设为首页 |  加入收藏
首页首页 期刊简介 消息通知 编委会 电子期刊 投稿须知 广告合作 联系我们
一种提高变体库多样性的寡核苷酸设计方法

Oligonucleotide design for improving the diversity of library of variants

作者: 唐瑞  郑浩然 
单位:中国科学技术大学计算机科学与技术学院(合肥 230027)<br />通信作者:郑浩然,副教授。E-mail: hrzheng@ustc.edu.cn
关键词: 基因变体库;定向进化;寡核苷酸;基因重组;同源性 
分类号:R318
出版年·卷·期(页码):2022·41·4(338-343)
摘要:

目的 基于定向进化的新特性蛋白质合成研究的关键在于基因变体库的生成以及目标蛋白的筛选,而目标蛋白筛选的成功率取决于基因变体库的多样性,针对采用从头寡核苷酸合成策略的基于基因芯片技术的基因变体库合成生物技术,本文研究并设计一种旨在提高基因变体库多样性的寡核苷酸设计方法。方法 首先采用了逆翻译算法提高基因序列的一致性,并利用在高同源区域的片段切割来提高基因合成过程中的重组率,进而提高基因变体库的多样性。结果 与DNAWorks和TmPrime设计后的基因序列相比,我们的方法能够达到更高的一致性,同时基因片段切割结果显示我们能够使切割后的前后片段末端具有非常高的相似度。最后设计的寡核苷酸经过基因芯片合成筛选得到了目标蛋白质。结论 本文提出的寡核苷酸设计方法充分利用了DNA序列间的基因重组,设计出的寡核苷酸序列经过基因芯片的合成证实了可行性,说明了本文提出的寡核苷酸设计算法能够有效地提高基因变体库的多样性。

Objective The key to the research of synthesis of protein with new characteristics based on directed evolution lies in the generation of library of variants and the screening of target proteins. The success rate of target protein screening depends on the diversity of library of variants. Therefore, for the library of variants synthetic biotechnology based on de novo oligonucleotide synthesis strategy and gene chip technology, we design and develop an oligonucleotide design tool to improve the diversity of library of variants. Methods We first apply the reverse translation algorithm to improve the identity of the gene sequences. And use the fragment cutting in the highly homologous region to improve the recombination rate in the process of gene synthesis, thereby increasing the diversity of library of variants. Results Compared with the gene sequences designed by DNAWorks and TmPrime, our method can achieve higher identity. At the same time, the long gene sequences cutting results show that we can make the ends of the fragments after the cutting process have a very high similarity. Finally, the designed oligonucleotides were synthesized and screened by gene chip to obtain the target protein. Conclusions The oligonucleotide design method proposed in this paper makes full use of the genetic recombination between DNA sequences. The designed oligonucleotide sequences have been synthesized by gene chip which verifies the feasibility of our method and illustrates the effectively of increasing the diversity of library of variants.

参考文献:

[1] Porter JL, Rusli RA, Ollis DL. Directed evolution of enzymes for industrial biocatalysis[J]. ChemBioChem, 2016, 17(3): 197-203.
[2] Kumar A, Singh S. Directed evolution: tailoring biocatalysts for industrial applications[J]. Critical Reviews in Biotechnology, 2013, 33(4): 365-378.
[3] Turner NJ. Directed evolution of enzymes for applied biocatalysis[J]. Trends in Biotechnology, 2003, 21(11): 474-478.
[4] Copp JN, Hanson-Manful P, Ackerley DF, et al. Error-prone PCR and effective generation of gene variant libraries for directed evolution[M]//Directed Evolution Library Creation. Methods in Molecular Biology (Methods and Protocols). New York: Springer, New York, NY, 2014,1179: 3-22.
[5] Neylon C. Chemical and biochemical strategies for the randomization of protein encoding DNA sequences: library construction methods for directed evolution[J]. Nucleic Acids Research, 2004, 32(4): 1448-1459.
[6] Stemmer WPC. Rapid evolution of a protein in vitro by DNA shuffling[J]. Nature, 1994, 370(6488): 389-391.
[7] Baweja M, Nain L, Kawarabayasi Y, et al. Current technological improvements in enzymes toward their biotechnological applications[J]. Frontiers in Microbiology, 2016, 7: 965.
[8] Joern JM, Meinhold P, Arnold FH. Analysis of shuffled gene libraries[J]. Journal of Molecular Biology, 2002, 316(3): 643-656.
[9] Acevedo-Rocha CG, Reetz MT. Assembly of designed oligonucleotides: a useful tool in synthetic biology for creating high-quality combinatorial DNA libraries[M]//Directed Evolution Library Creation. Methods in Molecular Biology (Methods and Protocols). New York: Springer, New York, NY, 2014,1179: 189-206.
[10] Ness JE, Kim S, Gottman A, et al. Synthetic shuffling expands functional protein diversity by allowing amino acids to recombine independently[J]. Nature Biotechnology, 2002, 20(12): 1251-1255.
[11] Bessette PH, Mena MA, Nguyen AW, et al. Construction of designed protein libraries using gene assembly mutagenesis[M]// Directed Evolution Library Creation. Methods in Molecular Biology. Clifton, NJ: Humana Press, 2003,231: 29-37.
[12] Zha D, Eipper A, Reetz MT. Assembly of designed oligonucleotides as an efficient method for gene recombination: a new tool in directed evolution[J]. ChemBioChem, 2003, 4(1): 34-39.
[13] Kosuri S, Eroshenko N, Leproust EM, et al. Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips[J]. Nature Biotechnology, 2010, 28(12): 1295-1299.
[14] Baker M. Microarrays, megasynthesis[J]. Nature Methods, 2011, 8(6): 457-460.
[15] Wan W, Li L, Xu Q, et al. Error removal in microchip-synthesized DNA using immobilized MutS[J]. Nucleic acids research, 2014, 42(12): e102.
[16] Wan W, Lu M, Wang D, et al. High-fidelity de novo synthesis of pathways using microchip-synthesized oligonucleotides and general molecular biology equipment[J]. Scientific Reports, 2017, 7: 6119.
[17] Hoover DM, Lubkowski J. DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis[J]. Nucleic Acids Research, 2002, 30(10): e43.
[18] Bode M, Khor S, Ye H, et al. TmPrime: fast, flexible oligonucleotide design software for gene synthesis[J]. Nucleic Acids Research, 2009, 37: W214-W221.
[19] Hogeweg P, Hesper B. The alignment of sets of sequences and the construction of phyletic trees: an integrated method[J]. Journal of Molecular Evolution, 1984, 20: 175-186.
[20] Tamaki FK. Directed evolution of enzymes[J]. Emerging Topics in Life Sciences, 2020, 4(2): 119-127.

服务与反馈:
文章下载】【加入收藏
提示:您还未登录,请登录!点此登录
 
友情链接  
地址:北京安定门外安贞医院内北京生物医学工程编辑部
电话:010-64456508  传真:010-64456661
电子邮箱:llbl910219@126.com