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. In this work, we describe a method for converting DNA libraries into "gene-linked RNA aptamer particles" (GRAPs) that each display ∼105 copies of a single RNA sequence alongside the DNA that encodes it. We then screen large libraries of GRAPs in a high-throughput manner using the FACS instrument based directly on their fluorescence-enhancing properties. Using this strategy, we demonstrate the capability to generate fluorescence-enhancing aptamers that produce a variety of different emission wavelengths upon binding the dye of interest.

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. Single-turnover experiments carried out with LDP and UV1C, relative to control experiments with LDP alone in single-stranded and double-stranded contexts, show that while UV1C does modestly promote thymine dimer formation, its major activity is indeed photoreactivation. Distinct photostationary states are reached for LDP in its three contexts: as a single strand, as a constituent of a double-helix, and as a 1:1 complex with UV1C. The above results on the cofactor-independent photoreactivation capabilities of a catalytic DNA reinforce a series of recent, unexpected reports that purely nucleotide-based photoreactivation is also operational within conventional double-helical DNA.

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. The cross-linking maps we obtain using chirally resolved substrates give us, for the first time, a stereochemical glimpse of the 8-17's active site. Thus, we identifiy the asymmetric positioning of the DNAzyme's C13 residue, which is catalytically indispensable. We also identify, for the first time, the previously unheralded C3 residue. On the basis of the latter's proximal location to the cleavage site and the impact of its mutation on the DNAzyme's catalytic rate, we hypothesize it may play an acid-base role in the catalysis of the 8-17 DNAzyme. Overall, the approach described in this paper should find wide application in the study of the tertiary folding of RNAs and DNAs, as well as of complexes formed by RNA and DNA with proteins and other ligands.

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. We have used different mapping procedures to identify a number of bases located in loops of the G-quadruplex fold of UV1C as the sites for cross-linking with LDPs. Mutation of one cross-linking adenine, in particular, leads to a major reduction in UVIC's catalytic activity. A map of these contact cross-linking sites enables us to refine an earlier structural-topological model for the folded UV1C.LDPs complex. The surprising facility with which these novel contact cross-links can be generated between a nucleic acid enzyme and its substrate's reaction site opens up a powerful new approach to mapping the active sites of different ribozymes and deoxyribozymes as well as enabling the dissection of key contacts within RNA-protein complexes.

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. Although the rate enhancements characteristic of the two DNAzymes were similar, the cofactor-requiring Sero1C repaired a substantially broader range of substrates compared to UV1C, including thymine, uracil, and a range of chimeric deoxypyrimidine and ribopyrimidine dimers. Similarities and differences in the properties of these two photolyase DNAzymes suggest, first, that the harnessing of less damaging UV light for the repair of photolesions may have been a primordial catalytic activity of nucleic acids, and, second, the broader substrate range of Sero1C may highlight an evolutionary advantage to coopting amino-acid-like cofactors by functionality-poor nucleic acid enzymes.