Sequence-Defined Scaffolding of Peptides on Nucleic Acid Polymers

Abstract

We have developed a method for the T4 DNA ligase-catalyzed DNA-templated polymerization of 5'-phosphorylated pentanucleotides containing peptide fragments. The polymerization proceeds sequence-specifically to generate DNA-scaffolded peptides in excellent yields. The method has been shown to tolerate peptides ranging from two to eight amino acids in length with a wide variety of functionality. We validated the capabilities of this system in a mock selection for the enrichment of a His-tagged DNA-scaffolded peptide phenotype from a library, which exhibited a 190-fold enrichment after one round of selection. This strategy demonstrates a promising new approach to allowing the generation and in vitro selection of high-affinity reagents based upon single-stranded DNA scaffolding of peptide fragments.

Enzymatic Synthesis of Sequence‐Defined Synthetic Nucleic Acid Polymers with Diverse Functional Groups

“…1 m m ATP resulted in an error rate of 4.7 %, which gradually increased to 5.9 % with decreasing ATP concentrations to 25 μ m (Supporting Information, Table S2); however, 25 μ m ATP was superior with respect to polymerization yield and codon bias. Increased efficiency of polymerization at lower ATP concentrations are likely the result of decreased inhibition of DNA binding and the minimization of an over‐adenylated system …”

“…The modifications were incorporated through amide bond formation between the hexylamine appended to the C8 position of adenosine and various carboxylic acid derivatives (Figure , inset). This modification site has been previously shown to be the most permissive for this system . Duplex DNA sequencing was performed on the products of LOOPER for each monofunctionalized library, and the yields, biases, and fidelities for each library are reported in Table .…”

Influence of Linker Length on Ligase‐Catalyzed Oligonucleotide Polymerization

“…Expansion beyond four modifications requires an alternative strategy that does not rely on polymerase‐mediated incorporation. To this end, Ligase‐catalyzed OligOnucleotide PolymERization (LOOPER) permits the expansion of chemical diversity in DNA beyond four different modifications (Scheme ) . LOOPER involves the copolymerization, catalyzed by T4 DNA ligase, of a library of modified oligonucleotides along a library of DNA templates.…”

Structure–activity relationships of the ATP cofactor in ligase-catalysed oligonucleotide polymerisations

“…15 Recently, our lab developed Ligase-catalysed OligOnucleotide PolymERisation (LOOPER), which enables the sequence-defined incorporation of multiple unique instances of functional groups along a single-stranded DNA (ssDNA) polymer (Scheme 1). 16–18 The method relies upon T4 DNA ligase to catalyse the DNA-templated polymerisation of modified 5′-phosphorylated oligonucleotides. LOOPER has been shown to proceed with excellent fidelities in a library context with codon sets as large as 256 members (codon set = NNNNT).…”

“…Furthermore, the system can also tolerate a host of chemical modifications on the pentanucleotide building blocks, ranging from small molecules 18 to peptide fragments. 16 Pentanucleotides are ideal for this LOOPER, as they polymerise efficiently, yet require a primer to initiate the polymerisation process, thus defining the reading frame. Longer oligonucleotides also decrease the density of functional group modifications on the polymer product.…”