Optimization of nucleic acid scaffold design using fluorescence measurements
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MRS Advances © 2019 Materials Research Society DOI: 10.1557/adv.2019.48
Optimization of nucleic acid scaffold design using fluorescence measurements Jessica Anderson1, McKenze Moss1, Nancy Nguyen1, Natalie Hughes1, Amira Gee1 and Mehnaaz F. Ali1 1
Department of Chemistry, Xavier University of Louisiana, 1, Drexel Drive, New Orleans LA 70125
Abstract
The current work focuses on optimizing aptamer scaffolds that are tailored to allow for the formation of binding pockets for both a redox active signaling molecule and the target miR92a. These newly designed allosteric nucleic acid systems are studied for efficacy to undergo a target based conformational switch. Two hairpin scaffolds were designed with differing stem stabilities and were explored using fluorescence quenching measurements. The dose dependent data for the detection of miR-92a shows the importance of scaffold design where the stability of the intra-molecular hairpin structure has to be optimized for target binding. Additional experiments explored the selectivity of the aptamer scaffolds in the presence of competing miR’s and mismatched sequences. These results provide an important precursor to constructing nucleic acid scaffolds for the detection of miR’s using label-free redox signaling.
INTRODUCTION: The design and exploration of structure switching nucleic acid scaffolds with functional molecular recognition domains is of significant interest due to their potential role as molecular switches, chemo-responsive sensors or genetic control elements.[1] The field of allosteric nucleic acids was preceded, by studying the functional dexterity of naturally occurring RNA polymers.[1] Conformational changes and allosteric transitions are ubiquitous for RNA structures that are involved in biological processes such as protein synthesis, viral and cellular gene expression and mRNA splicing.[2, 3] This previous field serves as a precursor for our current studies where we are using rational design to construct conformational switches using DNA scaffolds. The scaffolds are tailored to contain both a target binding region as well as a small-molecule binding
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loop. It is expected that this system will be used to bind flavin adenine dinucleotide (FAD) based molecules, which are redox active to serve as signaling molecules. We have made preliminary contributions to this field with our explorations on probing FAD as a ligand for nucleic acid binding.[4, 5] For the current work, the conformational change is being studied using fluorescence measurements. The presence of the target miR-92a leads to a conformational shift and unravels a hairpin shaped aptamer scaffold. The fluorescence measurements provide a facile mechanism to determine optimal scaffold design in order to facilitate a stable target binding probe. EXPERIM
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