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*****Glen Research Glen Report*****
tC and tCo: New Tricyclic Fluorescent Cytidine Analogues with a very Bright Future
Glen Research has offered fluorescent nucleosides for a long time: 2-Aminopurine deoxynucleoside and Etheno-dA have been in our catalog for more than 15 years. More recently, we have become interested in the potential for dC analogues to offer interesting fluorescence properties while maintaining excellent base pairing behavior with dG. In partnership with Berry and Associates, we introduced pyrrolo-dC1, which has intriguing properties that allow its exact position in a duplex to be determined. And, recently, we have pointed out the fluorescence properties of AP-dC (G-Clamp).2
Now we are happy to introduce the tricyclic fluorescent nucleoside analogues, 1,3-diaza-2-oxophenothiazine, tC, and 1,3-diaza-2-oxophenoxazine, tCo. Tricyclic dC base analogues 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.
Figure 2 shows the absorption and emission spectra of the oligo 5’-ATC GXT CAT GAT G-3’, where X is tC, as determined in house. The absorption maximum was found to be at 392nm with no shift in wavelength in duplex DNA. The emission maximum was found to be at 506nm. Figure 2 illustrates the melting curves for the tC oligo as well as the control where X = dC. We found that the Tm of this oligo was raised by 1.0° relative to the control oligo containing dC.
Figure 3 shows the absorption and emission spectra of the oligo 5’-ATC GXT CAT GAT G-3’, where X is tCo, as determined in house. The absorption maximum was found to be at 360nm, shifting to 365nm in duplex DNA. The emission maximum was found to be at 465nm. Figure 3 also shows the melting curves for the tCo oligo, as well as the control where X = dC. We found that the Tm of this oligo was raised by 0.5° relative to the control oligo containing dC.
Synthesis and Deprotection
tC- and tCo-CE Phosphoramidites couple optimally with a reaction time of 3 minutes. Deprotection can be carried out using ammonium hydroxide at room temperature (to avoid minor degradation at elevated temperatures), AMA at 65°C or potassium carbonate in methanol, as required for deprotection of the nucleobases.
We are happy to offer tC as a very bright and stable probe of DNA structure and tCo as the brightest and most promising internal fluorescent probe for DNA to date. In combination, they offer great potential for the analysis of DNA structure and function. We would also like to thank Marcus Wilhelmsson for his help in preparing this article.
1. D.A. Berry, et al., Tetrahedron Lett, 2004, 45, 2457-2461.
2. The Glen Report, 2007, 19, 8-9.
3. P. Sandin, et al., Nucleic Acids Res., 2008, 36, 157-167.
4. P. Sandin, et al., Nucleic Acids Res., 2005, 33, 5019-5025.
5. K.C. Engman, et al., Nucleic Acids Res., 2004, 32, 5087-5095.
Please contact Glen Research if you have any questions or comments!