Construction of a Highly Diverse mRNA Library for in vitro Selection of Monobodies

Materials and Reagents

1. Oligonucleotides (sequences and suppliers are listed in Table 1)

2. T4 DNA ligase (New England Biolabs, catalog number: M0202L)

3. Bsa I-HF^® (New England Biolabs, catalog number: R3535L)

   Note: This has been replaced with a new version (Bsa I-HFv2^®, catalog number: R3733L).

4. Pfu-S DNA polymerase prepared in-house (Wang et al., 2004; Kondo et al., 2020)

5. T7 RNA polymerase prepared in-house (Abramochkin and Shrader, 1995; Kondo et al., 2020)

6. ExcelBand 100-bp DNA Ladder (Smobio Technology, catalog number: DM2100)

7. 0.5 mol/L-EDTA Solution (pH 8.0) (Nacalai Tesque, catalog number: 14347-21)

8. Formamide (Nacalai Tesque, catalog number: 16229-95)

9. Glycerol (Nacalai Tesque, catalog number: 17018-25)

10. 40 (w/v) %-Acrylamide/Bis Mixed Solution (37.5:1) (Nacalai Tesque, catalog number: 06121-95)

11. Trimethylolaminomethane (Tris) (Nacalai Tesque, catalog number: 35406-75)

12. Polyethylene Glycol 6000 (PEG 6000) (FUJIFILM Wako Pure Chemicals, catalog number: 169-22945)

13. 1,4‐Dithiothreitol (DTT) (Nacalai Tesque, catalog number: 14128-62)

14. Magnesium Chloride Hexahydrate (MgCl₂) (Nacalai Tesque, catalog number: 20908-65)

15. Potassium Chloride (KCl) (Nacalai Tesque, catalog number: 28514-75

16. Sodium Chloride (NaCl) (Nacalai Tesque, catalog number: 31320-05)

17. Polyethylene Glycol Mono-p-isooctylphenyl Ether (Triton X-100) (Nacalai Tesque, catalog number: 12967-45)

18. Dimethyl Sulfoxide (DMSO) (Nacalai Tesque, catalog number: 13407-03)

19. Magnesium Sulfate Heptahydrate (Nacalai Tesque, catalog number: 06296-25)

20. Spermidine Trihydrochloride (Nacalai Tesque, catalog number: 32110-54)

21. 100 mM dNTP Set (NIPPON GENE CO., LTD., Custom order)

22. CTP, GTP, UTP (JENA BIOSCIENCE, Custom order)

23. ATP (FUJIFILM Wako Pure Chemicals, catalog number: 019-09672)

24. Phenol:Chloroform:Isoamyl Alcohol 25:24:1 Mixed, pH 6.7 (Nacalai Tesque, catalog number: 25967-74)

25. Chloroform (Nacalai Tesque, catalog number: 08402-55)

26. 2-Propanol (Nacalai Tesque, catalog number: 29113-95)

27. Ethanol (EtOH) (Nacalai Tesque, catalog number: 14713-95)

28. 2× Ligation solution (see Recipes)

29. 1× PCR solution (see Recipes)

30. 1× RNA Pol solution (see Recipes)

31. DNA loading buffer (see Recipes)

    
    

    Table 1. Oligonucleotide sequences and suppliers. NNK codons were prepared via trinucleotide phosphoramidite synthesis (20% Tyr, 10% Ser, 15% Gly, 10% Trp, and 3% each of all the remaining amino acids, with the exception of Cys). Abbreviations: SPC18, spacer 18; Pu, puromycin; HEX, hexachloro-fluorescein.
    

Equipment

1. T100 thermal cycler (Bio-Rad, catalog number: 1861096J1)

2. Gel Doc EZ (Bio-Rad, catalog number: 1708270)

3. Mini-PROTEAN System (Bio-Rad, catalog number: 1658000FC)

4. Cooling slab electrophoresis device (BIO CRAFT, catalog number: BE130G)

Procedure

1. Preparation of A-fragment DNA of the monobody library

   1. Prepare the following solution (35 μl), heat at 95°C for 2 min, and place at room temperature for 5 min:

      FN3F0.F83 (2 μM final concentration), FN3F1-2.F29(P) (2 μM final concentration), FN3FF1coR8.F73 (1 μM final concentration), FN3FF1coR10.F79 (1 μM final concentration), Fn3an1.R20(3NH2) (4 μM final concentration), and Fn3an2-1.R20(3NH2) (4 μM final concentration) in 10 mM Tris-HCl pH 7.5.

   2. Add 2× ligation solution (35 μl) to the solution described in (1) and incubate at 25°C for 1 h (see Figure 3A for analysis of the ligation reaction).

   3. Add the mixture to 1× PCR solution (15 ml) and dispense 77 µl resulting solution into each well of two 96-well PCR plates. Amplify the DNA using T7SD8M2.F44 and FN3BsaI.R40 under the following conditions: initial activation step at 94°C for 1 min, followed by seven cycles of heat denaturation at 94°C for 20 s, primer annealing at 55°C for 20 s, and primer extension at 72°C for 45 s (see Figure 3B for optimization of the number of thermal cycles).

   4. Add 3 M NaCl (1.5 ml).

   5. Mix 3 ml phenol:chloroform:isoamyl alcohol (25:24:1) with the resulting solution and centrifuge at room temperature for 5 min at 16,000 × g.

   6. Mix 6 ml chloroform with the water layer and centrifuge at room temperature for 5 min at 16,000 × g.

   7. Mix 16 ml 2-propanol with the water layer and centrifuge at room temperature for 15 min at 16,000 × g.

   8. After removing the supernatant, add 1 ml 70% EtOH and centrifuge at room temperature for 5 min at 16,000 × g.

   9. After removing the supernatant, dry the pellet and dissolve in 0.5 ml 10 mM Tris-acetic acid pH 7.8.

   10. Determine the DNA concentration by 12% polyacrylamide gel electrophoresis using the ExcelBand 100-bp DNA Ladder.




2. Preparation of B-fragment DNA of the monobody library

   1. Prepare the following solution (35 μl), heat at 95°C for 2 min, and place at room temperature for 5 min:

      FN3FF2co.F72(p) (2 μM final concentration), FN3F3coR10.F70(p) (1 μM final concentration), FN3F3coR12.F76(p) (1 μM final concentration), and Fn3an3.R20(3NH2) (4 μM final concentration) in 10 mM Tris-HCl pH 7.5.

   2. Add 2× ligation solution (35 μl) to the solution described in (1) and incubate at 25°C for 1 h (see Figure 3A for analysis of the ligation reaction).

   3. Add the mixture to 1× PCR solution (15 ml) and dispense 77 µl resulting solution into each well of two 96-well PCR plates. Amplify the DNA using FN3BsaI.F33 and FN3Pri2.R44 under the following conditions: initial activation step at 94°C for 1 min, followed by seven cycles of heat denaturation at 94°C for 20 s, primer annealing at 55°C for 20 s, and primer extension at 72°C for 45 s (see Figure 3B for optimization of the number of thermal cycles).

   4. Add 3 M NaCl (1.5 ml).

   5. Mix 3 ml phenol:chloroform:isoamyl alcohol (25:24:1) with the resulting solution and centrifuge at room temperature for 5 min at 16,000 × g.

   6. Mix 6 ml chloroform with the water layer and centrifuge at room temperature for 5 min at 16,000 × g.

   7. Mix 16 ml 2-propanol with the water layer and centrifuge at room temperature for 15 min at 16,000 × g.

   8. After removing the supernatant, add 1 ml 70% EtOH and centrifuge at room temperature for 5 min at 16,000 × g.

   9. After removing the supernatant, dry the pellet and dissolve in 0.5 ml 10 mM Tris-acetic acid pH 7.8.

   10. Determine the DNA concentration by 12% polyacrylamide gel electrophoresis using the ExcelBand 100-bp DNA Ladder.




3. Digestion of the A-fragment and B-fragment DNA with Bsa I

   1. Incubate the reaction mixture (1× CutSmart^® buffer, 2 μM A-fragment or B-fragment, 200 units/ml Bsa I-HF^®; in a total of 400 μl) at 37°C for 4 d.

   2. Add 3 M NaCl (40 µl).

   3. Mix 100 µl phenol:chloroform:isoamyl alcohol (25:24:1) with the resulting solution and centrifuge at room temperature for 5 min at 16,000 × g.

   4. Mix 200 µl chloroform with the water layer and centrifuge at room temperature for 5 min at 16,000 × g.

   5. Mix 400 µl 2-propanol with the water layer and centrifuge at room temperature for 15 min at 16,000 × g.

   6. After removing the supernatant, add 100 µl 70% EtOH and centrifuge at room temperature for 5 min at 16,000 × g.

   7. After removing the supernatant, dry the pellet and dissolve in 10 µl 10 mM Tris-acetic acid pH 7.8.

   8. Mix 10 µl resulting solution with 10 µl DNA loading buffer.

   9. Subject the solution to 12% polyacrylamide gel electrophoresis (10 cm × 7.3 cm × 0.75 mm, 300 V, 20 min).

   10. After cutting the band out of the gel, elute the DNA using 0.3 M NaCl (1.2 ml).

   11. Mix 1.2 ml 2-propanol with the solution and centrifuge at room temperature for 15 minutes at 16,000 × g.

   12. After removing the supernatant, add 100 µl 70% EtOH and centrifuge at room temperature for 5 minutes at 16,000 × g.

   13. After removing the supernatant, dry the pellet and dissolve in 100 µl 10 mM Tris-acetic acid pH 7.8.

   14. Determine the DNA concentration by 12% polyacrylamide gel electrophoresis using the ExcelBand 100 bp DNA Ladder.




4. Ligation of the digested A-fragment and B-fragment DNA

   1. Mix the solution containing 2 μM each A-fragment and B-fragment DNA (100 μl) with the 2× ligation solution (100 μl).

   2. Incubate the resulting mixture at 25°C for 1 h (see Figure 3C for analysis of the ligation reaction).




5. PCR amplification of the DNA fragment

   1. Add the mixture to 1× PCR solution (60 ml) and dispense 77 µl resulting solution into each well of eight 96-well PCR plates.

   2. Amplify the DNA fragment using T7SD8M2.F44 and G5S-4Gan21-3.R42 under the following conditions: initial activation step at 94°C for 1 min, followed by four cycles of heat denaturation at 94°C for 20 s, primer annealing at 55°C for 20 s, and primer extension at 72°C for 45 s (see Figure 3D for optimization of the number of thermal cycles).

   3. Add 3 M NaCl (6 ml).

   4. Mix 8 ml phenol:chloroform:isoamyl alcohol (25:24:1) with the resulting solution and centrifuge at room temperature for 5 min at 16,000 × g.

   5. Mix 16 ml chloroform with the water layer and centrifuge at room temperature for 5 min at 16,000 × g.

   6. Mix 66 ml 2-propanol with the water layer and centrifuge at room temperature for 15 min at 16,000 × g.

   7. After removing the supernatant, add 1 ml 70% EtOH and centrifuge at room temperature for 5 min at 16,000 × g.

   8. After removing the supernatant, dry the pellet and dissolve in 6 ml 10 mM Tris-acetic acid pH 7.8.

   9. Determine the DNA concentration by 12% polyacrylamide gel electrophoresis using the ExcelBand 100-bp DNA Ladder.




6. Preparation of the mRNA library

   1. Incubate a mixture of 1× RNA Pol and 50 nM amplified DNA (in a total of 11 ml) at 37°C for 6 h.

   2. Add 0.55 ml 0.5 M EDTA pH 8.0 and 1.2 ml 3 M NaCl.

   3. Mix 2 ml phenol:chloroform:isoamyl alcohol (25:24:1) with the resulting solution and centrifuge at room temperature for 5 min at 16,000 × g.

   4. Mix 4 ml chloroform with the water layer and centrifuge at room temperature for 5 min at 16,000 × g.

   5. Mix 9 ml 2-propanol with the water layer and centrifuge at room temperature for 15 min at 16,000 × g.

   6. After removing the supernatant, add 1 ml 70% EtOH and centrifuge at room temperature for 5 min at 16,000 × g.

   7. After removing the supernatant, dry the pellet and dissolve in 0.5 ml ultrapure water.

   8. Mix 20 µl resulting solution with 150 µl formamide.

   9. Subject the solution to 6 M urea 5% polyacrylamide gel electrophoresis (13.8 cm × 12 cm × 2 mm, 40 W, 40 min).

   10. After cutting the band out of the gel, elute the DNA using 0.3 M NaCl 50% formamide solution (18 ml).

   11. Mix 4 ml phenol:chloroform:isoamyl alcohol (25:24:1) with the resulting solution and centrifuge at room temperature for 5 min at 16,000 × g.

   12. Mix 8 ml chloroform with the water layer and centrifuge at room temperature for 5 min at 16,000 × g.

   13. Mix 18 ml 2-propanol with the water layer and centrifuge at room temperature for 15 min at 16,000 × g

   14. After removing the supernatant, add 1 ml 70% EtOH and centrifuge at room temperature for 5 min at 16,000 × g.

   15. After removing the supernatant, dry the pellet and dissolve in 20 µl ultrapure water.

   16. Determine the mRNA concentration by measuring the OD at 260 nm. Store at -80°C.




7. Preparation of the mRNA/HEX-PuL complex

   1. Before the first round of selection, heat the solution (25 mM HEPES-K pH 7.8, 200 mM potassium acetate, 4-8 μM RNA, and 4 μM HEX-PuL, in a total of 80 μl) at 95°C for 2 min, then cool to 25°C. Use the resulting complex for the first round of selection.

      
      

      
      Figure 3. PAGE analysis of the reaction in each procedure. A. Ligation of oligonucleotides during Procedures A and B. B. Optimization of the number of thermal cycles in Procedures A and B. The reaction was performed in 10 µl solution. C. Ligation of the BsaI-treated A- and B-DNA fragments in Procedure D. D. Optimization of the number of thermal cycles in Procedure E. The reaction was performed in 10 µl solution.

Data analysis

The 44 clones in the DNA library were cloned, and the sequences were analyzed by Sanger sequencing. Fourteen single-base deletions and 19 single-base insertions were observed in the backbone sequence; they were likely by-products of the oligonucleotides used in the construction of the DNA template. As a result, the reading frame was maintained in 19 out of 44 clones. The number of codons in the random sequences is summarized in Table 2. The observed frequency showed good agreement with the theoretical frequency.

Table 2. Analysis of the codons in the BC and FG loop sequences of 44 clones.

Notes

The theoretical diversity of the ssDNA, dsDNA, and mRNA libraries was calculated as follows:

# A-fragment in Procedure A: 1 µM oligonucleotides (75 µl scale); 75 pmol, 4.5 × 10¹³ molecules

# B-fragment in Procedure B: 1 µM oligonucleotides (75 µl scale); 75 pmol, 4.5 × 10¹³ molecules

# A-fragment in Procedure A: 43 nM dsDNA product after 7 cycles (15 ml scale); 0.64 nmol, 3.9 × 10¹⁴ molecules, 0.6 × 10¹³ diversity with 64 copies

# B-fragment in Procedure B: 38 nM dsDNA product after 7 cycles (15 ml scale); 0.57 nmol, 3.4 × 10¹⁴ molecules, 0.5 × 10¹³ diversity with 64 copies (only the complementary DNA was synthesized in the 1^st cycle)

# dsDNA product in Procedure D: 1 µM dsDNA (200 µl scale); 200 pmol, 1.2 × 10¹⁴ molecules

# dsDNA product in Procedure E: 33 nM dsDNA product after 4 cycles (60 ml scale), 2.0 nmol, 1.2 × 10¹⁵ molecules, 7.5 × 10¹³ diversity with 16 copies

# mRNA product in Procedure F: 50 nM DNA template (11 ml scale); 0.55 nmol, 3.3 × 10¹⁴ molecules, the diversity was limited by Procedure E.

Recipes

1. 2× Ligation solution
   100 mM Tris-HCl pH 7.5
   12 mM MgCl₂
   1 mM ATP
   16% PEG 6000
   20 mM DTT
   4,000 U/ml T4 DNA ligase
   
2. 1× PCR solution
   10 mM Tris-HCl pH 8.4
   100 mM KCl
   0.1% (v/v) Triton X-100
   2% (v/v) DMSO
   2 mM MgSO₄
   0.2 mM each dNTP
   0.375 μM each primer
   2 nM Pfu-S DNA polymerase
   
3. 1× RNA Pol solution
   40 mM Tris-HCl pH 8.0
   1 mM spermidine
   0.01% (v/v) Triton X-100
   10 mM DTT
   25 mM MgCl₂
   5 mM each NTP
   1 μM T7 RNA polymerase
4. DNA loading buffer
   80 mM Tris-acetate pH 8.0
   2 mM EDTA
   20% (v/v) glycerol

Acknowledgments

This work was supported by Grant-in-Aid for Scientific Research (A) (General) [Grant Number 15H02006 to H.M.], Grant-in-Aid for Scientific Research on Innovative Areas [Grant Number 20H04704 to G.H.], and Grant-in-Aid for Early-Career Scientists [Grant Number 18K14332 to T.F.] from the Japan Society for the Promotion of Science; and a donation from Dr. Hiroshi Murakami. This protocol was derived from our previous research paper (Kondo et al., 2020).

Competing interests

The authors declare no competing interests.

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