Each bears a pyridyl nucleobase scaffold but they are differentiated by methyl substitution, and were designed to vary both inter- and intra-strand packing within duplex DNA.
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To differentiate between these possibilities, we determined the effect of mispairing at other positions, including dG 1: Curve A Fully natural and correctly paired duplex N 4: Melting temperatures were determined via the derivative method using the Cary Win UV thermal application software. Overall, the data reveal that the nucleotides are selective for self pairing, with the stability of each self pair at least 2. Hypersensitization of the duplex to mispairing appears to result from global and interdependent solvation effects mediated by the unnatural nucleotide s and the mispair.
The use of the d 34DMPy self pair for increased hybridization fidelity might be useful for a variety of biotechnology applications, however it is limited to applications where the unnatural nucleotide is present in both hybridizing strands. The increased fidelity would be more generally useful for different applications if it were manifest with the unnatural nucleotide present in only one of the hybridizing strands, with the other strand being unmodified.
Overall, the data reveal that individually the self pair and the mispair each induce small or negligible decreases in duplex solvation, but when combined they cause a synergistic decrease in duplex solvation. The melting temperatures T m of the duplexes were determined and are used to evaluate the stability of the unnatural self pairs and mispairs with each natural nucleotide Table 1.
Stability and polymerase recognition of pyridine nucleobase analogues: Support Center Support Center. The effects of the unnatural base pairs on duplex stability demonstrate that the pyridine scaffold may be optimized for stable and selective pairing, and identify one self pair, the pair formed between two d34DMPy nucleotides, which is virtually as stable as a dA:
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