Fluorescence Quenchers
for Hydrazone and Oxime Orthogonal
Bioconjugation
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Abstract
We describe the synthesis and properties of new fluorescence
quenchers
containing aldehyde, hydrazine, and aminooxy groups, allowing convenient
bioconjugation as oximes or hydrazones. Conjugation to oligonucleotides
proceeded in high yield with aniline as catalyst. Kinetics studies
of conjugation show that, under optimal conditions, a hydrazine or
aminooxy quencher can react with aldehyde-modified DNA to form a stable
hydrazone or oxime adduct in as little as five minutes. The resulting
quencher-containing DNAs were assessed for their ability to quench
the emission of fluorescein in labeled complements and compared to
the commercially available dabcyl and Black Hole Quencher 2 (BHQ2),
which were conjugated as phosphoramidites. Results show that the new
quenchers possess slightly different absorbance properties compared
to dabcyl and are as efficient as the commercial quenchers in quenching
fluorescein emission. Hydrazone-based quenchers were further successfully
incorporated into molecular beacons and shown to give high signal
to background ratios in single nucleotide polymorphism detection <i>in vitro.</i> Finally, aminooxy and hydrazine quenchers were
applied to quenching of an aldehyde-containing fluorophore associated
with living cells, demonstrating cellular quenching within one hour