Abstract
Fluorescent probes that can distinguish different DNA topologies through changes in optical readout are sought after for DNA-based diagnostics. In this work, the 4,4-difluoro-4-bora-3a, 4a-diaza-s-indacene (BODIPY) chromophore attached to cyanophenyl substituents (BODIPY-CN) has been tethered to the 5′-end of the 15-mer thrombin binding aptamer (TBA) that contains the guanine (G) nucleobase. TBA folds into a unimolecular antiparallel G-quadruplex (GQ) upon binding thrombin and certain metal ions. The 5′-BODIPY-CN-TBA sample possesses a Stokes shift of ~40 nm with wavelengths of excitation/emission at 550/590 nm and exhibits a 2-fold increase in emission intensity compared to the free BODIPY-CN in aqueous buffer that possesses a brightness (εΦfl) of ~16,956 M−1. cm−1. However, when 5′-BODIPY-CN-TBA is base-paired to a complementary strand in the B-form duplex, the emission of the BODIPY-CN end-label increases 7-fold, 14-fold compared to the free-dye. This signal-on response enables the BODIPY-CN end-label to serve as a quencher-free fluorescent probe for monitoring duplex-GQ exchange. The visible end-label minimally perturbs GQ stability and thrombin binding affinity, and the modified TBA can act as a combinatorial logic circuit having INHIBIT logic functions. These attributes make BODIPY-CN a highly useful end-label for creating nanomolecular devices derived from G-rich oligonucleotides.
Highlights
In search for brighter visible alternatives capable of discerning duplex from GQs, we were encouraged by
Our results demonstrate the utility of 5′-BODIPY containing cyanophenyl substituents for monitoring duplex-GQ exchange in a quencher-free format
When attached to the 5′-end of the 15-mer thrombin binding aptamer (TBA) that contains the G nucleobase, the probe exhibits ~2-fold increase in fluorescence intensity compared to the free-dye in aqueous buffer
Summary
In search for brighter visible alternatives capable of discerning duplex from GQs, we were encouraged by. The best signal-off response (12-fold) occurred with the BODIPY FL attached to a 5′-C with hybridization to a complementary strand containing a 3′-GGG-overhang that is not base-paired within the duplex This suggested that a 5′-BODIPY may serve as a useful end-label for monitoring DNA duplex-GQ exchange in the absence of a second label, such as a commercial quencher. BODIPYs possess very high quantum yields, are often more photostable than fluorescein (FAM) and rhodamine analogs[25] and can readily be functionalized at desired positions to improve photophysical properties[26] and perhaps aptamer performance This prompted our laboratory to synthesize BODIPY probes to test performance at the 5′-end of TBA in order to make direct comparison to internal FBAs and 5′-FAM27. The BODIPY end-label is considerably brighter than internal FBAs used previously by our laboratory[15,27] and undergoes excitation in the visible region with a reasonably large Stokes shift for aptasensor applications
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