We tested this unique substrate out of curiosity,34) and thereby found that DNA polymerases sensitively recognize dDsT[P.sub.NH2] as a pairing partner of Pa, but not of Ds, in templates.
We still do not know the mechanism underlying the improved fidelity of the unnatural base pairing using the [gamma]-amidotriphosphates.
Using Method 2, we developed a quantification method to determine the misincorporation rate of the natural bases at the Ds-Pa pair position, and assessed the selectivity (>99% per replication) of the Ds-Pa pairing in PCR using Vent DNA polymerase ([exo.sup.+]).
We compared the selectivity of the Ds-Pa pairing and the misincorporation of the unnatural base substrates opposite the natural bases in transcription with those of the natural A-U pairing, by using biotin-linked Pa and U substrates (Biotin-PaTP and Biotin-UTP).
Therefore, we further improved the unnatural base pair system to bypass the need for the .amidotriphosphates, while maintaining the high selectivity (more than 99%) of the unnatural base pairing, in PCR.
The fidelity of unnatural base pairing in the polymerase reaction is determined by both the selectivity of the unnatural base substrates opposite the unnatural pairing partner bases and the misincorporation rate of the unnatural base substrates opposite the natural bases.
The selectivity of the Ds-modified-Px pairing is as high as 99.8-99.9% per replication, and the misincorporation rate of unnatural bases opposite the natural bases is 0.005% per base per replication.
(2009) Unnatural nucleosides with unusual base pairing properties.
(1997) Theoretical and experimental study of isoguanine and isocytosine: Base pairing in an expanded genetic system.
In general, the creation of unnatural base pairs is initiated by designing the molecular structures or modifying the A-T and G-C pairs, based on a certain idea or concept underlying the mechanism of the selective complementarity of the natural base pairings in polymerase reactions.