title: Optimization of fluorescent 8-heteroaryl-guanine probes for monitoring protein-mediated duplex → G-quadruplex exchange.
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 ((Pyr)dG), furyl ((Fur)dG), thienyl ((Th)dG), benzofuryl ((Bfur)dG), indolyl ((Ind)dG) and benzothienyl ((Bth)dG). 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 ((Th)dG) 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 (Th)dG probe versus multiple (Th)dG 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.
authors.affiliation: Department of Chemistry & Toxicology, University of Guelph, Guelph, ON, Canada N1G 2W1. rmanderv@uoguelph.ca.
authors.forename: Kaila L
authors.initials: KL
authors.lastname: Fadock
authors.affiliation: Department of Chemistry & Toxicology, University of Guelph, Guelph, ON, Canada N1G 2W1. rmanderv@uoguelph.ca.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Manderville
authors.affiliation: Department of Chemistry & Biochemistry, University of Lethbridge, Lethbridge, AB, Canada T1K 3M4. Stacey.Wetmore@uleth.ca.
authors.forename: Purshotam
authors.initials: P
authors.lastname: Sharma
authors.affiliation: Department of Chemistry & Biochemistry, University of Lethbridge, Lethbridge, AB, Canada T1K 3M4. Stacey.Wetmore@uleth.ca.
authors.forename: Stacey D
authors.initials: SD
authors.lastname: Wetmore
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry*; D054856:G-Quadruplexes; D006147:Guanine / Q000737:chemistry*; D056004:Molecular Dynamics Simulation; D055620:Optical Phenomena; D013696:Temperature; D013917:Thrombin / Q000378:metabolism*
chemical_list: D052157:Aptamers, Nucleotide; D005456:Fluorescent Dyes; D006147:Guanine; D013917:Thrombin