
1, is template with EGS clone (pET28a-EGS-clone), the EGS expression cassette is carried out pcr amplification (Fig. 5-1);
2, the PCR product with above-mentioned purifying is a template, and EGS is transcribed preparation (Fig. 5-1);
The preparation flow of EGS-clone is shown in Fig. 5-1.Pcr amplification EGS-clone-IVTT.The theoretical size of EGS-clone-IVTT is 826bp.The PCR product is carried out the agarose gel electrophoresis analysis---the clip size of EGS-clone-IVTT (Fig. 5-2) meets theoretical prediction.EGS-clone-IVTT with purifying is a template, prepares EGS-clone by in-vitro transcription.The theoretical size of EGS-clone is 71bp, and prepared EGS is carried out the agarose gel electrophoresis analysis---the clip size of EGS-clone (Fig. 5-3) meets theoretical prediction.
3, the in-vitro transcription product of above-mentioned purifying and target RNA (AIDS, the Disease-causing gene that liver cancer is relevant) and RNase P hybrid reaction, with in-vitro screening function EGS (Fig. 6-1);
3.1RNaseP preparation
RNaseP is made up of C5 albumen and M1 RNA, reference report (Lundblad EW, Xiao G, Ko JH, Altman S.Rapid selection of accessible and cleavable sites in RNA by Escherichia coli RNaseP and random external guide sequences.Proc.Natl.Acad.Sci.U.S.A 2008; 105:2354-2357.) method is prepared.
3.2 target RNA preparation
Obtain the in-vitro transcription template (5 ' end contains the T7 promotor) of target RNA, prepare target RNA by in-vitro transcription.
3.3RNA purifying
Utilize RNA purification kit (QIAGEN), the in-vitro transcription product is carried out purifying.
3.4 reaction conditions
3.5 reaction product analysis
Reaction product is carried out polyacrylamide gel electrophoresis (gum concentration is 8%, contains 7M urea) analysis, whether cut by specificity to judge target RNA.
Above-mentioned described experimental procedure is an EGS in-vitro screening strategy, shown in Fig. 6-1.
4, above-mentioned screen functional EGS transfection to corresponding cell model, and the phenotype (propagation suppresses, promotes apoptosis, suppresses virus infection) that it caused carried out check analysis (Fig. 6-2).
4.1, the EGS transfection
Utilize transfection reagent: Lipofectamine 2000 (Invitrogen), to the target cell, detail operations is seen the transfection reagent specification sheets by the EGS transfection.
4.2, cell proliferation detects
Utilize MTT cell proliferation and cytotoxicity detection kit (Beyotime) to carry out cell proliferation and detect, detail operations is seen the test kit specification sheets.
4.3 apoptosis detects
Utilize Annexin V-FITC apoptosis test regent box (Beyotime) to carry out apoptosis and detect, detail operations is seen the test kit specification sheets.
4.4 viral copy number detects (is example with HIV)
Utilize lentiviral gene group RNA copy number detection kit: Lenti-X
TMQRT-PCR Titration Kit detects HIV geneome RNA copy number, to estimate the propagation situation of HIV.
Above-mentioned described experimental procedure is an authentication policy in the EGS body, shown in Fig. 6-2
Two, EGS library screening result
By above-mentioned screening procedure, in-vitro screening is to the EGS-c-myc SEQ ID NO.3 of liver cancer targeting c-myc oncogene; Experimental result shows in the body: EGS-c-myc can effectively disturb the expression of c-myc among the hepatoma cell line HepG2, and induces HepG2 that apoptosis (Fig. 7) takes place.
1, the EGS-c-myc of in-vitro screening target c-myc oncogene (Fig. 7-1)
Utilize above-mentioned in-vitro screening program, screen the EGS-c-myc (Fig. 7-1) that the c-myc oncogene is effectively discerned cutting at the external RNase of inducing P, EGS-c-myc can carry out the specificity cutting to c-myc mRNA at the external RNaseP of guidance, and its cleaved products is identified shown in Fig. 7-3.
2, negative control: EGS-D-c-myc (Fig. 7-2) in the body of preparation EGS-c-myc
Utilize the method for preparing EGS-c-myc to prepare EGS-D-c-myc, but when the EGS-c-myc expression cassette is carried out pcr amplification, introduce base mutation (UUC → AAG).
The structural representation of EGS-D-c-myc is shown in Fig. 7-2.
3, EGS-c-myc and EGS-D-c-myc are distinguished transfection to the HepG2 cell
Utilize transfection reagent: Lipofectamine 2000 (Invitrogen), to the target cell, detail operations is seen the transfection reagent specification sheets by the EGS transfection.
4, EGS-c-myc and EGS-D-c-myc are to the influence of c-myc expression level
4.1 the fluorescence real-time quantitative PCR analysis (Fig. 7-4-1)
A, HepG2 cell by EGS transfection 72h after, utilize Trizol reagent (Invitrogen) to extract total RNA, detail operations is seen the reagent specification sheets;
B, reverse transcription test kit (QuantiTect Rev.Transcription Kit, QIAGEN) RNA to said extracted carries out reverse transcription, and detail operations is seen the test kit specification sheets;
C, utilize Q-PCR (SYBR method) test kit (QuantiTect SYBR Green PCR Kits, QIAGEN) β-actin and c-myc gene are carried out quantitatively, test kit comprises β-actin and c-myc gene is carried out quantitative Q-PCR primer, β-actin is as confidential reference items, and detail operations is seen the test kit specification sheets.
Fig. 7-4-1 knot shows that EGS-c-myc compares its negative control EGS-D-c-myc and suppresses the expression of c-myc oncogene on transcriptional level significantly.
4.2Western the blot analysis (Fig. 7-4-2)
A, HepG2 cell by EGS transfection 72h after, utilize Western and IP cell pyrolysis liquid (Beyotime) preparation protein example, detail operations is seen the test kit specification sheets;
B, utilize BCA determination of protein concentration test kit (Beyotime) that above-mentioned protein example is carried out concentration determination, detail operations is seen the test kit specification sheets;
C, above-mentioned protein example is carried out SDS-PAGE, applied sample amount is 10 μ g;
D, engram analysis: blotting membrane is a nitrocellulose filter, and one anti-is: β-Actin (9): sc-130301, and mousemonoclonal, Santa Cruz 1: 500, is used to detect β-Actin; C-Myc (A-14): sc-789, rabbit polyclonalSanta Cruz, 1: 500, be used to detect c-Myc, two is anti-: the goat anti-rabbit igg of HRP-mark (sc-2004, Santa Cruz, 1: 2000), the sheep anti-mouse igg (sc-2005, Santa Cruz, 1: 5000) of HRP-mark; Chemiluminescence detection kit: BeyoECL Plus (Beyotime), detail operations is seen the reagent specification sheets.
Fig. 7-4-2 result shows that EGS-c-myc compares its negative control EGS-D-c-myc and suppresses the expression of c-myc oncogene on protein level significantly.
5, the effect of EGS-c-myc and EGS-D-c-myc cell death inducing
((effect of cell death inducing of Fig. 7-5-2), detail operations is seen the test kit specification sheets for Fig. 7-5-1) and EGS-D-c-myc to utilize Annexin V-FITC apoptosis test regent box (Beyotime) to detect EGS-c-myc.
The result shows that (Fig. 7-5-1) compares its negative control EGS-D-c-myc, and (Fig. 7-5-2) promotes the apoptosis of HepG2 cell to EGS-c-myc significantly.
The above results shows, EGS-c-myc of the present invention can be used for preparation and prevent and/or treat c-myc oncogene high expression level disease medicament, c-myc oncogene high expression level disease includes but not limited to osteogenic sarcoma, chondrosarcoma, chordoma, liposarcoma, rhabdosarcoma, liver cancer.
SEQUENCE?LISTING
＜110〉Guangzhou Jinqi Gene Technology Research Development Centre
＜120〉a kind of new outer guide sequence (EGS) library (LEGS) constructing technology
<130>US6057153
<160>2
<170>PatentIn?version?3.2
<210>1
<211>58
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(31)..(37)
<223>n?is?a，c，g，or?t
<400>1
tctccggggt?tccecaatac?gattttacca?nnnnnnnctt?cctaagcttg?gaagcttc 58
<210>2
<211>58
<212>DNA
＜213〉artificial sequence
<220>
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<223>n?is?a，c，g，or?t
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tcgatgacgg?cagatttaga?gtcgctnnnt?ttggctatag?tgagtcgtat?taatttcg 58
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Claims (5)
1. method for screening external-direction sequence libraries comprises:
A) guiding sequence clone in addition is template, externally guides sequence S expression cassette to carry out pcr amplification;
B) be template with the PCR product, externally guide sequence to transcribe preparation;
C) transcription product and target RNA and RNase P hybrid reaction, guide sequence outward with the in-vitro screening function.
2. the described method for screening external-direction sequence libraries of claim 1, wherein to guide sequence clone at home and abroad be pET28a-EGS-clone to step a), shown in Fig. 5-1.
3. the described method for screening external-direction sequence libraries of claim 1, wherein target RNA is AIDS or the relevant Disease-causing gene of liver cancer in the step c).
4. the described gene of claim 3 is the c-myc oncogene.
5. the described method for screening external-direction sequence libraries of claim 1, wherein RNaseP is made up of C5 albumen and M1RNA in the step c).
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997033991A1 (en) * | 1996-03-14 | 1997-09-18 | Innovir Laboratories, Inc. | Short external guide sequences |
| US6057153A (en) * | 1995-01-13 | 2000-05-02 | Yale University | Stabilized external guide sequences |
| WO2000055341A1 (en) * | 1999-03-17 | 2000-09-21 | Oxford Biomedica (Uk) Limited | Anti-viral vectors |
| US6248525B1 (en) * | 1998-03-30 | 2001-06-19 | Yale University | Method for identifying essential or functional genes |
| CN101463351A (en) * | 2009-01-13 | 2009-06-24 | 暨南大学 | External leader sequence for guiding RNase P ribozyme and use thereof in anti-HCMV medicament preparation |
-
2009
- 2009-08-12 CN CN200910162321A patent/CN101634047A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6057153A (en) * | 1995-01-13 | 2000-05-02 | Yale University | Stabilized external guide sequences |
| WO1997033991A1 (en) * | 1996-03-14 | 1997-09-18 | Innovir Laboratories, Inc. | Short external guide sequences |
| US6248525B1 (en) * | 1998-03-30 | 2001-06-19 | Yale University | Method for identifying essential or functional genes |
| WO2000055341A1 (en) * | 1999-03-17 | 2000-09-21 | Oxford Biomedica (Uk) Limited | Anti-viral vectors |
| CN101463351A (en) * | 2009-01-13 | 2009-06-24 | 暨南大学 | External leader sequence for guiding RNase P ribozyme and use thereof in anti-HCMV medicament preparation |
Non-Patent Citations (1)
| Title |
|---|
| QITAO YAN ET AL: "generation of an external guide sequence library for a reverse genetic screen in caenorhabditis elegans", 《BMC BIOTECHNOLOGY》 * |
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