Reducing Product Inhibition in Nucleic Acid‐Templated Ligation Reactions: DNA‐Templated Cycligation

Abstract

Programmable interactions allow nucleic acid molecules to template chemical reactions by increasing the effective molarities of appended reactive groups. DNA/RNA-triggered reactions can proceed, in principle, with turnover in the template. The amplification provided by the formation of many product molecules per template is a valuable asset when the availability of the DNA or RNA target is limited. However, turnover is usually impeded by reaction products that block access to the template. Product inhibition is most severe in ligation reactions, where products after ligation have dramatically increased template affinities. We introduce a potentially generic approach to reduce product inhibition in nucleic acid-programmed ligation reactions. A DNA-triggered ligation-cyclization sequence ("cycligation") of bifunctional peptide nucleic acid (PNA) conjugates affords cyclic ligation products. Melting experiments revealed that product cyclization is accompanied by a pronounced decrease in template affinity compared to linear ligation products. The reaction system relies upon haloacetylated PNA-thioesters and isocysteinyl-PNA-cysteine conjugates, which were ligated on a DNA template according to a native chemical ligation mechanism. Dissociation of the resulting linear product-template duplex (induced by, for example, thermal cycling) enabled product cyclization through sulfur-halide substitution. Both ligation and cyclization are fast reactions (ligation: 86 % yield after 20 min, cyclization: quantitative after 5 min). Under thermocycling conditions, the DNA template was able to trigger the formation of new product molecules when fresh reactants were added. Furthermore, cycligation produced 2-3 times more product than a conventional ligation reaction with substoichiometric template loads (0.25-0.01 equiv). We believe that cyclization of products from DNA-templated reactions could ultimately afford systems that completely overcome product inhibition.

Templated chemistry for bioorganic synthesis and chemical biology

“…Careful optimization of the ligation site such as the replacement of a glycine‐cysteine by a glycine‐isocysteine junction allowed improvements of turnover numbers . In another report, we described bifunctional PNA conjugates, which reacted in a two‐step reaction . Templated NCL was followed by a cyclization reaction.…”

Platforms for Bioorthogonal Oligonucleotide-templated Reactions: Translating Concepts into Devices

“…To overcome this issue of sensitivity and enable the detection of low copy numbers of endogenous NA biomarkers, strategies that prevent product inhibition have been developed where the NA target can dissociate from the OTR product and be recycled for catalytic turnover to occur [35]. This can be achieved via either thermocycling (alternating heat cycles to favour product formation and product dissociation) and/or the use of additional reagents [36].…”

Templated native chemical ligation: peptide chemistry beyond protein synthesis

“…This analysis prompted Roloff et al . to propose a method for reducing product inhibition in ligation reactions . They assumed that global changes in the product structure would be required to destabilize the product‐template complex and designed a reaction system involving a post‐ligation cyclization step (cycligation).…”