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Albany 2001

category image Biomolecular
Stereodynamics
SUNY at Albany
June 19-23, 2001

Interaction of EtBr and NMM-porphyrin with the parallel-stranded DNA formed by 3'-d(GTGTGTGTGG)-pO(CH2CH2O)3p-d(GGTGTGTGTG)-3' oligonucleotide

The GT repeats are abundant in telomeres, microsatellites and regulatory regions of genomes. The formation of quadruplex structure by two self-folded oligonucleotides 3'-d(GT)5-pO(CH2CH2O)3p-d(GT)5-3' have been reported [1]. Conformational diversity of the GT repetitive sequence has been observed earlier [2], where the substitution of the terminal T for G in d(GT)5 strand led to formation of a novel parallel-stranded (ps) intramolecular double helix with GG and TT bases (hp-GT) [2]. In the present work we continued investigation of the conformational features of the hp-GT ps-DNA. The ps double helix was probed with the intercalator EtBr and porphyrin (NMM) [4]. The DNA duplexes both with parallel (ps-t1) [3] and antiparallel (aps) strands were used as the references.

The following oligonucleotides were studied:
3'-d(GTGTGTGTGG)-L-dGGTGTGTGTG)-3' (hp-GT);
3'-d(TC)6-L-d(isoGA)6-3' (ps-t1);
5'-d(CATGCTAACT)-L-d(AGTTAGCATG)-3' (dsARB),
where L = pO(CH2CH2O)3p.

All experiments were run in 10 mM Na-phosphate buffer, pH 7, 0.1 M NaCl, at 3¼C. The EtBr binding isotherms were obtained by fluorescence emission, which is known to originate from intercalated EtBr molecules with a high quantum yield [2-3]. The parameters of EtBr anticooperative binding to the ps and aps hairpins were determined using a statististical mechanical treatment for binding ligands with exclusion length to a finite lattice [3]. The free energy of EtBr binding was found to be close for the both type duplexes: DG(hp-GT) = (6.6±0.1) kcal/mol; D G (ps-tl) = (6.8±0.1) kcal/mol; D G (dsARB) = (6.4±0.1) kcal/mol. Unlike this, the mean EtBr exclusion length (n) of the two ps duplexes was found to be less than that of the aps duplex dsARB: n (hp-GT) = (2.4±0.1) bp; n(ns-tl) = (2.1±0.1) bp; n(dsARB) = (3.0±0.2) bp. These data imply that the ps DNA conformational flexibility permits a maximal (~50%) extension of the structure upon binding of the intercalator.

The NMM porphyrin ligand is known to be a specific ligand for a DNA tetraplex structure [4]. Its binding to the aps Watson-Crick duplex is negligible. Here we have shown that the ps DNA hp-GT practically does not interact with NMM, similar to the aps double helix.

References and Footnotes
  1. O.F. Borisova, A K. Shchyolkina, E.N. Timofeev, V.L. Florentiev, FEBS Letters, 306, 140-142, 1992.
  2. A.K. Shchyolkina, O.F. Borisova, M.A. Livshits, R. Klement, T. Jovin, J. Biomol. Struct. Dynam., 18, 2001, in press.
  3. A.K. Shchyolkina, O.F. Borisova, T. Jovin, Nucleosides and Nucleotides, 18, 1555-1562, 1999.
  4. H. Arthanary, S. Basu, T.L. Kawano, P.H. Bolton, Nucl. Acids Res., 26, 3724-3728, 1998.

Olga F. Borisova and Anna K. Shchyolkina.

Engelhardt Institute of Molecular Biology RASc, Vavilova 32, Moscow, 119991, Russia.
Tel: 7095-1359789; Fax: 7095-1351405; E-mail: borisova@genome.eimb.relarn.ru