Nucleobase intrinsic quenchers: A fluorescence off switch

Abstract

Molecular beacons are important bioanalytical probes most often constructed from a single-stranded oligonucleotide which has been labeled at opposite termini with a fluorophore and a quencher. When the fluorophore and quencher are in close proximity, ideally no fluorescence is observed due to Fluorescence Resonance Energy Transfer (FRET). 4-Dimethylaminoazobenzene-4′-carboxylic acid (DABCYL) is a commonly used quencher in molecular beacons; however, DABCYL is unable to form a base-pair and is conventionally installed as an overhanging residue. In this arrangement, the DABCYL moiety has substantial mobility which affects the accuracy of FRET-related distance/orientation measurements and also limits the types of other conjugates that can be prepared. To address these limitations, we have synthesized peptide nucleic acid (PNA) analogues possessing DMPAU (5-[(4-dimethylaminophenyl)diazenyl]uracil) and NPhpC (6-(4-nitrophenyl)pyrrolocytosine) nucleobases which act as dark fluorophores, i.e. quenchers, while retaining the ability to base pair by canonical hydrogen bonding. This will allow more flexible design and construction of molecular beacons based on the insertion of nucleobase fluorophores and quenchers into the stem sequence. We have quantified the quenching ability of DMPAU and NPhpC PNA analogues by determining the Stern-Volmer constants against the intrinsically fluorescent nucleobase, phenylpyrrolocytosine, and other blue emitting fluorophores (pyrene, acridone). The hydrogen bonding ability of DMPAU with adenine, as determined by 1H NMR titration, is relatively unperturbed compared to uracil and thymine.