The tricyclic fluorescent nucleoside analogues, 1,3-diaza-2-oxophenothiazine, tC, and 1,3-diaza-2-oxophenoxazine, tCo, are deoxycytidine analogs that have been shown to base pair faithfully with dG with virtually no disruption of the normal duplex structure.3-5 This means that the stability of the DNA duplex is not compromised as compared to the control regardless of DNA sequence. The fluorescence quantum yield of tC is essentially unchanged between single stranded and double stranded DNA - 0.21 for single stranded DNA and 0.19 for duplex DNA. Also, the fluorescence characteristics of tC are not sensitive to neighboring base combinations. tCo has been shown to be the brightest fluorescent nucleoside analogue in duplex context reported so far and even retains the majority of its fluorescence when surrounded by guanine residues. Indeed, tCo has been reported to be 25-50 times brighter than 2-aminopurine.
The base analogue tCnitro is a FRET-acceptor together with tCO (or tC) as the donor molecule. This constitutes the first ever description of a nucleobase FRET-pair. This novel FRET-pair provides a unique tool for investigations of nucleic acid containing systems. tCnitro is virtually non-fluorescent under normal conditions.
Coupling: No changes needed from standard method recommended by synthesizer manufacturer.
Deprotection: As required by nucleobases. Compatible with Ammonium Hydroxide for 17 hours at room temperature, Ammonium hydroxide/40% methylamine 1:1 (AMA) for 10 minutes at 65°C or UltraMild deprotection conditions.
Freezer storage, -10 to -30°C, dry
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