The Internation Conference on Photochemistry vol:26th pages:491-491
The International Conference on Photochemistry edition:26th location:Leuven date:21-26 July 2013
Aptamers are short ssDNA or RNA oligonucleotides that bind targets with high affinity and specificity by folding into defined tertiary structures. Thanks to these properties aptamers represent attractive bio-receptors. Traditionally new aptamers are obtained through SELEX, an iterative process in which sequences are selected against target molecules. Generally, aptamer-target interactions are detected in indirect ways relying on labeling of either target or aptamer. However, labeling is not always desirable and label-free detection would be valuable for sensitive detection of binding events. This work explores the use of fluorescent silver nanoclusters (AgNCs) to develop an innovative system for monitoring aptamer-protein interactions.
AgNCs are complexes between few Ag atoms and a specific DNA sequence template to stabilize the clusters. In most cases, AgNCs are synthesized either at the 3’ or 5’ end of the template. Our results show that AgNCs can successfully be generated from a template embedded in the middle of a hybridization probe, used for the isothermal amplification of aptamers, called rolling circle amplification (RCA). RCA uses circular oligonucleotide probes to generate long, ssDNA molecules containing periodic repeats of the circular probe. Previous works show that overexpression of aptamers by RCA increases target binding efficiency compared to monovalent aptamers. The RCA concatemer combines both the aptamer and the fluorescent AgNC template. Subsequently synthetized AgNCs exhibit strong, robust and tunable fluorescence, eliminating the need for labeling. Importantly, it has been shown that aptamer-AgNCs retain the same specificity and affinity for the cognate protein and that target binding results in a drastic decrease of nanocluster fluorescence.
This work explores the integration of an AgNC-aptamer sequence into a circular probe to generate intrinsically fluorescent aptamer concatemers with improved binding efficiency. Possible applications are monitoring target binding during SELEX and label-free ultrasensitive detection of proteins.