Title
Optimization of fluorescent 8-heteroaryl-guanine probes for monitoring protein-mediated duplex → G-quadruplex exchange
Author
Kaila L. Fadock, Richard A. Manderville, Purshotam Sharma, Stacey D. Wetmore
Year
2016
Journal
Organic and Biomolecular Chemistry
Abstract
In this study, we describe the thermal and optical properties of the thrombin binding aptamer (TBA) that has been modified at syn-G-tetrad positions with fluorescent 8-heteroaryl-2′-deoxyguanosine derivatives consisting of pyrrolyl (PyrdG), furyl (FurdG), thienyl (ThdG), benzofuryl (BfurdG), indolyl (InddG) and benzothienyl (BthdG). Insertion of the modified base into the syn-G5 position of TBA decreases duplex stability, but enhances stability of the antiparallel G-quadruplex (GQ) structure produced by TBA in the presence of K+ ion and its molecular target, thrombin. The resulting modified TBA (mTBA) oligonucleotides have been employed in duplex → GQ exchange to monitor thrombin binding affinity and rates of GQ formation driven by thrombin binding. Our studies demonstrate that 8-heteroaryl-dG bases can be inserted into syn-G-tetrad positions of TBA without perturbing thrombin binding affinity and that the 8-thienyl-dG (ThdG) analog is particularly useful as an emissive probe for monitoring duplex → GQ exchange due to its heightened emissive sensitivity to change in DNA topology compared to the other 8-heteroaryl-dG analogs. The positional impact of a single ThdG probe versus multiple ThdG incorporation at syn-G sites of TBA highlight an advantage for di-substituted mTBA oligonucleotides for increased emission intensity and rates of duplex → GQ exchange that can be vital for diagnostics through aptamer detection strategies.
Instrument
J-815
Keywords
Circular dichroism, Biochemistry