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Oct 2020
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Construction of a Highly Diverse mRNA Library for in vitro Selection of Monobodies
体外选择单体的高度多样性mRNA文库的构建   

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Abstract

Recently, we developed transcription/translation coupled with the association of puromycin linker (TRAP) display as a quick in vitro selection method to obtain antibody-like proteins. For the in vitro selection, it is important to prepare mRNA libraries among which the diversity is high. Here, we describe a method for the preparation of monobody mRNA libraries with greater than 1013 theoretical diversity. First, we synthesized two long single-stranded DNAs that corresponded to fragments of monobody DNA, with random codons in the BC and FG loops. These oligonucleotides were ligated by T4 DNA ligase with the support of guide oligonucleotides containing 3′ ends that were protected by a modification. After amplifying the product DNAs by PCR, one end of each DNA fragment was digested with the type II restriction enzyme BsaI, and the resulting DNA fragments were ligated using T4 DNA ligase. After amplification of the DNA product, mRNAs were synthesized by T7 RNA polymerase. This method is simple and could be used for the preparation of mRNA libraries for various antibody-like proteins.


Graphic abstract:



Construction of a highly diverse mRNA library.


Keywords: In vitro selection (体外选择), TRAP display (陷阱显示), mRNA library (mRNA文库), Antibody-like protein (抗体样蛋白), Monobody (单体)

Background

Various antibody-like proteins (ALPs) have been obtained using an in vitro selection approach and employed in the biological and medical fields (Schumacher et al., 2018; Simeon and Chen 2018). During this in vitro selection, initial library construction is an important step in obtaining high-affinity binders. For example, to develop a nanobody, the VHH domains of camel antibodies obtained after immunization were used for the construction of an initial library (Arbabi Ghahroudi et al., 1997). Alternatively, the VHH domains from naïve genes or a semi-synthetic or synthetic library could be used as the initial library (Goldman et al., 2006; Monegal et al., 2009; Yan et al., 2014).


For other non-immunoglobulin scaffolds, synthetic libraries were used exclusively as the initial library (Nord et al., 1997; Koide et al., 1998; Beste et al., 1999; Binz et al., 2003; Grabulovski et al., 2007; Olson et al., 2008). Oligonucleotides containing random codons were assembled by extension PCR and ligated to other DNA fragments for synthesis of the DNA library. As randomized codons, NNK codons (with N representing A, C, G, or T; and K representing T or G) were generally used because they cover all 20 amino acids and can reduce the redundancy of codons and the frequency of stop codons. Alternatively, direct codon synthesis using trinucleotide phosphoramidites (Virnekäs et al., 1994) was used to mimic the amino acid frequency in the CDR3 sequences of antibodies (Zemlin et al., 2003; Wojcik et al., 2010; Koide et al., 2012).


The construction of medium-sized libraries (1010 diversity) is relatively simple, whereas that of large-sized libraries (1013 to 1014 diversity) requires special care to maintain the diversity (Olson and Roberts 2007). For example, when a 50 nM DNA product is obtained after 10 cycles of PCR, the concentration of the original template can be calculated as 50 pM. If we use only 100 µl reaction mixture, the maximum diversity that can be achieved using the conditions described above is limited to 3 × 109.


Recently, by modifying the traditional mRNA display (Nemoto et al., 1997; Roberts and Szostak 1997) and the original version of the TRAP display (Ishizawa et al., 2013; Kawakami et al., 2013), we developed an improved TRAP display for the quick in vitro selection of ALPs from a large library (>1013 theoretical diversity), from which we selected high-affinity monobodies (sub-nM KD) against the SARS-CoV-2 spike protein (Kondo et al., 2020). In the present article, we describe the preparation of a monobody mRNA library with >1013 diversity (Figures 1 and 2). We first divided the monobody gene into two fragments, one containing 8 or 10 random codons in the BC loop sequence (A-fragment) and the other containing 10 or 12 random codons in the FG loop sequence (B-fragment). As a random codon, we used a codon mix with the following ratios: 20% Tyr, 10% Ser, 15% Gly, 10% Trp, and 3% each of all remaining amino acids, with the exception of Cys, which was similar to the original cocktail (30% Tyr, 15% Ser, 10% Gly, 5% Phe, 5% Trp, and 2.5% each of all remaining amino acids, with the exception of Cys) (Wojcik et al., 2010; Koide et al., 2012). To prepare a template DNA for each of the two fragments, we ligated 2 to 3 oligonucleotides assembled by guide DNAs using the T4 DNA ligase. We added a modification at the 3 end of the guide DNAs to prevent them from becoming a primer in the following amplification step. After amplifying the product DNAs by large-scale PCR with minimum amplification cycles (15 ml, 7 cycles), these DNA fragments were digested with BsaI and ligated using T4 DNA ligase. After amplification of the DNA product by large-scale PCR (60 ml, 4 cycles), mRNAs were synthesized by T7 RNA polymerase. This procedure would be useful for the preparation of high-diversity mRNA libraries for various ALPs in the future.



Figure 1. Monobody library sequences. A. DNA sequence of the monobody library [T7 promoter, green; codon mix (20% Tyr, 10% Ser, 15% Gly, 10% Trp, and 3% each of all remaining amino acids, with the exception of Cys), yellow; an21 sequence (the 21-mer sequence for annealing to HEX-PuL), cyan]. B. Protein sequence of the monobody library (randomized residues X, yellow).



Figure 2. Scheme used for preparation of the monobody mRNA library.

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.

Amino acids Number of observed codons Observed frequency (%) Theoretical frequency (%)
Ala 25 3.3 3
Cys 0 0.0 0
Asp 26 3.4 3
Glu 31 4.1 3
Phe 22 2.9 3
Gly 98 12.8 15
His 39 5.1 3
Ile 22 2.9 3
Lys 17 2.2 3
Leu 16 2.1 3
Met 28 3.7 3
Asn 17 2.2 3
Pro 18 2.4 3
Gln 26 3.4 3
Arg 18 2.4 3
Ser 52 6.8 10
Thr 30 3.9 3
Val 52 6.8 3
Trp 65 8.5 10
Tyr 162 21.2 20
Stop 0 0.0 0

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 1st 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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  24. Zemlin, M., Klinger, M., Link, J., Zemlin, C., Bauer, K., Engler, J. A., Schroeder, H. W. and Kirkham, P. M. (2003). Expressed murine and human CDR-H3 intervals of equal length exhibit distinct repertoires that differ in their amino acid composition and predicted range of structures. J Mol Biol 334(4): 733-749.

简介

[摘要]最近,我们开发了结合嘌呤霉素接头 (TRAP) 显示的转录/翻译,作为获得抗体样蛋白的快速体外选择方法。对于体外选择,它以制备mRNA的文库是重要其中所述diversit ý是高的。在这里,我们描述了一种制备具有大于10 13理论多样性的单体 mRNA 文库的方法。首先,我们合成了两个,其相应于monobody DNA的片段长的单链DNA ,具有随机密码子中的BC和FG环。这些寡核苷酸在含有受修饰保护的 3' 末端的引导寡核苷酸的支持下通过 T4 DNA 连接酶连接。通过PCR扩增的DNA产物后,每个DNA片段的一端消化用的II型限制性内切酶的BSA我,并使用T4DNA连接酶将所得的DNA片段连接。DNA 产物扩增后,通过 T7 RNA 聚合酶合成 mRNA。该方法简单,可用于制备各种抗体样蛋白的mRNA文库。


图文摘要:

构建高度多样化的 mRNA 文库。

[背景]各种抗体样使用蛋白(ALPS)已经获得的体外选择方法和雇用在生物和医学领域ED (舒马赫等人,2018;和西麦陈2018) 。在此体外选择,初始文库构建是一个重要的步骤中获得高亲和力的结合剂。例如,为了开发纳米抗体,免疫后获得的骆驼抗体的 VHH 结构域用于构建初始文库(Arbabi Ghahroudi等,1997)。或者,来自原始基因或半合成或合成文库的 VHH 结构域可用作初始文库(Goldman等人,2006 年;Monegal等人,2009 年;Yan等人,2014 年)。

对于其他非免疫球蛋白支架,合成文库仅用作初始文库(Nord等,1997;Koide等,1998;Beste等,1999;Binz等,2003;Grabulovski等,2007 年;奥尔森等人,2008 年)。含有随机密码子的寡核苷酸通过延伸 PCR 组装并连接到其他 DNA 片段以合成DNA 文库。作为随机密码子,通常使用NNK密码子(N代表A、C、G或T;K代表T或G),因为它们覆盖了所有20个氨基酸并且可以减少密码子的冗余和终止密码子的频率。或者,使用三核苷酸亚磷酰胺的直接密码子合成(Virnekäs等,1994)用于模拟抗体 CDR3 序列中的氨基酸频率(Zemlin等,2003;Wojcik等,2010;Koide等,2012) 。

施工的中-大小d库(10 10多样性)比较简单,而那大-大小d库(10 13至10 14集),需要特别注意保持多样性(Olson和2007罗伯茨)。例如,当 PCR 10 个循环后得到 50 nM 的 DNA 产物时,原始模板的浓度可以计算为 50 pM。如果我们仅使用100μ升反应混合物,将格言嗯可以使用来实现分集条件小号上述被限制为3 × 10 9 。

最近,通过修改传统的 mRNA 展示(Nemoto等人,1997;Roberts 和 Szostak 1997)和原始版本的 TRAP 展示(Ishizawa等人,2013;Kawakami等人,2013),我们开发了改进的 TRAP 展示为了从大型文库(>10 13理论多样性)中快速体外选择 ALP ,我们从中选择了针对 SARS-CoV-2 刺突蛋白的高亲和力单体(sub-nM K D )(Kondo等人,2020)。在本文中,我们描述了具有 >10 13多样性的单体 mRNA 文库的制备(图 1 和 2)。我们首先将单体基因分成两个片段,一个在BC 环序列中包含 8 或 10 个随机密码子(A 片段),另一个在FG 环序列中包含 10 或 12 个随机密码子(B 片段)。作为随机密码子,我们使用了以下比例的密码子混合物:20% Tyr、10% Ser、15% Gly、10% Trp 和 3% 的所有剩余氨基酸,除了 Cys,它们是相似的到原始鸡尾酒(30% Tyr、15% Ser、10% Gly、5% Phe、5% Trp 和 2.5% 的所有剩余氨基酸,Cys 除外)(Wojcik等人,2010 年;Koide等人,2012 年)。为了为这两个片段中的每一个准备模板 DNA,我们使用 T4 DNA 连接酶连接了由引导 DNA 组装的2到3 个寡核苷酸。我们在引导 DNA的 3 末端添加了一个修饰,以防止它们在接下来的扩增步骤中成为引物。通过大规模PCR以最小的扩增循环(15扩增产物的DNA后米升,7个周期),这些DNA片段进行消化用BSA我并连接使用T4DNA连接酶。通过大规模 PC R(60 ml ,4 个循环)扩增 DNA 产物后,通过 T7 RNA 聚合酶合成 mRNA。该程序将有助于在未来为各种 ALP 准备高多样性 mRNA 文库。

图 1. Monobody 文库序列。一个。单体文库的DNA序列 [T7 启动子,绿色;密码子混合物(20% Tyr、10% Ser、15% Gly、10% Trp 和所有剩余氨基酸各 3%,Cys 除外),黄色;an21 序列(用于与HEX-PuL退火的 21 聚体序列),青色]。乙。单体文库的蛋白质序列(随机残基 X,黄色)。

˚Figure 2.计划用于制备monobody基因库

关键字:体外选择, 陷阱显示, mRNA文库, 抗体样蛋白, 单体

材料和试剂
 
1.寡核苷酸(序列和供应商列于表1)      
2. T4 DNA连接酶(New England Biolabs,目录号:M0202L)      
3. BSA I-HF ® (新英格兰生物实验室,目录号:R3535L)      
注:此公顷š被替换用新的版本(BSA I-HF V2 ® ,目录号:R3733L) 。
4.内部制备的Pfu-S DNA 聚合酶(Wang et al. , 2004; Kondo et al. , 2020)      
5.内部制备的 T7 RNA 聚合酶(Abramochkin 和 Shrader ,1995;Kondo等,2020)      
6. ExcelBand 100 - bp的DNA梯(š mobio Ť童占梅,目录号:DM2100)      
7. 0.5 mol/ L -EDTA 溶液(pH 8.0)(Nacalai T esque ,目录号:14347-21)      
8.甲酰胺(Nacalai T esque ,目录号:16229-95)      
9.甘油(Nacalai Tesque ,目录号:17018-25)      
10. 40(w/v)%-丙烯酰胺/双混合溶液(37.5:1)(Nacalai Tesque ,目录号:06121-95)   
11.三羟甲基氨基甲烷(Tris)(Nacalai Tesque ,目录号:35406-75)   
12.聚乙二醇6000(PEG 6000)(FUJIFILM Wako Pure Chemicals,目录号:169-22945)   
13. 1,4 -二硫苏糖醇(DTT)(ナTesque公司,目录号:14128-62)   
14.六水氯化镁(MgCl 2 )(Nacalai Tesque ,目录号:20908-65)   
15.氯化钾(K Cl)(Nacalai Tesque ,目录号:28514-75   
16.氯化钠(NaCl)(Nacalai Tesque ,目录号:31320-05)   
17.聚乙二醇单对异辛基苯基醚(Triton X-100)(Nacalai Tesque ,目录号:12967-45)   
18.二甲基亚砜(DMSO)(Nacalai Tesque ,目录号:13407-03)   
19.七水硫酸镁(Nacalai Tesque ,目录号:06296-25)   
20.亚精胺三盐酸盐(Nacalai Tesque ,目录号: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,目录号:019-09672)   
24.苯酚:氯仿:异戊醇25:24:1混合,pH 6.7(Nacalai Tesque ,目录号:25967-74)   
25.氯仿(Nacalai Tesque ,目录号:08402-55)   
26. 2-丙醇(Nacalai Tesque ,目录号:29113-95)   
27.乙醇(EtOH)(Nacalai Tesque ,目录号:14713-95)   
28. 2 ×连接溶液 [100 mM Tris-HCl pH 7.5、12 mM MgCl 2 、1 mM ATP、16% PEG 6000、20 mM DTT 和 4,000 U/ml T4 DNA 连接酶]   
29. 1 × PCR 溶液 [10 mM Tris-HCl pH 8.4, 100 mM K Cl, 0.1% (v/v) Triton X-100, 2% (v/v) DMSO, 2 mM MgSO 4 , 0.2 mM 每个 dNTP , 0.375 μM 每个引物和 2 nM Pfu-S DNA 聚合酶]   
30. 1 × RNA聚合酶溶液[40毫摩尔的Tris-HCl pH 8.0中,1mM的亚精胺,0.01%(V / V)的Triton X-100,10mM的DTT,25毫摩尔MgCl 2 ,5mM的各NTP,和1μMT7 RNA聚合酶]   
31. DNA 上样缓冲液 [ 80 mM Tris-醋酸盐 pH 8.0,2 mM EDTA,20% (v/v) 甘油]   
 
表1 。寡核苷酸序列和供应商。NNK 密码子通过三核苷酸亚磷酰胺合成制备(20% Tyr、10% Ser、15% Gly、10% Trp 和 3% 的所有剩余氨基酸,Cys 除外)。缩写:SPC18,垫片18;Pu,嘌呤霉素;HEX,六氯荧光素。
 
设备
 
T100热循环仪(Bio-Rad,目录号:1861096J1 )
Gel Doc EZ(Bio-Rad,目录号:1708270 )
Mini-PROTEAN 系统(Bio-Rad,目录号:1658000FC )
冷却板电泳装置(BIO CRAFT,目录号:BE130G )
 
程序
 
单体文库A片段 DNA 的制备
制备以下溶液(35 μl),95°C加热2分钟,室温放置5分钟:
FN3F0.F83(2 μM 终浓度)、FN3F1-2.F29(P)(2 μM 终浓度)、FN3 FF1coR8.F73(1 μM 终浓度)、FN3FF1coR10.F79(1 μM 终浓度)、Fn3an1.R20( 3NH2)(4 μM 终浓度)和 Fn3an2-1.R20(3NH2)(4 μM 终浓度)在10 mM Tris-HCl pH 7.5 中。
将 2 ×连接溶液 (35 μl) 添加到 (1) 中描述的溶液中,并在 25°C 下孵育 1 小时(连接反应分析见图 3A )。
将混合物加入 1× PCR溶液(15毫升)和分配77微升所得的溶液加入每个孔中的2 96 -Well PCR板。在以下条件下使用 T7SD8M2.F44 和 FN3BsaI.R40 扩增 DNA :在 94°C 下进行 1 分钟的初始激活步骤,然后在 94°C 下进行 7 个循环热变性 20 秒,在 55°C 下进行引物退火 20 s,以及在72°C 下引物延伸45 秒(参见图 3B 以优化热循环次数)。
添加 3 M NaCl (1.5 ml )。
将 3 ml 苯酚:氯仿:异戊醇 (25:24:1) 与所得溶液混合,并在室温下以 16,000 × g离心 5分钟。
将 6 ml 氯仿与水层混合,在室温下以 16,000 × g离心 5分钟。
将 16 ml 2-丙醇与水层混合,并在室温下以 16,000 × g离心 15分钟。
取出后的上清液,在室温下加入1ml 70%乙醇,离心5分钟,在16000×克。
取出后的上清液,干燥沉淀,并在0.5ml的10mM Tris-乙酸pH值溶解7.8。
使用ExcelBand 100 - bp DNA Ladder通过 12% 聚丙烯酰胺凝胶电泳确定 DNA 浓度。
 
单体文库 B 片段 DNA 的制备
制备以下溶液(35 μl),95°C加热2分钟,室温放置5分钟:
FN3FF2co.F72(p)(2 μM 终浓度)、FN3F3coR10.F70(p)(1 μM 终浓度)、FN3F3coR12.F76(p)(1 μM 终浓度)和 Fn3an3.R20(3NH2)(4 μM 终浓度)浓度)在10 mM Tris-HCl pH 7.5 中。
将 2 ×连接溶液 (35 μl) 添加到 (1) 中描述的溶液中,并在 25°C 下孵育 1 小时(连接反应分析见图 3A )。
该混合物添加到1 × PCR溶液(15毫升)和分配77微升所得的溶液加入每个孔中的2 96 -Well PCR板。在以下条件下使用 FN3BsaI.F33 和 FN3Pri2.R44 扩增 DNA :在 94°C 下进行 1 分钟的初始激活步骤,然后在 94°C 下进行 7 个循环热变性 20 秒,在 55°C 下进行引物退火 20 s,以及在72°C 下引物延伸45 秒(参见图 3B 以优化热循环次数)。
添加 3 M NaCl (1.5 ml )。
将 3 ml 苯酚:氯仿:异戊醇 (25:24:1) 与所得溶液混合,并在室温下以 16,000 × g离心 5分钟。
将 6 ml 氯仿与水层混合,在室温下以 16,000 × g离心 5分钟。
将 16 ml 2-丙醇与水层混合,并在室温下以 16,000 × g离心 15分钟。
取出后的上清液,在室温下加入1ml 70%乙醇,离心5分钟,在16000×克。
取出后的上清液,干燥沉淀,并在0.5ml的10mM Tris-乙酸pH值溶解7.8。
使用ExcelBand 100 - bp DNA Ladder通过12% 聚丙烯酰胺凝胶电泳确定 DNA 浓度。
 
用Bsa I消化 A 片段和 B 片段 DNA
孵育反应混合物(1 × CutSmart ®缓冲液,2μMA-片段或B片段,200个单位/ ml BSA I-HF ® ;在一个总的400微升),在37℃下进行4 d。
添加 3 M NaCl (40 µl )。
将 100 µl 苯酚:氯仿:异戊醇 (25:24:1) 与所得溶液混合,并在室温下以 16,000 × g离心 5分钟。
将 200 µl 氯仿与水层混合,并在室温下以 16,000 × g离心 5分钟。
将 400 µl 2-丙醇与水层混合,并在室温下以 16,000 × g离心 15分钟。
取出后的上清液,在室温下添加100微升70%乙醇,离心5分钟,在16000×克。
取出后的上清液,干燥沉淀,并在10微升溶解的10mM Tris-乙酸pH值7.8。
将 10 µl 所得溶液与 10 µl DNA 上样缓冲液混合。
受试者的溶液至12%的聚丙烯酰胺凝胶电泳(10厘米× 7.3厘米× 0.75毫米,300V,20分钟)。
从凝胶上切下条带后,使用 0.3 M NaCl 溶液 (1.2 ml) 洗脱 DNA。
将 1.2 ml 2-丙醇与溶液混合,并在室温下以 16,000 × g离心 15 分钟。
取出后的上清液,以16,000×在室温下添加100微升70%乙醇,离心5分钟克。
取出后的上清液,干燥沉淀,并在100微升溶解的10mM Tris-乙酸pH值7.8。
使用ExcelBand 100 bp DNA Ladder通过12% 聚丙烯酰胺凝胶电泳确定 DNA 浓度。
 
连接消化后的 A 片段和 B 片段 DNA
中号IX含有2μM与2各自A-片段和B片段的DNA(100微升)溶液×连接溶液(100微升)。
将所得混合物在 25°C 下孵育 1 小时(参见图 3C 以分析结扎反应)。
 
DNA片段的PCR扩增
添加混合物至1 × PCR溶液(60毫升)和分配77微升所得的溶液加入每个孔中的8 96 -Well PCR板。
在以下条件下使用 T7SD8M2.F44 和 G5S-4Gan21-3.R42 扩增 DNA 片段:在 94°C 下进行 1 分钟的初始激活步骤,然后在 94°C 下进行四次热变性 20 秒,引物退火在55°C 20 秒,72°C 引物延伸 45 秒(参见图 3D 以优化热循环次数)。
添加 3 M NaCl (6 ml )。
将 8 ml 苯酚:氯仿:异戊醇 (25:24:1) 与所得溶液混合,并在室温下以 16,000 × g离心 5分钟。
将 16 ml 氯仿与水层混合,并在室温下以 16,000 × g离心 5分钟。
将 66 ml 2-丙醇与水层混合,并在室温下以 16,000 × g离心 15分钟。
取出后的上清液,在室温下加入1ml 70%乙醇,离心5分钟,在16000×克。
取出后的上清液,干燥沉淀,并在6毫升的10mM Tris-乙酸pH值溶解7.8。
使用ExcelBand 100 - bp DNA Ladder通过 12% 聚丙烯酰胺凝胶电泳确定 DNA 浓度。
 
制备的基因库
孵育一混合物的1 × RNA聚合酶溶液和50nM扩增的DNA; 总共 11 毫升)在 37°C 下放置 6 小时。
添加 0.55 ml 0.5 M EDTA pH 8.0 和 1.2 ml 3 M NaCl。
将 2 ml 苯酚:氯仿:异戊醇 (25:24:1) 与所得溶液混合,并在室温下以 16,000 × g离心 5分钟。
将 4 ml 氯仿与水层混合,并在室温下以 16,000 × g离心 5分钟。
将 9 ml 2-丙醇与水层混合,并在室温下以 16,000 × g离心 15分钟。
取出后的上清液,在室温下加入1ml 70%乙醇,离心5分钟,在16000×克。
取出后的上清液,干燥沉淀,并在0.5ml超纯水溶解。
将 20 µl 所得溶液与 150 µl 甲酰胺混合。
受试者的溶液至6M尿素5%聚丙烯酰胺凝胶电泳(13.8厘米× 12厘米× 2毫米,40 W,40分钟)。
从凝胶上切下条带后,使用 0.3 M NaCl 50% 甲酰胺溶液(18 ml)洗脱 DNA。
将 4 ml 苯酚:氯仿:异戊醇 (25:24:1) 与所得溶液混合,并在室温下以 16,000 × g离心 5分钟。
将 8 ml 氯仿与水层混合,在室温下以 16,000 × g离心 5分钟。
将 18 ml 2-丙醇与水层混合,并在室温下以 16,000 × g离心 15分钟
取出后的上清液,在室温下加入1ml 70%乙醇,离心5分钟,在16000×克。
取出后的上清液,干燥沉淀,并在20μl超纯水溶解。
通过测量260 nm 处的OD确定 mRNA 浓度。储存在- 80°C。
 
mRNA/HEX-PuL 复合物的制备
在第一轮选择之前,将溶液(25 mM HEPES-K pH 7.8、200 mM 醋酸钾、4 - 8 μM RNA 和 4 μM HEX-PuL;总共 80 μl)在 95°C 下用于2 分钟,然后冷却至 25°C。使用生成的复合体进行第一轮选择。
 
 
在每一过程中的反应的图3. PAGE分析。一个。期间连接寡核苷酸P rocedures A和B.乙。在热循环的数目的优化P rocedures A和10进行反应B.微升溶液。Ç 。所述的结扎BSA中和I处理的A-B-DNA片段P rocedure D. d 。在热循环的数目的优化P rocedure E.将反应物在10执行微升溶液。
 
数据分析
 
对DNA文库中的44个克隆进行克隆,并通过Sanger测序分析序列。在主链序列中观察到14 个单碱基缺失和 19 个单碱基插入,这可能是用于构建 DNA 模板的寡核苷酸的副产物。结果,在 44 个克隆中的 19 个中保持了阅读框。随机序列中的密码子数量总结在表2 中。观察到的频率与理论频率显示出良好的一致性。
表2 。的密码子的分析在44个克隆的BC和FG环的序列
 
笔记
 
ssDNA、dsDNA 和 m RNA 文库的理论多样性计算如下:
#在A-片段P rocedure答:1个μMoligonucle otides(75微升尺度); 75 pmol, 4.5 × 10 13分子
#B-片段在P rocedure B:1点μM的寡核苷酸(75微升尺度); 75 pmol, 4.5 × 10 13分子
#在A-片段P rocedure答:7个循环(15毫升刻度)后为43nm的dsDNA产物; 0.64 nmol, 3.9 × 10 14分子, 0.6 × 10 13多样性64 个拷贝
#在B-片段P rocedure B:38 nM的双链DNA 7个周期(15毫升刻度)后产物; 0.57 nmol, 3.4 × 10 14分子,0.5 × 10 13多样性,64个拷贝(第一次循环仅合成互补DNA )
#双链DNA产物在P roced URE d:1μM的dsDNA(200微升尺度); 200 pmol, 1.2 × 10 14分子
#在双链DNA产物P rocedure E:4个周期(60毫升刻度),2.0纳摩尔后33 nM的双链DNA产物,1.2 × 10 15个分子,7.5 × 10 13与16份多样性
#的mRNA产物在P rocedure ˚F :50nM的DNA模板(11毫升规模); 0.55纳摩尔,3.3 × 10 14分子,分集被限制P rocedure E.
 
致谢
 
这项工作是由格兰特在急救的科学研究(A)支持(一般)授权号15H02006到HM],格兰特在急救科学研究的创新领域[授权号20H04704到GH] ,和格兰特,在- 来自日本学术振兴会的早期职业科学家援助 [授予编号 18K14332 到 TF];以及村上浩博士的捐赠。该协议源自我们之前的研究论文(Kondo等人,2020 年)。
 
利益争夺
 
作者声明没有竞争利益。
 
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Kondo, T., Eguchi, M., Tsuzuki, N., Murata, N., Fujino, T., Hayashi, G. and Murakami, H. (2021). Construction of a Highly Diverse mRNA Library for in vitro Selection of Monobodies. Bio-protocol 11(16): e4125. DOI: 10.21769/BioProtoc.4125.
  2. Kondo, T., Iwatani, Y., Matsuoka, K., Fujino, T., Umemoto, S., Yokomaku, Y., Ishizaki, K., Kito, S., Sezaki, T., Hayashi, G. and Murakami, H. (2020). Antibody-like proteins that capture and neutralize SARS-CoV-2.Sci Adv 6(42).
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