Birkmose Nanna, Frydendahl Emilie U, Knudsen Charlotte R
Department of Molecular Biology and Genetics, Aarhus University, Universitetsbyen 81, Aarhus C DK-8000, Denmark.
Biochemistry. 2024 Dec 17;63(24):3273-3286. doi: 10.1021/acs.biochem.4c00013. Epub 2024 Dec 6.
DNA-encoded libraries hold great potential for discovering small, cyclized peptides with drug potential. Split-intein circular ligation of peptides and proteins (SICLOPPS) is a well-established method for selection of cyclic peptides targeting specific intracellular components. However, the method has mainly been used in prokaryotic cells. In contrast, selection studies performed directly in eukaryotic cells allow for the identification of cyclic peptides promoting a functional outcome, without the need to define a specific cellular target. Here, we report the construction of a -specific SICLOPPS library of 80 million members, via careful optimization of several steps to increase the size of the library. Individual library members were shown to be correctly expressed and processed in yeast. High-throughput sequencing was conducted on the randomized primer used for library construction and the pure yeast SICLOPPS library isolated from . A distinct guanine insertion bias was observed in the peptide-encoding, randomized sequence, which was primarily attributed to the degenerate primer used to introduce the randomized sequence. Moreover, high-throughput sequencing was performed on the library before and after the induction of cyclic peptide expression in yeast. Importantly, expression of the SICLOPPS library in caused only a marginal further sequence bias. Our work paves the way for selection studies using a large and diverse library to identify cyclic peptides of therapeutic interest that promote a specific phenotypic outcome in eukaryotic organisms, with yeast representing a beneficial model system due to its high transformation efficiency.
DNA编码文库在发现具有药物潜力的小分子环化肽方面具有巨大潜力。肽与蛋白质的分裂内含子环化连接（SICLOPPS）是一种成熟的用于筛选靶向特定细胞内成分的环化肽的方法。然而，该方法主要用于原核细胞。相比之下，直接在真核细胞中进行的筛选研究能够鉴定出促进功能结果的环化肽，而无需确定特定的细胞靶点。在此，我们报告通过仔细优化几个步骤以增加文库规模，构建了一个包含8000万个成员的特异性SICLOPPS文库。单个文库成员在酵母中被证明能够正确表达和加工。对用于文库构建的随机引物以及从酵母中分离出的纯SICLOPPS文库进行了高通量测序。在编码肽的随机序列中观察到明显的鸟嘌呤插入偏向，这主要归因于用于引入随机序列的简并引物。此外，在酵母中诱导环化肽表达前后对文库进行了高通量测序。重要的是，SICLOPPS文库在酵母中的表达仅引起轻微的进一步序列偏向。我们的工作为利用庞大且多样的文库进行筛选研究铺平了道路，以鉴定在真核生物中促进特定表型结果的具有治疗意义的环化肽，酵母因其高转化效率而成为一个有益的模型系统。
