Photocaged G-Quadruplex DNAzyme and Aptamer by Post-Synthetic Modification on Phosphodiester Backbone

Abstract

G-quadruplex-containing DNAzymes and aptamers are widely applied in many research fields because of their high stability and prominent activities versus the protein counterparts. In this work, G-quadruplex DNAs were equipped with photolabile groups to construct photocaged DNAzymes and aptamers. We incorporated TEEP-OH (thioether-enol phosphate, phenol substituted) into phosphodiester backbone of G-quadruplex DNA by a facile post-synthetic method to achieve efficient photocaging of their activities. Upon light irradiation, the peroxidase-mimicking activity of the caged G-quadruplex DNAzyme was activated, through the transformation of TEEP-OH into a native DNA phosphodiester without any artificial scar. Similarly, the caged G-quadruplex thrombin-binding aptamer also showed light-induced activation of thrombin inhibition activity. This method could serve as a general strategy to prepare photocaged G-quadruplex DNA with other activities for noninvasive control of their functions.

Chemo‐Enzymatic Synthesis of Position‐Specifically Modified RNA for Biophysical Studies including Light Control and NMR Spectroscopy

“…Incorporation of as ingle photoprotected nucleotide at the ligand binding site prevents ligand binding ( Figure S66). 15 N-labelled 2'dG facilitates observation of the base paired imino proton of the ligand. Only after uncaging,t ertiary interactions of the ligand-bound state were detected by reporter signals (highlighted in red;F igure 2b).…”

“…[8] Theenzyme undergoes multiple incorporations.T oextend at the 3'-end with as ingle nucleotide,r eaction conditions need to be optimized and are not obligatorily available. [14] Recent studies using these strategies have focused on structural and kinetic studies of Gquadruplexes, [15] riboswitches, [16] bistable RNAs, [17] ribozymes, [18] miRNA, [19] splice-switching oligonucleotides [20] and CRISPR/Cas9. [10] Studies by Uhlenbeck and others [10,11] have shown that nucleoside 3',5'-bisphosphates with modifications at the sugar-, phosphate-, or base-site can be incorporated in an ATP-dependent reaction.…”

“…Restriction to only as ingle incorporation is given since the 3'-phosphate group blocks further extension. Schematic representation of the three-stepe nzymatic procedure for synthesis of RNA labelled with an azobenzene C-nucleoside, aphotocage, or a 13 C, 15 N-enriched nucleoside. [13] Photoremovable protecting groups or photoswitches attached to RNAs are important for structural and functional studies because light as trigger signal can be applied under high spatiotemporal control.…”

“…C,15 N-labelled adenosine bisphosphate ( 13 C, 15 N-pAp;S upporting Information, Section 3.8) was incorporated to the 3'-end of RNA. The 13 C-…”

Chemo‐Enzymatic Synthesis of Position‐Specifically Modified RNA for Biophysical Studies including Light Control and NMR Spectroscopy

“…[13] Photoremovable protecting groups or photoswitches attached to RNAs are important for structural and functional studies because light as trigger signal can be applied under high spatiotemporal control. [14] Recent studies using these strategies have focused on structural and kinetic studies of Gquadruplexes, [15] riboswitches, [16] bistable RNAs, [17] ribozymes, [18] miRNA, [19] splice-switching oligonucleotides [20] and CRISPR/Cas9. [21] By solid-phase synthesis,i ntroduction of photocages is cost-and time-consuming.A lso postsynthetic introduction of photocages in RNAusing convertible nucleosides is limited to primary amines.…”