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Compositional Bias in Naïve and Chemically-modified Phage-Displayed Libraries uncovered by Paired-end Deep Sequencing.
Scientific Reports ( IF 4.6 ) Pub Date : 2018-01-19 , DOI: 10.1038/s41598-018-19439-2
Bifang He , Katrina F. Tjhung , Nicholas J. Bennett , Ying Chou , Andrea Rau , Jian Huang , Ratmir Derda

Understanding the composition of a genetically-encoded (GE) library is instrumental to the success of ligand discovery. In this manuscript, we investigate the bias in GE-libraries of linear, macrocyclic and chemically post-translationally modified (cPTM) tetrapeptides displayed on the M13KE platform, which are produced via trinucleotide cassette synthesis (19 codons) and NNK-randomized codon. Differential enrichment of synthetic DNA {S}, ligated vector {L} (extension and ligation of synthetic DNA into the vector), naïve libraries {N} (transformation of the ligated vector into the bacteria followed by expression of the library for 4.5 hours to yield a "naïve" library), and libraries chemically modified by aldehyde ligation and cysteine macrocyclization {M} characterized by paired-end deep sequencing, detected a significant drop in diversity in {L} → {N}, but only a minor compositional difference in {S} → {L} and {N} → {M}. Libraries expressed at the N-terminus of phage protein pIII censored positively charged amino acids Arg and Lys; libraries expressed between pIII domains N1 and N2 overcame Arg/Lys-censorship but introduced new bias towards Gly and Ser. Interrogation of biases arising from cPTM by aldehyde ligation and cysteine macrocyclization unveiled censorship of sequences with Ser/Phe. Analogous analysis can be used to explore library diversity in new display platforms and optimize cPTM of these libraries.

中文翻译:

配对末端深度测序发现的天真和经化学修饰的噬菌体展示库中的成分偏倚。

了解基因编码(GE)库的组成对于配体发现的成功至关重要。在本手稿中,我们研究了M13KE平台上展示的线性,大环和化学翻译后修饰(cPTM)四肽在GE库中的偏倚,这些肽是通过三核苷酸盒体合成(19个密码子)和NNK随机密码子产生的。合成DNA {S}的差异富集,连接的载体{L}(合成DNA的延伸和连接),未加工的文库{N}(将连接的载体转化为细菌,然后在4.5小时内表达该文库)产生一个“天真的”文库),并通过醛连接和半胱氨酸大环化{M}化学修饰的文库,其特征在于双末端深度测序,检测到{L}→{N}的多样性显着下降,但是{S}→{L}和{N}→{M}的成分差异很小。在噬菌体蛋白pIII的N末端表达的文库可检查带正电的氨基酸Arg和Lys。pIII结构域N1和N2之间表达的文库克服了Arg / Lys审查,但引入了对Gly和Ser的新偏向。通过醛连接和半胱氨酸大环化对cPTM产生的偏倚的询问揭示了对Ser / Phe序列的审查。可以使用类比分析来探索新显示平台中的库多样性,并优化这些库的cPTM。pIII结构域N1和N2之间表达的文库克服了Arg / Lys审查,但引入了对Gly和Ser的新偏向。通过醛连接和半胱氨酸大环化对cPTM产生的偏倚的询问揭示了对Ser / Phe序列的审查。可以使用类比分析来探索新显示平台中的库多样性,并优化这些库的cPTM。pIII结构域N1和N2之间表达的文库克服了Arg / Lys审查,但引入了对Gly和Ser的新偏向。通过醛连接和半胱氨酸大环化对cPTM产生的偏倚的询问揭示了对Ser / Phe序列的审查。可以使用类比分析来探索新显示平台中的库多样性,并优化这些库的cPTM。
更新日期:2018-01-19
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