Molecular basis of action of HyBeacon fluorogenic probes: a spectroscopic and molecular dynamics study.
HyBeacons, novel DNA probes for ultra-rapid detection of single nucleotide polymorphisms, contain a fluorophore covalently attached via a linker group to an internal nucleotide. As the probe does not require a quencher or self-complementarity to function, this study investigates the molecular-level mechanism underlying the increase of fluorescence intensity on hybridization of HyBeacons with target DNA. Spectroscopic ultraviolet-visible and fluorimetric studies, combined with molecular dynamics simulations, indicate projection of the fluorophore moiety away from the target-probe duplex into aqueous solution, although specific linker-DNA interactions are populated. Based on evidence from this study, we propose that for HyBeacons, the mechanism of increased fluorescence on hybridization is due to disruption of quenching interactions in the single-stranded probe DNA between the fluorophore and nucleobases. Hybridization leads to an extended linker conformation, removing the fluorophore from the immediate vicinity of the DNA bases.
Marks
Abigail H R
AH
Wolfson Centre for Structure-Based Rational Design of Molecular Diagnostics, School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester M13 9PL, UK.
Bhadra
Pranab K
PK
McDowell
David G
DG
French
David J
DJ
Douglas
Kenneth T
KT
Bichenkova
Elena V
EV
Bryce
Richard A
RA
D001483:Base Sequence; D004247:DNA / Q000737:chemistry; D015342:DNA Probes / Q000737:chemistry*; D004277:DNA, Single-Stranded / Q000737:chemistry; D005456:Fluorescent Dyes / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D008856:Microscopy, Fluorescence; D008956:Models, Chemical; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000737:chemistry; D020641:Polymorphism, Single Nucleotide; D011487:Protein Conformation; D012984:Software; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet; D055672:Static Electricity; D013696:Temperature; D014466:Ultraviolet Rays
D015342:DNA Probes; D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D009841:Oligonucleotides; D004247:DNA