Direct Selection of Fluorescence-Enhancing RNA Aptamers.

RNA aptamers that generate a strong fluorescence signal upon binding a nonfluorescent small-molecule dye offer a powerful means for the selective imaging of individual RNA species. Unfortunately, conventional in vitro discovery methods are not efficient at generating such fluorescence-enhancing aptamers, because they primarily exert selective pressure based on target affinity-a characteristic that correlates poorly with fluorescence enhancement. Thus, only a handful of fluorescence-enhancing aptamers have been reported to date.

DNA Repair by DNA: The UV1C DNAzyme Catalyzes Photoreactivation of Cyclobutane Thymine Dimers in DNA More Effectively than Their de Novo Formation.

UV1C, a 42-nt DNA oligonucleotide, is a deoxyribozyme (DNAzyme) that optimally uses 305 nm wavelength light to catalyze photoreactivation of a cyclobutane thymine dimer placed within a gapped, unnatural DNA substrate, TDP. Herein we show that UV1C is also capable of photoreactivating thymine dimers within an authentic single-stranded DNA substrate, LDP. This bona fide UV1C substrate enables, for the first time, investigation of whether UV1C catalyzes only photoreactivation or also the de novo formation of thymine dimers.

A stereochemical glimpse of the active site of the 8-17 deoxyribozyme from iodine-mediated cross-links formed with the substrate's scissile site.

A powerful approach for defining the active site of a complexly folded ribozyme or deoxyribozyme (DNAzyme) is to map the contact cross-links formed between the substrate's reaction site and component residues of the enzyme. Here, we use a novel iodine- and phosphorothioate-mediated method for generating contact cross-links to define key residues of the 8-17 DNAzyme most proximal to the scissile phosphodiester of its bound substrate. Substitution of a phosphorothioate for the scissile phosphodiester renders that site chiral.

Unusual DNA-DNA cross-links between a photolyase deoxyribozyme, UV1C, and its bound oligonucleotide substrate.

UV1C is a photolyase deoxyribozyme that repairs thymine dimers in a DNA oligonucleotide substrate. We report that treatment with iodine generates specific DNA-DNA cross-links between UV1C and a bound substrate analogue, LDPs, in which a single phosphate at the photoreactivation site has been replaced with a phosphorothioate. Although iodine has been reported to generate lysine-cysteine cross-links within a protein, the formation of DNA-DNA cross-links is both unexpected and novel.

A deoxyribozyme, Sero1C, uses light and serotonin to repair diverse pyrimidine dimers in DNA.

An in vitro selection search for DNAs capable of catalyzing photochemistry yielded two distinctive deoxyribozymes (DNAzymes) with photolyase activity: UV1C, which repaired thymine dimers within DNA using a UV light of >300 nm wavelength and no extraneous cofactor, and Sero1C, which required the tryptophan metabolite serotonin as cofactor in addition to the UV light. Catalysis by Sero1C conformed to Michaelis-Menten kinetics, and analysis of the action spectrum of Sero1C confirmed that serotonin did indeed serve as a catalytic cofactor rather than as a structural cofactor. Sero1C and UV1C showed strikingly distinct wavelength optima for their respective photoreactivation catalyses.