Single base interrogation by a fluorescent nucleotide: each of the four DNA bases identified by fluorescence spectroscopy

Abstract

Nucleoside C, which contains a rigid nitroxide spin label, is effectively reduced in DNA by sodium sulfide to the corresponding amine, yielding a fluorescent probe (Cf) that can report the identity of its base-pairing partner in duplex DNA.

Large flanking sequence effects in single nucleotide mismatch detection using fluorescent nucleoside Çf

“…All duplexes are expected to be stable at 20°C, based on T M values of 46-63°C for the oligonucleotide with flanking sequence 5 0 -d(GÇ f A). 23 The relative fluorescence intensities of the 64 duplexes at 20°C is shown in Figure 3.…”

“…24 Upon reduction of the spin labelled DNA with Na 2 S, highly fluorescent DNA was obtained, which was attributed to the amine derivative (Ç f ) of the spin label. 23 As the amine is a precursor for the nitroxide, we sought to incorporate the phosphoramidite of Ç f directly, avoiding the oxidation and subsequent reduction steps (Scheme 1). A dimethoxytrityl group was introduced at the 5 0 -OH group of the fluoroside, followed by phosphitylation of the 3 0 -OH.…”

“…1), which, after incorporation into duplex DNA, is able to uniquely identify each of the four DNA bases in duplex DNA. 23 Upon further examination it became evident that flanking sequence had an effect on the mismatch detection. 23 Here we report a systematic study of the effects of all the flanking sequences on mismatch detection using fluorescent nucleoside Ç f .…”

“…23 Upon further examination it became evident that flanking sequence had an effect on the mismatch detection. 23 Here we report a systematic study of the effects of all the flanking sequences on mismatch detection using fluorescent nucleoside Ç f . We found a large variation in the fluorescence intensities of the mismatches between different flanking sequences.…”

Chemical RNA Modifications for Studies of RNA Structure and Dynamics

“…[102] The DNA spin label 42 is not only highly valuable for EPR spectroscopy but has also been shown to be readily convertible into a fluorescent nucleoside analogue, thereby opening the possibility for multiple studies with complementary spectroscopic techniques. [103] Given the recent developments in EPR methodology and advancements in chemical synthesis of modified nucleic acids, it is expected that novel strategies for labeling of RNA with rigid spin labels will be explored in the near future. The design of suitable spin labels for attachment to RNA residues should take into account their impact on the overall structure and stability of the molecule, especially on the base-pairing and stacking interactions of nucleobases located near the labeled sites.…”