Diverse size approach to incorporate and extend highly fluorescent unnatural nucleotides into DNA.

We have prepared a series of size-diverse unnatural nucleotides containing fluorescent ( dApyrTP , dUpyrTP , dUantTP , dUthiTP ) and quencher ( dUazoTP ) units, as well as nucleotides presenting small functional groups ( dAethTP , dAoctTP , dUethTP , dUiodTP ), all based on deoxyadenosine and deoxyuridine, and examined their suitability for use in enzymatic incorporation and extension into DNA. We observed a size-dependence of the incorporation and extension capability (following the order dUiodTP = dUethTP = dUthiTP > dUazoTP > dUpyrTP > dUantTP ) during primer extension. This result was supported by circular dichroism (CD) spectra, which revealed a trend in the different B-form DNA structures depending on the size of the unit at the 5-position of the deoxyuridine ( dUiodTP > dUethTP > dUthiTP > dUpyrTP ), obtained from the PCR products. Interestingly, dUthiTP could be incorporated and extended into long DNA strands during primer extension and even PCR amplification, with CD spectroscopy confirming a stable secondary B-form duplex DNA structure. We observed full-length extension products even when combining dUthiTP with a template containing 24 continuous dA units during the primer extension. Thus, we believe that dUthiTP is a promising fluorescent nucleotide for a diverse range of biological applications requiring multiple incorporation and extension directly without disruption of B-form DNA structures. [ABSTRACT FROM AUTHOR]