Label-free functional nucleic acid sensors for detecting target agents

摘要

A general methodology to design label-free fluorescent functional nucleic acid sensors using a vacant site approach and an abasic site approach is described. In one example, a method for designing label-free fluorescent functional nucleic acid sensors (e.g., those that include a DNAzyme, aptamer or aptazyme) that have a tunable dynamic range through the introduction of an abasic site (e.g., dSpacer) or a vacant site into the functional nucleic acids. Also provided is a general method for designing label-free fluorescent aptamer sensors based on the regulation of malachite green (MG) fluorescence. A general method for designing label-free fluorescent catalytic and molecular beacons (CAMBs) is also provided. The methods demonstrated here can be used to design many other label-free fluorescent sensors to detect a wide range of analytes. Sensors and methods of using the disclosed sensors are also provided.

主权项

1. A sensor, comprising
a catalytic nucleic acid molecule specific for a target agent comprising an enzyme nucleic acid strand and a substrate nucleic acid strand, wherein the enzyme nucleic acid strand comprises a 3′-end and a 5′-end and an active site specific for a target agent, wherein the substrate nucleic acid strand comprises a 3′-end and a 5′-end, wherein the substrate nucleic acid strand comprises nucleotides at the 5′-end of the substrate nucleic acid strand that permits formation of a loop of at least six nucleotides at the 5′-end of the substrate nucleic acid strand, and wherein the substrate nucleic acid strand hybridizes with the enzyme nucleic acid strand, thereby forming a vacant site between the 3′-end of the enzyme nucleic acid strand and the 5′-end of the substrate nucleic acid strand, wherein the vacant site is opposite to a cytosine present in the substrate nucleic acid strand and wherein the vacant site can bind to a fluorophore, wherein in the absence of the target agent, fluorescence of the fluorophore when bound to the vacant site is quenched, and wherein in the presence of the target agent, catalytic cleavage of substrate nucleic acid strand perturbs the vacant site and releases the fluorophore bound to the vacant site resulting in increased fluorescence.