Nucleic Acid Templated Reactions for Chemical Biology

Abstract

Nucleic acid directed bioorthogonal reactions offer the fascinating opportunity to unveil and redirect a plethora of intracellular mechanisms. Nano- to picomolar amounts of specific RNA molecules serve as templates and catalyze the selective formation of molecules that 1) exert biological effects, or 2) provide measurable signals for RNA detection. Turnover of reactants on the template is a valuable asset when concentrations of RNA templates are low. The idea is to use RNA-templated reactions to fully control the biodistribution of drugs and to push the detection limits of DNA or RNA analytes to extraordinary sensitivities. Herein we review recent and instructive examples of conditional synthesis or release of compounds for in cellulo protein interference and intracellular nucleic acid imaging.

Keywords: in situ drug release; in situ drug synthesis; nucleic acid encoded reactions; nucleic acid sensing.

Figure 1

Nucleic acid triggered reactions proceeding with turnover.

Figure 2

DNA/mRNA‐triggered peptidyl transfer by NCL proceeds with turnover at nanomolar amounts of template, enabling the formation of full‐length bioactive peptides. Transferred peptidyl units are shown in red.

Figure 3

Logic circuit leading to the release of nitric oxide. Inset: multivalent chimeric signal transducer.

Figure 4

A) Interaction of peptide–PNA probe with single‐strand DNA modulates peptide binding to Src‐SH2. Left: extended peptide conformation displays increased affinity for Src‐SH2. Further interaction with scavenger DNA fully complementary to the activator DNA disassembles the chimera, releasing the unstructured peptide–PNA complex. Right: seamless hybridization leads to constrained peptides with low affinity. B) Activation of Src kinase by a PNA–phosphopeptide chimera. The inactive‐closed kinase conformation is stabilized by the interaction between pTyr527 and the SH2 domain. Addition of activator DNA/RNA induces strand exchange to form an activated chimera complex with high affinity for SH2, thereby displacing pTyr527 from the SH2 binding pocket. This triggers the activation of Src kinase. C) A hairpin‐structured peptide–PNA conjugate that signals the presence of the Src‐SH2 protein. Left: red labels are pyrene units.

Figure 5

DNA hairpin system for sensing of mono‐ and bivalent proteins.

Figure 6

Allosteric control of binding to human carbonic anhydrase II (hCA‐II) through strand displacement.

Figure 7

Strain‐promoted cycloaddition yields peptide–PSAO conjugates. The conjugates comprise an antisense oligophosphorothiate for downregulation of c‐flip mRNA (master regulator of the extrinsic pathway of apoptosis) and a Smac peptidomimetic for antagonizing the inhibitor of apoptosis proteins (IAPs; master regulators of the intrinsic pathway of apoptosis).

Figure 8

A) p‐Azidobenzyl‐based immolative linker for the uncaging of functional molecules. B) 23S rRNA‐dependent IPTG release system in bacterial cells triggers GFP expression. C) Photoinduced cleavage of a phenacyl ester linker triggers the release of biotin. D) Uncaging of siRNA to modulate gene expression in HeLa cells. Red/green light‐responsive photosensitizer catalyzes ¹O₂ formation, which cleaves the 9‐alkoxyanthracenyl‐based quencher, thereby releasing active siRNA.

Figure 9

A) Templated Staudinger reduction of azides to unleash fluorescence signal. B) Introduction of an immolative linker to trigger fluorescence signal. C),D) Tetrazine‐mediated transfer (TMT) to uncage a fluorescence signal. E) Catalyzed transfer of a reporter group by templated acyl transfer. Red labels: quenchers; blue labels: quenched fluorophores; purple labels: free fluorophores; white circles: reacted/transferred quencher.

Figure 10

A) Templated fluorophore synthesis and host–guest interaction lead to fluorescence exacerbation. B) Quadruplex‐templated aldol condensation yields fluorogenic Cy3 dyes.