北京生物醫(yī)學(xué)工程

一種提高變體庫多樣性的寡核苷酸設(shè)計方法

Oligonucleotide design for improving the diversity of library of variants

作者：唐瑞鄭浩然

單位：中國科學(xué)技術(shù)大學(xué)計算機科學(xué)與技術(shù)學(xué)院（合肥 230027）<br />通信作者：鄭浩然,副教授。E-mail: [email protected]

分類號：R318

出版年·卷·期（頁碼）：2022·41·4（338-343）

摘要：

目的基于定向進化的新特性蛋白質(zhì)合成研究的關(guān)鍵在于基因變體庫的生成以及目標(biāo)蛋白的篩選，而目標(biāo)蛋白篩選的成功率取決于基因變體庫的多樣性，針對采用從頭寡核苷酸合成策略的基于基因芯片技術(shù)的基因變體庫合成生物技術(shù)，本文研究并設(shè)計一種旨在提高基因變體庫多樣性的寡核苷酸設(shè)計方法。方法首先采用了逆翻譯算法提高基因序列的一致性，并利用在高同源區(qū)域的片段切割來提高基因合成過程中的重組率，進而提高基因變體庫的多樣性。結(jié)果與DNAWorks和TmPrime設(shè)計后的基因序列相比，我們的方法能夠達到更高的一致性，同時基因片段切割結(jié)果顯示我們能夠使切割后的前后片段末端具有非常高的相似度。最后設(shè)計的寡核苷酸經(jīng)過基因芯片合成篩選得到了目標(biāo)蛋白質(zhì)。結(jié)論本文提出的寡核苷酸設(shè)計方法充分利用了DNA序列間的基因重組，設(shè)計出的寡核苷酸序列經(jīng)過基因芯片的合成證實了可行性，說明了本文提出的寡核苷酸設(shè)計算法能夠有效地提高基因變體庫的多樣性。

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.

參考文獻：

[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.

服務(wù)與反饋：

【文章下載】【加入收藏】

提示：您還未登錄，請登錄！點此登錄

51黑料吃瓜在线观看,51黑料官网|51黑料捷克街头搭讪_51黑料入口最新视频