Label-Free Fluorescent Functional DNA Sensors Using Unmodified DNA: A Vacant Site Approach

Abstract

A general methodology to design label-free fluorescent functional DNA sensors using unmodified DNA via a vacant site approach is described. By extending one end of DNA with a loop, a vacant site that binds an extrinsic fluorophore, 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND), could be created at a selected position in the DNA duplex region of DNAzymes or aptamers. When the vacant site binds ATMND, ATMND’s fluorescence is quenched. This fluorescence can be recovered when one strand of the duplex DNA is released through either metal ion-dependent cleavage by DNAzymes or analyte-dependent structural-switching by aptamers. Through this design, label-free fluorescent sensors for Pb²⁺, UO₂²⁺, Hg²⁺, and adenosine have been successfully developed. These sensors have high selectivity and sensitivity; detection limits as low as 3 nM, 8 nM, 30 nM, and 6 μM have been achieved for UO₂²⁺, Pb²⁺, Hg²⁺ and adenosine, respectively. Control experiments using vacant-site-free DNA duplexes and inactive variants of the functional DNAs indicate that the presence of the vacant site and the activity of the functional DNAs are essential for the performance of the proposed sensors. The vacant site approach demonstrated here can be used to design many other label-free fluorescent sensors to detect a wide range of analytes.