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Nucleic Acids Research, 2001, Vol. 29, No. 5 1132-1143
© 2001 Oxford University Press

A nicked duplex decamer DNA with a PEG6 tether

L. Kozerski1,2,*, A. P. Mazurek1, R. Kawecki2, W. Bocian1, P. Krajewski2, E. Bednarek1, J. Sitkowski1,2, M. P. Williamson3, A. J. G. Moir3 and P. E. Hansen4

1Drug Institute, 00-725 Warszawa, Chetmska 30/34, Poland, 2Institute of Organic Chemistry, Polish Academy of Sciences, 01-224 Warszawa, Kasprzaka 44, Poland, 3Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK and 4Department of Life Sciences and Chemistry, Roskilde University, DK-4000 Roskilde, Denmark

A dumbbell double-stranded DNA decamer tethered with a hexaethylene glycol linker moiety (DDSDPEG), with a nick in the centre of one strand, has been synthesised. The standard NMR methods, E.COSY, TOCSY, NOESY and HMQC, were used to measure 1H, 31P and T1 spectral parameters. Molecular modelling using rMD-simulated annealing was used to compute the structure. Scalar couplings and dipolar contacts show that the molecule adopts a right-handed B-DNA helix in 38 mM phosphate buffer at pH 7. Its high melting temperature confirms the good base stacking and stability of the duplex. This is partly attributed to the presence of the PEG6 linker at both ends of the duplex that restricts the dynamics of the stem pentamers and thus stabilises the oligonucleotide. The inspection of the global parameters shows that the linker does not distort the B-DNA geometry. The computed structure suggests that the presence of the nick is not disturbing the overall tertiary structure, base pair geometry or duplex base pairing to a substantial extent. The nick has, however, a noticeable impact on the local geometry at the nick site, indicated clearly by NMR analysis and reflected in the conformational parameters of the computed structure. The 1H spectra also show much sharper resonances in the presence of K+ indicating that conformational heterogeneity of DDSDPEG is reduced in the presence of potassium as compared to sodium or caesium ions. At the same time the 1H resonances have longer T1 times. This parameter is suggested as a sensitive gauge of stabilisation.

* To whom correspondence should be addressed at: Institute of Organic Chemistry, Polish Academy of Sciences, 01-224 Warszawa, Kasprzaka 44, Poland. Tel: +48 22 632 3221; Fax: +48 22 632 6681; Email: lkoz@ichf.edu.pl


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