Influence of Linker Length on Ligase-Catalyzed Oligonucleotide Polymerization

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.