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SELEX Library Construction.

RA Ryo Amano
YN Yoshikazu Nakamura
Masaki Takahashi Masaki Takahashi

Last updated date: Jan 24, 2023 Views: 525 Forks: 0

original An abbreviated version of this protocol was published in Proceedings of the National Academy of Sciences of the United States of America in Apr, 2021
Nucleic acid ligands act as a PAM and agonist depending on the intrinsic ligand binding state of P2RY2
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SELEX Library Construction

Ryo Amano1, Yoshikazu Nakamura1, 2, Masaki Takahashi1, *

 

1Project Division of RNA Medical Science, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan

2RIBOMIC Inc., Tokyo, Japan 

*For correspondence: tmasaki@g.ecc.u-tokyo.ac.jp

 

[Abstract] This protocol describes the procedure to prepare the single-stranded RNA library with 2’-ribose modifications for SELEX.

 

Materials and Reagents

Note: other brands of these materials and reagents may be used if necessary.

 

Materials

  1. Single-stranded DNA (ssDNA) B02 library (5′-GTACGCTAGGCGTTAGTCTC-N40-ATCGTACGACGGTCGTACCC-3′, N40 represents a random sequence of 40 nucleotides) was chemically synthesized and purchased from GeneDesign Inc. (Osaka, Japan)
  2. B02 forward primer (5′-TAATACGACTCACTATAGGGTACGACCGTCGTACGAT-3′, T7 promoter sequence is underlined) was synthesized and purchased from FASMAC Co. Ltd (Kanagawa, Japan).
  3. Amicon®️ Ultra -0.5 mL Centrifugal Filter Unit, column molecular weight cutoff (MWCO) of 30 kDa (Merck Millipore (MA, USA), catalog number: 25967-74)

 

Reagents

  1. Thermostable DNA polymerase for PCR (e.g., TaKaRa EX Taq®️ (TaKaRa Bio Inc. (Shiga, Japan), catalog number: RR001A))
  2. Labo-made recombinant mutant of T7 RNA polymerase (Y639F)
  3. 2′-fluoro-CTP and 2′-fluoro-UTP (GeneACT Inc. (Fukuoka, Japan), catalog number: FU010 and FC010, respectively)
  4. 2′-deoxy-ATP and 2′-hydroxy-GTP (TaKaRa Bio Inc., catalog number: 4026 and 4042, respectively)
  5. Phenol/chloroform/isoamyl alcohol, 25:24:1 v/v (Nacalai Tesque, Inc. (Kyoto, Japan), catalog number: 25967-74)
  6. Chloroform (Nacalai Tesque, Inc., catalog number: 08401-65)
  7. Sodium acetate (Nacalai Tesque, Inc., catalog number: 31137-25)
  8. Acetic acid (Nacalai Tesque, Inc., catalog number: 00211-95)
  9. Ethanol (Nacalai Tesque, Inc., catalog number: 14712-05)
  10. 40(w/v)%‐Acrylamide/bis mixed solution, 29:1 (Nacalai Tesque, Inc., catalog number: 06119-45)
  11. Tris(hydroxymethyl) aminomethane (Tris) (Nacalai Tesque, Inc., catalog number: 35406-75)
  12. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Nacalai Tesque, Inc., catalog number: 17546-05)
  13. Sodium chloride (Nacalai Tesque, Inc., catalog number: 31319-45)
  14. Potassium chloride (Nacalai Tesque, Inc., catalog number: 28513-85)
  15. Magnesium chloride hexahydrate (Merck Sigma Aldrich (MO, USA), catalog number: 255777-25G)
  16. Calcium chloride (Nacalai Tesque, Inc., catalog number: 06729-55)
  17. Polyoxyethylene sorbitan monolaurate (Tween 20) (Santa Cruz Biotechnology, Inc. (TX, USA), catalog number: SC-29113)
  18. Boric acid (Nacalai Tesque, Inc., catalog number: 05215-05)
  19. Hydrochloric acid (35%) (Nacalai Tesque, Inc., catalog number: 18321-05)
  20. Sodium hydroxide (Nacalai Tesque, Inc., catalog number: 31511-05)
  21. Triton X-100 (Nacalai Tesque, Inc., catalog number: 12967-32)
  22. Spermidine (Merck Sigma Aldrich, catalog number: S2626-1G)
  23. Dithiothreitol (DTT) (Nacalai Tesque, Inc., catalog number: 14128-62)
  24. Ethylenediaminetetraacetic acid (EDTA) (Nacalai Tesque, Inc., catalog number: 15105-35)
  25. Manganese(II) chloride tetrahydrate  (FUJIFILM Wako Pure Chemical Co. (Osaka, Japan), catalog number: 133-00725)
  26. Urea (FUJIFILM Wako Pure Chemical Co. catalog number: 215-00616)
  27. N,N,N',N'-tetramethylethylenediamine (TEMED)     (Nacalai Tesque, Inc., catalog number: 33401-72)
  28. Ammonium persulfate (APS) (Nacalai Tesque, Inc., catalog number: 02627-34)
  29. Ethidium bromide solution(10mg/ml) (Nacalai Tesque, Inc., catalog number: 14631-94)
  30. Loading Dye Brilliant Color(6×) (Nacalai Tesque, Inc., catalog number: 11943-91)
  31. Xylene cyanol FF (XC) (Nacalai Tesque, Inc., catalog number: 36629-64)
  32. Bromophenol blue (BPB) (Nacalai Tesque, Inc., catalog number: 05808-61)
  33. O'RangeRuler 10 bp DNA Ladder, ready-to-use (Thermo Fisher Scientific Thermo Scientific (MA, USA), catalog number: SM1313
  34. DynaMarker® RNA Low Ⅱ (BioDynamics Laboratory Inc. (Tokyo, Japan), catalog number: DM152)
  35. SYBR® Gold Nucleic Acid Gel stain (10,000X Concentrate in DMSO) (Thermo Fisher Scientific Invitrogen, catalog number: S11494)

 

Solutions

Note: all solutions should be prepared under ribonuclease (RNase) free conditions.

  1. Mixture for preparing double-stranded DNA (dsDNA) (see Recipes)
  2. Mixture for in vitro transcription (see Recipes)
  3. 10 × in vitro transcription buffer (see Recipes)
  4. Modified NTPs mixture (see Recipes)
  5. 6% polyacrylamide gel in 1 × TBE
  6. 8% polyacrylamide gel in 1 × TBE containing 7 M Urea
  7. 2 × denaturing buffer
  8. 10 × SELEX buffer (with Tween20) (see Recipes)

 

Recipes

1. Mixture for preparing dsDNA

Reagent            

Final concentration

Amount      

10 × PCR buffer (supplemented by TaKaRa Ex Taq®️)

1 × 

30 mL

dNTPs (each 2.5 mM, supplemented by TaKaRa Ex Taq®️)

each 0.2 mM

24 mL

B02 forward primer (100 mM)

6 mM

18 mL

ssDNA B02 library (100 mM)

1 mM

3 mL

Ex Taq (5 U/mL, supplemented by TaKaRa Ex Taq®️)

0.17 U/ mL

10 mL

DNase/RNase-free distilled water

n/a

215 mL

Total

n/a

300 mL

 

2. Mixture for in vitro transcription 

Reagent            

Final concentration

Amount

10 × in vitro transcription buffer

1 × 

20 mL

Modified NTPs mixture (each 25 mM)

each 2.5 mM

20 mL

DTT (100 mM)

10 mM

20 mL

Manganese(II) Chloride (50 mM)

2.5 mM

10 mL

dsDNA 

n/a

50 mL

DNase/RNase-free distilled water

n/a

67.5 mL

Y639F T7 RNA polymerase (0.5 mg/mL)

40 ng/mL

12.5 mL

Total

n/a

200 mL

 

3. 10 × in vitro transcription buffer

Reagent            

Final concentration

Amount

HEPES NaOH (1 M, pH 8.0)

        400 mM

20 mL

Sodium chloride (5 M)

200 mM

2 mL

Magnesium chloride (1 M)

200 mM

10 mL

Spermidine (1 M)

10 mM

0.5 mL

Triton X-100

0.1%

0.05 mL

DNase/RNase-free distilled water

n/a

17.45 mL

Total

n/a

50 mL

 

4. Modified NTPs mixture

Reagent            

Final concentration

Amount

2′-deoxy-ATP (100 mM, pH 7.5)

25 mM

100 mL

2′-fluoro-UTP (100 mM, pH 7.5)

25 mM

100 mL

2′-fluoro-CTP (100 mM, pH 7.5)

25 mM

100 mL

2′-hydroxy-GTP (100 mM, pH 7.5)

25 mM

100 mL

Total

n/a

400 mL

 

5. 6% polyacrylamide gel in 1 × TBE

Reagent            

Final concentration

Amount

40(w/v)%‐Acrylamide/bis mixed solution, 29:1

6%

1.5 mL

10 × TBE

1 ×

1 mL

10% APS

2%

200 mL

TEMED

n/a

3.5 mL

DNase/RNase-free distilled water

n/a

Up to 10 mL

Total

n/a

10 mL

 

6. 8% polyacrylamide gel in 1 × TBE containing 7 M Urea

Reagent            

Final concentration

Amount

40(w/v)%‐Acrylamide/bis mixed solution, 29:1

8%

2 mL

10 × TBE

1 ×

1 mL

Urea

8 M

4.2 g

10% APS

0.2%

200 mL

TEMED

n/a

3.5 mL

DNase/RNase-free distilled water

n/a

Up to 10 mL

Total

n/a

10 mL

 

7. 2 × denaturing buffer

Reagent            

Final concentration

Amount

Urea

8 M

4.8 g

10 × TBE

1 ×

1 mL

XC (1%)

0.1%

1 mL

BPB (1%)

0.1%

1 mL

DNase/RNase-free distilled water

n/a

Up to 10 mL

Total

n/a

10 mL

 

8. 1 × SELEX buffer (with Tween20)

Reagent            

Final concentration

Amount

Tris-HCl (1 M, pH 7.6)

20 mM

5 mL

Sodium chloride (5 M)

145 mM

7.25 mL

Potassium chloride (3 M)

5.4 mM

450 mL

Magnesium chloride (1 M)

0.8 mM

200 mL

Calcium chloride (1 M)

1.8 mM

450 mL

Tween20

0.01%

25 mL

DNase/RNase-free distilled water

n/a

Up to 250 mL

Total

n/a

250 mL

 

Equipment

  1. Thermal cycler (Thermo Fisher Scientific Applied systems)
  2. High-speed refrigerated micro centrifuge (TOMY Seiko Co., Ltd., Tokyo, Japan)
  3. Incubator (EYELA TOKYO RIKAKIKAI Co., Ltd, Tokyo, Japan) 
  4. Mini gel slab electrophoretic device (BIO CRAFT Co., Ltd., Tokyo, Japan) 
  5. High-voltage electrophoresis power supply (ATTO Corporation, Tokyo, Japan)
  6. Shaker (BIO CRAFT Co., Ltd.)
  7. Gel imager (NIPPON Genetics Co., Ltd., Tokyo, Japan)
  8. GEL-TRAY UV TRANSMITTING (Analytik Jena US LLC, CA, USA)
  9. UV spectrometer (Thermo Fisher Scientific Thermo Scientific)

 

Procedure

Note: all experiments should be performed under DNase/RNase-free conditions.

A. Library design

  1. Choose the length of the random region. The length of the random region is typically 30-100 nucleotides (nt). Theoretically, libraries containing a long random region increase the probability of successful identification of aptamers (Ohuchi, S., 2014). Usually, libraries with the random region ranging from 30 to 40 nt are used for efficient identification of relatively short length aptamers (~40 nt).
  2. Design the forward and reverse primer sequences. Generally, the length of primers is approximately 20 nt because of the appropriate melting temperature for reverse transcription and PCR (55-60°C) and the ease of chemical synthesis. We prefer to use primers of 15-18 nt in length. The primer sequences should be designed by avoiding a secondary structure formation or self-association that could lead to the production of primer dimer (Hall, B., et al. 2009). Moreover, the T7 promoter sequence containing GGG, which is essential for efficient in vitro transcription with T7 RNA polymerase, is added to the 5′ end of the forward primer. 
  3. Our library and primer sequences are described below. 
    B02 library: 5′-GTACGCTAGGCGTTAGTCTC-N40-ATCGTACGACGGTCGTACCC-3′ (N40 represents a random sequence of 40 nt)
    B02 forward primer: 5′-TAATACGACTCACTATAGGGTACGACCGTCGTACGAT-3′ (T7 promoter sequence is underlined)
    B02 reverse primer: 5′-GTACGCTAGGCGTTAGTCTC-3′

 

B. dsDNA pool preparation by primer extension reaction

  1. Prepare 300 mL of the mixture for preparing dsDNA (see Recipes).
  2. Perform extension reaction by the thermal cycler as the following process: 
    Denaturing at 95°C for 3 min
    Annealing at 55°C for 5 min 
    Extension at 68°C for 20 min 
    Storage at 4°C until use
  3. Check the dsDNA products by 6% native PAGE:
    1. Prepare a 6% polyacrylamide gel in 1 × TBE (see Recipes).
    2. Add 1 mL of Loading Dye Brilliant Color (6×) to 5 mL of the dsDNA.
    3. load the mixture and 5 mL of O’RangeRuler 10 bp DNA Ladder onto the gel.
    4. After electrophoresis, soak the gel with ethidium bromide (at a final concentration of 0.1 mg/mL) and gently shake the gel for 10 min to stain the dsDNA and analyze the band image using a gel imager.
  4. Purify the dsDNA product by phenol/chloroform extraction and ethanol precipitation as follows:
    1. Transfer 300 mL of the supernatant to a new 1.5 mL microtube.
    2. Add an equal volume (300 mL) of phenol/chloroform/isoamyl alcohol to the tube and vortex well.
    3. Centrifuge at 14,000 × g for 2 min at room temperature (RT).
    4. Transfer 300 mL of the supernatant to a new microtube.
    5. Add an equal volume (300 mL) of chloroform to the microtube and vortex well.
    6. Centrifuge at 14,000 × g for 2 min at RT.
    7. Transfer 300 μL of the supernatant to a new microtube, add one-tenth volume (30 mL) of 3 M sodium acetate (pH 5.2), vortex well, and spin down the mixture.
    8. Add two times the volume of 100% ethanol (660 mL), vortex well, and chill at -80°C for 10 min.
    9. Centrifuge at 14,000 × g at 4°C for 20 min.
    10. Discard the supernatant by micropipette and rinse with 1 mL of 70% ethanol.
    11. Centrifuge at 14,000 × g at 4°C for 2 min.
    12. Discard the supernatant by micropipette and dry up completely.
  5. Dissolve the purified dsDNA in 50 mL DNase/RNase-free distilled water.
     

C. Oligonucleotide pool preparation by in vitro transcription.

  1. Prepare 200 mL of the mixture for in vitro transcription (see Recipes) and incubate at 37°C overnight. 
  2. Purify the single-stranded nucleic acid library by phenol/chloroform extraction (for protein removal) and ultrafiltration with Amicon®️ Ultra -0.5 mL Centrifugal Filter Unit 30 K (for NTP removal) as follows:
    1. Add 500 mL of DNase/RNase-free distilled water to the device.
    2. Centrifuge at 14,000 × g for 2 min at RT. 
    3. Discard the supernatant and flow through and add 300 mL of DNase/RNase-free distilled water to the device.
    4. Transfer 200 mL of the phenol/chloroform-treated transcript to the device.
    5. Centrifuge at 14,000 × g for 5 min at RT.
    6. Discard the flow through and add up to 500 mL of DNase/RNase-free distilled water to the device.
    7. Repeat the washing procedure (steps e and f) five times in total.
    8. Transfer the supernatant to a new 1.5 mL microtube by micropipette.
  3. Determine the pool concentration by measuring the absorbance at 260 nm using a UV spectrometer.
    Note: the quantity of the single-stranded nucleic acid library per preparation is usually 30-40 mg (1.5-2 mg/ml, 20mL).
  4. Check the single-stranded nucleic acid library by 8% denaturing PAGE:
    1. Prepare an 8% polyacrylamide gel in 1 × TBE containing 7 M Urea (see Recipes).
    2. Add 5 mL of the 2 × denaturing buffer (see Recipes) to 5 mL of 50 ng/ mL single-stranded nucleic acid library and DynaMarker® RNA Low Ⅱ.
    3. Heat their mixtures at 95°C for 5 min and load them onto the gel quickly.
    4. After electrophoresis, the gel is soaked into 1 × TBE buffer supplemented with SYBR® Gold Nucleic Acid Gel stain (at a final concentration of 1 ×) and gently agitated with shaker for 10 min to stain the single-stranded nucleic acid library, and then subjected to visualization of the band image using a gel imager.

 

D. Refolding of the single-stranded nucleic acid library

Note: this procedure should be performed just before the SELEX experiment. 

  1. Dilute 20 mg (≒ 800 pmol; 4.8 × 1014 molecules) of the purified pool with 1 × SELEX buffer (with Tween20) (see Recipes) to 100 mL.
  2. Heat at 95°C for 5 min and snap cool on ice, and then store the microtube at RT until use.

 

Acknowledgments

This protocol was adapted from a previously published paper by Takahashi, M et al. (2021).

 

Competing interests

The authors have no competing interests to disclose.

 

References

  1. Ohuchi, S. (2014). Identification of RNA Aptamers Against Recombinant Proteins with a Hexa-Histidine Tag. Method Mol. Biol. 1111, 41–56. https:// doi: 10.1007/978-1-62703-755-6_4.
  2. Hall, B., Micheletti, M. J., Satya, P., Ogle, K. Pollard, J., & Ellington, D. A. (2009). Design, synthesis, and amplification of DNA pools for in vitro selection. Curr Protoc Mol Biol. Chapter 24, Unit 24.2. https://doi: 10.1002/0471142727.mb2402s88.

 

Copyright: Content may be subjected to copyright.
How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Amano, R, Nakamura, Y and Takahashi, M(2023). SELEX Library Construction.. Bio-protocol Preprint. bio-protocol.org/prep2130.
  2. Takahashi, M., Amano, R., Ozawa, M., Martinez, A., Akita, K. and Nakamura, Y.(2021). Nucleic acid ligands act as a PAM and agonist depending on the intrinsic ligand binding state of P2RY2. Proceedings of the National Academy of Sciences of the United States of America 118(18). DOI: 10.1073/pnas.2019497118

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