Department of Chemistry, University of Georgia, 140 Cedar Street, Athens, GA, 30602, USA.
Department of Chemistry, Centre for Research on Biomolecular Interactions, York University, 4700 Keele Street, Toronto, ON, M3J 1P3, Canada.
Chembiochem. 2019 Mar 15;20(6):793-799. doi: 10.1002/cbic.201800616. Epub 2019 Feb 5.
Ligase-catalyzed oligonucleotide polymerization (LOOPER) that enables the sequence-defined generation of DNA with up to 16 different modifications has recently been developed. This approach was used to develop new classes of diversely modified DNA aptamers for molecular recognition through in vitro evolution. The modifications in LOOPER are appended by use of a long hexane-1,6-diamine linker, which could negatively impact binding thermodynamics. Here we explore the incorporation of modifications with the aid of shorter linkers and the use of commercially available phosphoramidites and assess their efficiency and fidelity of incorporation. We observed that shorter linkers are less tolerated during LOOPER, with very short linkers providing high levels of error and sequence bias. An ethane-1,2-diamine linker was found to be optimal in terms of yield, efficiency, and bias; however, codon adjustment was necessary. This shorter-linker anticodon set for LOOPER should prove valuable in exploring the impact of diverse chemical modifications on the molecular function of DNA.
最近开发了一种通过连接酶催化的寡核苷酸聚合（LOOPER）技术，可以序列定义地生成多达 16 种不同修饰的 DNA。该方法被用于通过体外进化开发用于分子识别的新型多样化修饰 DNA 适体。LOOPER 中的修饰是通过使用长的己烷-1,6-二胺连接子来附加的，这可能会对结合热力学产生负面影响。在这里，我们探索了在较短的连接子的辅助下进行修饰的掺入，并评估了它们的掺入效率和保真度。我们观察到，在 LOOPER 中较短的连接子不太耐受，非常短的连接子会导致高水平的错误和序列偏向。发现乙烷-1,2-二胺连接子在产率、效率和偏向方面是最佳的；然而，需要进行密码子调整。对于 LOOPER，这种较短连接子的反密码子集应该有助于探索各种化学修饰对 DNA 分子功能的影响。
