Sequence-Specific Conjugates of Oligo(2'-O-methylribonucleotides) and HairpinOligocarboxamideMinor-GrooveBinders:Design,Synthesis,and BindingStudieswithDouble-StrandedDNA by DaryaS.Novopashina a ), AlexandreN.Sinyakov b ), Vladimir A. Ryabinin b ), AlyaG.Venyaminova a ), Ludovic Halby c ), Jian-Sheng Sun c ), and AlexandreS.Boutorine* c ) a ) Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences, pr. Lavrentieva 8, 630090 Novosibirsk, Russia (e-mail: danov@niboch.nsc.ru) b ) State Research Center of Virology and Biotechnology -Vector×, 630559 Kol×tsovo, Novosibirsk region, Russia (e-mail: sinyakov@niboch.nsc.ru) c ) USM 0503 Re ¬gulation et Dynamique des Genomes, Muse ¬um National d× Histoire Naturelle, INSERM U565 ± CNRS URA 5153, F-75231 Paris Cedex, France (phone:  33-140-79-36-96; fax:  33-140-79-37-05; e-mail: alexandre.boutorine@mnhn.fr) New conjugates of triplex-forming pyrimidine oligo(2'-O-methylribonucleotides) with one or two -head-to- head× hairpin oligo( N-methylpyrrole carboxamide) minor-groove binders (MGBs) attached to the terminal phosphate of the oligonucleotides with a oligo(ethylene glycol) linker were synthesized. It was demonstrated that, under appropriate conditions, the conjugates form stable complexes with double-stranded DNA (dsDNA) similarly to triplex-forming oligo(deoxyribonucleotide) (TFO) conjugates containing 5-methylated cytosines. Kinetic and thermodynamic parameters of the complex formation were evaluated by gel-shift assay and thermal denaturation. Higher melting temperatures ( T m ), faster complex formation, and lower dissociation constants ( K d ) of the triple helices (6 ± 7 nm) were observed for complexes of MGB-oligo(2'-O-methylribonucleotide) conjugates with the target dsDNA compared to the nonconjugated individual components. Interaction of MGB moieties with the HIV proviral DNA fragment was indicated by UV/VIS absorption changes at 320 nm in the melting curves. The introduction of thymidine via a3',3'-type -inverted× phosphodiester linkage at the 3'-end of oligo(2'-O-methylribonucleotide) conjugates (3'-protection) had no strong influence on triplex formation, but slightly affected complex stability. At pH 6.0, when one or two hairpin MGBs were attached to the oligonucleotide, both triplex formation and minor-groove binding played important roles in complex formation. When two -head-to-head× oligo( N-methylpyrrole) ligands were attached to the same terminal phosphate of the oligonucleotide or the linker, binding was observed at pH > 7.5 and at high temperatures (up to 748). However, under these conditions, binding was retained only by the MGB part of the conjugate. 1. Introduction. ± Molecules specifically interacting with double-stranded DNA (dsDNA) attract considerable interest because of their possible applications as biologically active compounds capable of acting on genomic DNA. Awide variety of in vitro and in vivo applications such as site-specific cleavage of DNA [1], mapping of genomic DNA, and selective regulation of gene expression could be envisaged [2][3]. Besides natural, specific peptides, two classes of synthetic molecules are known that recognize and bind specifically to double-stranded DNA sequences: triple-helix- forming oligonucleotides (TFOs) [4±6] and oligo( N-methylpyrrole)- and oligo( N- methylimidazole) carboxamides as minor-groove binders (MGBs) [7][8]. However, these two classes of sequence-specific agents have certain drawbacks. Oligocarboxamide MGBs with hairpin structures (two oligocarboxamide blocks CHEMISTRY & BIODIVERSITY ± Vol. 2 (2005) 936 ¹ 2005 Verlag Helvetica Chimica Acta AG, Z¸rich