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

• Published in: Bioorganic & medicinal chemistry 2017-07, Vol.25 (14), p.3591-3596
• Main Authors: Le, Binh Huy, Koo, Ja Choon, Joo, Han Na, Seo, Young Jun
• Format: Article
• Language: English
• Subjects: Circular Dichroism; DNA - chemistry; DNA - metabolism; DNA polymerase; DNA, B-Form - chemistry; DNA, B-Form - metabolism; Enzymatic synthesis; Fluorescent Dyes - chemistry; Fluorescent unnatural nucleotide; Nucleic Acid Conformation; Nucleotides - chemistry; Nucleotides - metabolism; PCR; Polymerase Chain Reaction; Primer extension; Pyrenes - chemistry
• ISSN: 0968-0896; 1464-3391
• DOI: 10.1016/j.bmc.2017.03.045

A series of size-diverse functional nucleotide triphosphates containing fluorescent units (dApyrTP, dUpyrTP, dUantTP, dUthiTP) and an azo quenching unit (dUazoTP) has been used to prepare DNA containing highly incorporated and extended fluorescent unnatural nucleotides. [Display omitted] •Synthesis of a series of size-diverse fluorescent nucleotides for incorporation and extension into DNA.•Size-dependence of the incorporation and extension capability of the functional unnatural nucleotides into DNA.•The fluorescent nucleotide dUthiTP could be incorporated and extended into long DNA strands.•dUthiTP appears to be a promising fluorescent nucleotide for a diverse range of biological applications. 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.