DNA Ligase-Mediated Translation of DNA Into Densely Functionalized Nucleic Acid Polymers

Abstract

We developed a method to translate DNA sequences into densely functionalized nucleic acids by using T4 DNA ligase to mediate the DNA-templated polymerization of 5′-phosphorylated trinucleotides containing a wide variety of appended functional groups. This polymerization proceeds sequence specifically along a DNA template and can generate polymers of at least 50 building blocks (150 nucleotides) in length with remarkable efficiency. The resulting single-stranded highly modified nucleic acid is a suitable template for primer extension using deep vent (exo-) DNA polymerase, thereby enabling the regeneration of template DNA. We integrated these capabilities to perform iterated cycles of in vitro translation, selection, and template regeneration on libraries of modified nucleic acid polymers.

Flow-cell based technology for massively parallel characterization of base-modified DNA aptamers

“…Second, during the paired-end turnaround step, C8-alkyne-dUTP is substituted for native dTTP during bridge PCR using the KOD-XL polymerase, which can incorporate this modification with high fidelity (step 2). 19,20 This produces aptamer clusters with alkyne click handles on the flow-cell, which are compatible with post-synthesis modification via a click chemistry reaction. 8,9 This enables us to modify every T with virtually any chemical functional group that we wish to incorporate.…”

Generation of Synthetic Copolymer Libraries by Combinatorial Assembly on Nucleic Acid Templates

“…86 The approach relies upon T4 DNA ligase to catalyze DNA-templated phosphodiester bond formation between adjacent 5′-phosphorylated trinucleotides adorned with functional groups attached to the nucleobase (Figure 14a). This method effectively enables the incorporation of a much larger number of modifications, theoretically up to 64 in a trinucleotide codon set, that would not be possible using single nucleotide incorporation.…”

Towards applications of synthetic genetic polymers in diagnosis and therapy

“…Notable advances from David Liu's lab include a system for the templated replication and evolution of substituent bearing nucleotide triplets, potentially allowing for a ''code'' of up to 64 distinct chemical groups [52]. Indeed, in a separate publication, the same group showed that templated replication need not be limited to polymers in the chemical vicinity of nucleic acids [53 ].…”