SELEX screening experiments of randomized RNA libraries can be used for (1) in vitro diagnosis; (2) in vivo therapy, etc.
Operation method
SELEX screening of randomized RNA libraries
Principle
SELEX technology is a combinatorial chemistry technique that utilizes random oligonucleotide (DNA or RNA) libraries to screen for specific ligands of target molecules. Random RNA libraries are most commonly used, because in addition to G-C and A-U base pairs, RNA molecules also have G-U and other variant base pairs, which can form a variety of spatial structures, such as hairpins, pseudoknots, convex rings, G-quadruplexes, etc., which can be chimeric or encapsulated to form stable complexes with the target molecules through the interaction of hydrogen bonding, van der Waals forces, and so on. Even a small segment of RNA molecules can form a fairly stable rigid structure. A random RNA library containing 25 random sequences can theoretically produce 425-1015 RNA molecules with different spatial structures, which has great potential for screening.
Materials and Instruments
X-ray film Removable enzyme-linked plate Protein A sepharose gel micropure-EZ desalting and microcon-desalting tubes pGKM-T carrier RiboMAXTM Large Volume Transcription Kit AMV reverse transcriptase T4 DNA ligase pfu Taq DNA polymerase r Taq DNA polymerase Move I. Materials and equipment Caveat Prevents contamination of RNAase. Common Problems I. Measures to prevent RNAase contamination 1. All glassware should be dry-roasted at 180℃ for 6hr or longer before use. 2. 2. Plastic containers can be soaked in 0.1% DEPC water or rinsed with chloroform (Note: Plexiglas utensils can be corroded by chloroform, so they can not be used). 3. 3. Plexiglass electrophoresis tanks can be washed with detergent, rinsed with double-distilled water, dried with ethanol, immersed in 3% H2O2 at room temperature for 10min, and then rinsed with 0.1% DEPC water and air-dried. 4. The prepared solution should be treated with 0.1% DEPC as much as possible, at 37°C for more than 12hr. Then autoclave to remove the residual DEPC. reagents that can not be autoclaved, should be prepared with DEPC-treated sterile double-distilled water, and then filtered through a 0.22μm membrane to remove bacteria. 5. Operators should wear disposable masks, hats and gloves, and the gloves should be changed frequently during the experimental process. 6. Set up a special laboratory for RNA operation, and all instruments should be specialized. Commonly used RNAase inhibitors 1. Diethyl pyrophosphate (DEPC): It is a kind of strong but incomplete RNAase inhibitor. It inhibits the activity of RNAase by binding to the imidazole ring of histidine, the active group of RNAase, and denaturing the protein. 2. 2. Guanidine isothiocyanate: Currently considered the most potent RNAase inhibitor, it inactivates RNAase while cleaving tissue. It both destroys cellular structures to dissociate nucleic acids from nucleoproteins and has a strong denaturing effect on RNA enzymes. 3. oxovanadium ribonucleoside complex: a complex formed by oxovanadium ions and ribonucleosides, which combines with RNAase to form a transition state-like substance that almost completely inhibits the activity of RNAase. 4. protein inhibitor of RNAase (RNasin): acidic glycoprotein extracted from rat liver or human placenta, RNasin is a non-competitive inhibitor of RNAase and can bind to a variety of RNAases and inactivate them. 5. Others: SDS, urea, diatomaceous earth, etc. also have a certain inhibitory effect on RNA enzymes. For more product details, please visit Aladdin Scientific website.
dNTP Mix rNTP Mix DNA Gel Elution Buffer RNA Gel Elution Buffer SHMCK Buffer Blocking Solution Filter Membrane Elution NT2 Buffer RNA Binding Buffer Protease K Buffer
Electrophoresis PCR Instrument Centrifuge Gel Imager Liquid Scintillator Long Wave UV Lamp UV Spectrophotometer Nucleic Acid Quantifier pH Meter Incubator Oscillator Clean Bench
1. Instruments and equipment: electrophoresis instrument, PCR instrument, centrifuge, gel imager, liquid scintillator, long-wave ultraviolet lamp, ultraviolet spectrophotometer, nucleic acid correlation meter, pH meter, incubator, oscillator, clean bench.
2. Materials: X-ray film (X-Omat blue Kodak), detachable enzyme plates (NUNC, Denmark), HAWP membrane (13 mm, 0.45 μm, Millipore), protein A sepharose gel (HiTRAP), (Millipore), micropure-EZ desalting and microcon desalting tubes. (Millipore), micropure-EZ desalting tubes and microcon-desalting tubes.
3. pGKM-T vector, RiboMAXTM Large Volume Transcription Kit, AMV reverse transcriptase, T4 DNA ligase (Promega).
4. pfu Taq DNA polymerase, r Taq DNA polymerase (TaKaRa).
5. dNTP mix: 25 nmol/L dNTP (25 nmol/L for each of dATP, dCTP, dGTP, and dTTP).
6. rNTP Mix: 25 nmol/L rNTP (25 nmol/L for each of ATP, CTP, GTP, TTP).
7. DNA gel elution buffer: 0.5 mol/L NH4Ac, 10 mmol/L MgAc2, 0.1% SDS, 1 mmol/L EDTA (pH 8.0).
8. RNA gel elution buffer: 0.2% SDS, 0.5 mol/L NH4Ac, 1 mmol/L EDTA (pH 8.0).
9. SHMCK buffer: 20 mmol/L HEPES (pH 7.35), 120 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L CaCl2, 1 mmol/L MgCl2.
10. Closure solution: SHMCK buffer + 0.1% gelatin.
11. Wash and elution buffer: SHMCK buffer + 0.1% gelatin + 0.05% Tween-20.
12. Filter membrane eluent: 7 mol/L urea, 0.5 mol/L NH4Ac, 0.2% SDS, 1 mmol/L EDTA (pH 8.0).
13. NT2 buffer: 50 mmol/L Tris-HCI (pH 7.4), 150 mmol/L NaCl, 1 mmoI/L MgCl2, 0.05% NP-40.
14. RNA binding buffer: 50 mmol/L Tris-HCl (pH 7.4), 100 mmol/L NaCl, 4 mmol/L EDTA, 2 mmoI/L MgCl2, 0.05% NP 40, 0.4% Vanadyl ribomicleoside complex, 200 U/ml RNasin, 100 μg/ml poly A, 100 μg/ml tRNA, 50 μg/ml BSA.
15. 2X Protease K buffer: 20 mmol/L Tris-HCl (pH 7.8), 10 mmol/L EDTA, 1% SDS.
II.
1. Construction of random RNA library and primer synthesis
Design a random RNA library containing 25 random sequences, 85 bases in length, with fixed sequences at both ends of the library. Primers can be designed for PCR amplification of the library. Based on this design, the first step is to synthesize the ssDNA template of the random library and its two primers.
The template and primer sequences are shown below:
5' primer: 5'-TAATACGACTCACTATAGCAATGGTACGGTACTTCC-3';
3' primer: 3'-TTAGCAAAGTAGCGTGCACTTTTG-3';
ssDNA-G template: 5'-TAATACGACTCACTATAGCATGGTACGGTACTTCC (25N) CAAAAGTGCACGCTACTTTGCTAA-3';
where N stands for random base and the underlined part is the T7 promoter sequence, which is used for transcription and preparation of random RNA library.
2. Establishment of the starting RNA library
(1) PCR amplification of ssDNA template is dsDNA, which is used to amplify ssDNA.
The PCR reaction system was as follows: 10 μl of 10X PCR buffer, 8 μl of dNTP mix, 2 μl of 5' primer (25 pmol/μl), 2 μl of 3' primer (25 pmol/μl), 0.25 μg of ssDNA template, 1 μl of pfu Taq DNA polymerase, and 100 μl of deionized water to make up the total.
PCR reaction conditions: pre-denaturation at 95°C for 5 min, denaturation at 4°C for 1 min, annealing at 37°C for 2 min, extension at 72°C for 2 min, amplification for 3 cycles, final extension at 72°C for 5 min.
After PCR of the synthesized ssDNA template of at least 2OD260 under optimized conditions, the PCR products were concentrated and purified by ultrafiltration as follows: Micmpure-EZ desiccation tubes, Microcon-3 desalting tubes and recovery tubes were mounted in top-to-bottom order; the PCR products were transferred to the desiccation tubes, approximately 200 μl of PCR products were added to each tube, and the products were centrifuged to Mkrocon® at 13,000 r/min. The PCR product was transferred to the demineralization tubes and approximately 200 μl of product was added to each tube and centrifuged at 13,000 r/min until approximately 50 μl of product remained in the Mkrocon-3 desalting tubes.
(2) In vitro transcription
① Transcription process.
Add each reaction solution in the following order: 5X T7 transcription buffer 4 μl, rNTP mixture 6 μl, DNA template 1~2 μg, T7 RNA polymerase 2 μl, and make up to 20 μl with RNase-free DEPC water.
The whole transcription system was reacted at 37°C for 3~4 h, and then DNA enzyme was added and reacted at 37°C for 30~60 min to digest the DNA template (1U of DNA enzyme was added for every 1 μg of DNA). 8% acrylamide gel electrophoresis containing 7 mol/L urea was used for identification analysis and cutting of gels for modeling and collection, and the transcription products were denatured at 95°C for 5 min before electrophoresis, and then put in an ice bath for 5 min. The transcripts were denatured at 95°C for 5 min and then placed in an ice bath for 5 min.
The transcripts were denatured at 95°C for 5 min before electrophoresis and then placed in an ice bath for 5 min.
After electrophoresis of the transcribed RNA products on an 8% urea denaturing PAGE gel, the corresponding RNA bands are cut off and transferred to a microcentrifuge tube. 2 times the volume of RNA gel elution buffer is added, and 1 unit of RNAase inhibitor is added to each 400 μl of eluate. The disposable pipette tip is swirled around in the tube and pressed against the side wall of the tube to break up the gel fragments, and then the tube is sealed.
The next day, carefully pipette the eluate into a new 1.5 ml centrifuge tube (do not aspirate the gel), add 2.5 times the volume of anhydrous ethanol, 1/10 of the volume of 3 mol/L sodium acetate, pH 5.2, and let stand at -70°C for 3 h. Then centrifuge the tube at 12,000 r/min at 4°C for 30 min, discard the supernatant, and rinse the precipitate with 0.5-1 ml of 70% ethanol, pre-cooled at 4°C. The precipitate was washed with 0.5~1 ml of 70% ethanol pre-cooled at 4℃, then centrifuged and the supernatant was discarded, and the precipitate was dried at room temperature. Finally, the dried RNA was dissolved in appropriate amount of DEPC water without RNAase and frozen at -20℃ or -70℃.
③ Determine the affinity parameter for the target protein.
The relationship between the concentration of the target protein and the amount of RNA library binding can be plotted by the filter membrane method or gel blocking method. The relationship is usually an S-curve when the logarithm of the protein concentration is plotted, and reaches a plateau at higher concentrations, suggesting that it is the saturated binding amount. From the graph, 5%~10% of the protein concentration needed to reach saturation is calculated as the protein concentration to be used in the following screening experiments.
3. Screening of RNA ligands
There are various methods to isolate RNA molecules bound to target molecules, including microtiter plate method, nitrocellulose membrane method, gel-shift method, immunoprecipitation method, and column binding method, etc. The advantage of the microtiter plate method is that it is easy to operate, and it can be used for the screening of RNA molecules. The advantage of the microtiter plate method is that it is relatively simple to perform, but the natural conformation of the target molecule may be affected due to the semi-solidified nature of the reaction system; the advantage of the membrane and gel-shift methods is that enrichment data can be obtained during the screening process; and immunoprecipitation can be used to screen for complex or impure target molecules.
(1) Microtiter plate method
① Screening process
A. Packing plate. Operate on a detachable enzyme labeling plate, set up two wells A and B. B wells were coated with 200 μl target protein solution (dissolved in PBS, pH 7.4); A wells were added with 200 μl PBS only, and incubated at 4℃ overnight.
B. Blocking. Discard the supernatant, add 200 μl of SHMCK blocking buffer to each well, and incubate at 37℃ for 2 hours.
C. Screening. Wash 6 times with 200 μl/well wash buffer, 3 min/time; dissolve denaturing and cooling processed RNA in 200 μl binding buffer, add to well A, then incubate at 37℃ for 45 min; wash well B; transfer the supernatant from well A to well B, incubate at 37℃ for 2 h, then wash. (This step includes the process of reverse sieving, i.e., removing random sequences bound to the microplate media.)
② RT-PCR
A. Reverse transcription process. The plate was denatured at 95℃ for 5 min and then placed at 4℃ for 5 min, then the components of the reverse transcription reaction were added: 10 μl of 5X AMV buffer, 5 μl of dNTP mixture, 1 μl of AMV reverse transcriptase (10 U/μl), 5 μl of 3' primer (25 pmol/μl), and then the plate was made up to 50 μl of DEPC water without RNAase, and then the reaction was repeated at 42℃ for 1 h. The reaction was repeated at 42℃ for 1 h. The plate was denatured at 95℃ for 5 min and then placed at 4°C for 5 min.
B. PCR amplification: The PCR reaction system was as follows: 10 μl of 10X PCR reaction buffer, 8 μl of dNTP mixture, 1 μl of 5' primer (25 pmol/μl), 1 μl of 3' primer (25 pmol/μl), 50 μl of reverse transcription system, 1 μl of Taq DNA polymerase (2 U/μl), and make up the total amount to 100 μl with deionized water.
PCR reaction conditions: pre-denaturation at 95°C for 5 min, amplification of 5 cycles, each cycle of 94°C denaturation for 1 min, annealing at 55°C for 2 min, extension at 72°C for 1 min, and finally extension at 72°C for 5 mim.
C. Repeat PCR to determine the number of cycles required for final amplification: 10 μl of the above PCR product was used as template for repeat PCR amplification, and samples were analyzed by 10% PAGE every 3 cycles. The number of cycles with high PCR yield and no heterozygous bands was chosen as the optimal number of cycles, and all of the remaining 90 μl of product was subjected to PCR amplification.
D. Recover the PCR amplified product by ethanol precipitation. If spurious bands are present, it is necessary to cut the gel to recover the correct bands (refer to RNA Cutting and Recovery Method).
(iii) In vitro transcription
Use the double-stranded DNA as the template for in vitro transcription, and cut and recover the RNA as the library for the next round of screening.
④ Repeat screening
Repeat the process of screening, isolation, RT-PCR and in vitro transcription for a total of n rounds of screening.
⑤ Analyze the specificity of RNA ligands by RT-PCR.
In the nth round of screening, there are screening group and control group at the same time, and target protein is added to the screening group, while no target protein is added to the control group. Both groups were incubated with equal amount of target protein, screened and RT-PCR, and then analyzed by 10% PAGE for identification and comparison of the binding status.
(2) Nitrocellulose filter method
① Screening procedure
A. Incubation. Denature the purified RNA at 100°C for 5 min and immediately place it in an ice bath for 5 min; gently mix the RNA with the target molecules in 200 μl of SHMCK buffer, and incubate for 1 h at 37°C to allow the RNA and target molecules to fully interact.
B. Separation. Turn on the negative pressure device of the sand core funnel and rinse the sand core funnel with SHMCK buffer; carefully place the HAWP filter membrane in the middle of the sand core funnel and pre-wet the membrane with SHMCK buffer; spot the incubation mixture on the membrane and wash it with 1 ml of SHMCK buffer under gentle negative pressure.
C. Recovery of RNA bound to target proteins. Put the membrane into a 1.5 ml centrifuge tube, cut the membrane with small scissors that are not contaminated with RNAase, add 200 μl of membrane eluent, and boil at 100℃ for 5 min; transfer the supernatant into a new centrifuge tube, add 200 μl of membrane eluent to the original tube and repeat the elution, then combine the two eluents and precipitate with ethanol.
The solution is the same as the microtiter plate method (②~4).
(3) Gel blocking method
The gel blocking method is similar to the nitrocellulose membrane method, except that the separation method is PAGE gel electrophoresis. The combination of proteins and the corresponding oligonucleotides to form a complex will change the molecular mass and charge of the oligonucleotides, and thus the electrophoretic mobility in the PAGE electrophoresis system is changed, and the rate of swimming is much slower than that of the free oligonucleotide fragments, and the slow RNA will be recovered by gel cutting, RT-PCR, and put into the next round of screening. The slow-swimming RNA will be recovered by cutting the gel, RT-PCR, and put into the next round of screening.
(4) Immunoprecipitation
①Screening process
A. Swell protein A Sepharose gel beads (bead) with excess NT2 buffer for 10 min; wash beads with 1 ml of NT2 buffer, centrifuge at 12000 g for 10 s, wash three times in total; the amount of beads should be sufficient to bind 2 mg of the target protein and prepare the same amount of beads at the same time to be used for reverse screening of the library.
B. Add the appropriate volume of antibody, the amount of antibody added is determined by the binding ability of the target protein to the antibody, and it is necessary to add a slightly larger amount of antibody; incubate at 4 ℃ for 1 h to allow the antibody to bind to the beads; and then wash the beads with 3X 1 ml of NT2 buffer.
C. Inverse Sieving. Split the antibody-bound gel beads into two equal portions and add equal volumes of RNA binding buffer, add the RNA library to one portion, incubate at 4℃ for 30 min, centrifuge and collect the supernatant for use.
D. Transfer the other gel bead to a new centrifuge tube, add 1-10 μg of target protein (for a 50 kDa target protein, 10 μg is already 0.2 mmol, which is sufficient even for the first round of screening), incubate at 4°C for 10 min, and rinse with 3X 1 ml NT2 buffer.
E. Add RNA prepared in step C. Incubate at 4°C for 10 min and wash with 5X 1 ml NT2 buffer.
F. In the last bead wash, take 100 μl of NT2 retention sample, add 100 μl of DEPC water, 200 μl of 2X Protease K buffer, 5 μl of protease, incubate at 37℃ for 15 min and then add PCI (Phenol: Chloroform: Isoamyl alcohol = 25:24:1), vortex for 30 s, centrifuge at 12000 g for 1 min, and then transfer the supernatant into a new centrifuge tube to precipitate with ethanol. Precipitation.
The supernatant was transferred to a new centrifuge tube and precipitated with ethanol. ②~④ were the same as ②~④ of the microtiter plate method.
4. Analysis of specific RNA ligands obtained after screening
(1) Analysis of affinity and specificity, determination of equilibrium dissociation constant Kd
① Radioisotope labeling of RNA
The total in vitro transcription system was 20 μl: 4 μl of 5X T7 transcription buffer, 1.5 μl each of 25 mmol/L ATP, CTP and GTP, 0.15 μl of 25 mmol/L UTP, 2.5 μl of [ α-32P ] UTP (3000 Ci/mmol), 1~2 μg of DNA template, 2 μl of T7 RNA polymerase, and make up to 20 μl with DEPC water without RNase. DEPC water without RNase was used to make up to 20 μl.
The rest of the reaction process was the same as the transcription process. The transcription products were electrophoresed by urea denaturing gel, then cut and recovered, and then subjected to radioactivity intensity detection: 1 μl of recovered solubilized α-32P-labeled RNA was diluted with 99 μl of DEPC water and then spotted on the membrane, which was then baked and dried under the incandescent lamp, and then the membrane was clamped into a liquid flash bottle, and then added with 3 ml of scintillation solution, and then counted in the liquid flash apparatus.
② Determination of Kd
A. Filter membrane method. After denaturing and cooling 5~10 pmol of α-32P-labeled RNA (about 80,000 cpm), mix it with different concentrations of proteins in 20 μl of SHMCK buffer, and incubate for 30 min at 37 ℃; pre-wet the HAWP membrane with SHMCK buffer, and place the membrane on a Millipore microfilter with small, dry-roasted tweezers; place the sample in the center of the membrane, and place the sample in the center of the filter. The sample was carefully placed in the center of the membrane and the lid of the filter was tightened; the membrane was washed under pressure with SHMCK buffer in a disposable 5 ml syringe; the membrane was removed, baked dry, and placed in a scintillation cup, and scintillation solution was added to measure the cpm value and analyze the value of the dissociation constant using Origin 4.0 graphical analysis. The equilibrium dissociation constant Kd is used to indicate the strength of RNA binding to the target protein, Kd = [ RNA ] X [ target protein ] / [ RNA target protein complex ], when the concentration of the target protein is much larger than the concentration of the RNA, the Kd value is approximately equal to the concentration of the protein when the amount of RNA bound is half of the maximum amount, usually at the nmol/L level.
B. Gel Block Method. This method is similar to the filter membrane method in that only PAGE gel is used to separate bound and free RNA, followed by radioautography.
(2) Clone sequencing
After several rounds of screening, a subpopulation of RNA ligands is subjected to RP-PCR, and then cut and recovered; the recovered DNA fragments are ligated with pGEM-T vector and transformed into sensory bacteria. The appropriate number of clones were selected for PCR identification, and the clones with the correct size bands were further sequenced, usually more than 20 clones.
(3) Sequence and biological activity analysis
These sequences were analyzed for RNA secondary structure prediction (using RNAstructure or the online tool Mfold http://hininfo.match.rpi.edu/~Mfold/rna/forml.cgi) and the conservativeness of random regions. The most representative sequences were selected and their equilibrium dissociation constants were further determined to obtain the highest affinity RNA ligands.
Depending on the biological function of the target molecule, the biological activity of the obtained RNA ligands can be analyzed.
(4) Further Applications
These RNA ligands can also be modified, such as by removing homologous sequences at both ends, to further analyze the interaction of the RNA ligand with the target protein.
The purpose of modification of oligonucleotide ligands is twofold: one is to further improve the specificity of the interaction of the ligand with the target protein; the other is to improve the bioavailability of the ligand. Generally, the half-life of oligonucleotides in vivo is very short, and modification of the ligands is necessary to utilize them in clinical diagnosis and disease treatment. There are two ways of modification: one is to incorporate modified nucleoside triphosphates into the oligonucleotide library before screening, and the commonly used modification positions are the phosphate group, the 5' position of the pyrimidine ring, the 8' position of the purine ring, and the 2' position of the five-carbon sugar. Another modification is to apply the above modifications to the screened oligonucleotide ligands. In order to prolong the retention of the ligand in vivo, polyethylene glycol can be attached to certain positions of the ligand, or the ligand can be bound to the surface of the membrane vesicle of the liposome. The oligonucleotide ligands after the above modifications can essentially meet the clinical requirements.