Fluorescent Base Analog for DNA or RNA Synthesis

Bio-Synthesis provides fluorescent nucleoside base oligonucleotide synthesis. These nucleoside base analog is highly dependent of the environment of teh base and the measurement of its fluorescence is a powerful and sensitive tool fo rthe analysis of DNA and RNA structure. It is especially useful for analyzing the interaction of DNAand RNA with their correspondingbindingproteins. Fluorescence measuremens allow real-time probing fo these structural interaction. Bio-Synthesis offers

Pyrrollo-dC and Pyrrollo C bases, these fluroescent analogues code efficiently as C, it is an ideal probe of DNA structure and dynamics.

•  It base-pairs as a normal dC nucleotide. An oligo fully substituted with pyrrolo-dC has the same Tm as the control dC oligo with the same specificity for dG
•  Its small size does not perturb the structure of the DNA helix and it is well tolerated by a number of DNA and RNA polymerases.
•  It is highly fluorescent and its excitation and emission are wellto the red of most fluorescent nucleotide analogs, which eliminates or reduces background fluorescence from proteins.

2-Aminopurine is another  fluorescent base which has found significant use in probing DNA structures, It is especially useful in that it is capable of hybridizing to T.

The spectrral properties of pyrrollo-dC, coupled with its unique base-pairing ability, making this fluorescent analog extremely valuable in probing DNA structure. When the pyrrolo-dC is base-paired, its fluorescence is significantly quenched through waht is most likely base stacking or dG interaction.

	QY	λ	ε(L/mol.cm)
Single-stranded	0.07	260 nm	4000
		347 nm	3700
double-stranded	0.02

The quantum yield of fluorescence for pyrrolo-dC is quite sensitive to its hybridization state, making it ideally suited8,9 for probing the dynamic structure of DNA. Work by Liu and Martin has shown9 that, when the pyrrolo-dC is mismatched in an otherwise duplex hybrid, the fluorescence is higher than the single-stranded species when the mismatched base is adenosine. This most likely arises from efficient energy transfer from the adenosine to the pyrrolo-dC. This unusual behavior also allows differentiation in situ between a DNA-DNA duplex and a DNA-RNA heteroduplex.

The quenching of pyrrolo-dC allows local structural changes to be probed with great sensitivity. Using pyrrolo-dC, Liu and Martin⁸ have characterized the transcription bubble in elongation complexes of T7 RNA Polymerase to single-base resolution by observing roughly a two-fold increase in fluorescence as the polymerase induces melting. By starving the T7 RNA Polymerase of specific nucleoside triphosphates, the enzyme could be stalled at specific sites, producing 'fluorescence snapshots' of the complex, and yielding detailed information on the nature of the transcription bubble and heteroduplex.

Work is still progressing in evaluating the effect of this modified fluorescent nucleoside in biological systems and will be reported. However, a few comments on our findings to date may be of interest. Oligonucleotides containing pyrrolo-dC act as efficient primers and the PCR products appear to be identical for primers with 0 to 5 pyrrolo-dC residues replacing dC. Preliminary data indicate that pyrrolo-dC codes as dC in PCR experiments. And very preliminary evidence indicates that pyrrolo-dC triphosphate is incorporated efficiently by Taq polymerase and is incorporated specifically opposite dG.

References

1. S.C. Srivastava, S.K. Raza, and R. Misra, Nucleic Acids Res, 1994, 22, 1296-304.
2. M.E. Hawkins, W. Pfleiderer, O. Jungmann, and F.M. Balis, Anal Biochem, 2001, 298, 231-240.
3. M.E. Hawkins, W. Pfleiderer, F.M. Balis, D. Porter, and J.R. Knutson, Anal Biochem, 1997, 244, 86-95.
4. S.L. Driscoll, M.E. Hawkins, F.M. Balis, W. Pfleiderer, and W.R. Laws, Biophys J, 1997, 73, 3277-86.
5. R. Charubala, et al., Nucleos Nucleot, 1997, 16, 1369-1378.
6. J.M. Jean and K.B. Hall, Proc Natl Acad Sci U S A, 2001, 98, 37-41.
7. J. Woo, R.B. Meyer, Jr., and H.B. Gamper, Nucleic Acids Res, 1996, 24, 2470-5.
8. C. Liu and C.T. Martin, J Mol Biol, 2001, 308, 465-75.
9. C. Liu and C.T. Martin, J Biol Chem, 2002, 277, 2725-31.