Published online 27 September 2004
Nucleic Acids Research, Vol. 32 No. 17 © Oxford University Press 2004; all rights reserved
DNA adopts normal B-form upon incorporation of highly fluorescent DNA base analogue tC: NMR structure and UV-Vis spectroscopy characterization
Department of Chemistry, Göteborg University, PO Box 462, SE-40530 Gothenburg, Sweden, 1 Physical Chemistry Section, Department of Chemistry and Bioscience, Chalmers University of Technology, SE-41296 Gothenburg, Sweden and 2 School of Chemistry, University of Southampton, Highfield, Southampton SO17 1BJ, UK
* To whom correspondence should be addressed. Tel: +46 (0) 31 7723051; Fax: +46 (0) 31 7723858; Email: mawi{at}chembio.chalmers.se
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
Received June 29, 2004; Revised and Accepted September 3, 2004
The influence of the highly fluorescent tricyclic cytosine base analogue (tC) on duplex DNA conformation is investigated. The duplex properties are characterized by absorbance and circular dichroism (CD) for all combinations of neighbouring bases to tC, and an NMR structure is determined for one tC-containing sequence. For the oligonucleotides with one tC incorporated instead of cytosine, the melting temperature is increased on average by 2.7°C above that for the unmodified ones. CD spectra are practically identical for modified and unmodified sequences, indicating an unperturbed B-DNA conformation. The NMR structure determination of the self-complementary sequence 5'-CTC(tC)ACGTGGAG shows a DNA conformation consistent with B-form for the whole duplex. The root-mean-square distance for the nucleotides of the eight central base pairs between the 10 structures with lowest CYANA target functions and a mean structure is 0.45 ± 0.17 Å. The NMR data confirm correct base pairing for tC by the observation of both intrastrand and interstrand imino proton NOEs. Altogether, this suggests that tC works well as a cytosine analogue, i.e. it is situated in the base stack, forming hydrogen bonds with G in the complementary strand, without distorting the DNA backbone conformation. This first example of an artificial, highly fluorescent DNA base that does not perturb the DNA conformation could have valuable applications for the study of the structure and dynamics of nucleic acid systems.
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