Solid-phase synthesis and NMR studies of modified oligonucleotides forming triplex helix and of oligonucleopeptides mimicking the topoisomerase I-DNA covalent complex

Report for the scientific sojourn carried out at the Institut de Biologia Molecular de Barcelona of the CSIC –state agency – from april until september 2007. Topoisomerase I is an essential nuclear enzyme that modulates the topological status of DNA, facilitating DNA helix unwinding during replication and transcription. We have prepared the oligonucleotide-peptide conjugate Ac-NLeu-Asn-Tyr(p-3’TTCAGAAGC5’)-LeuC-CONH-(CH2)6-OH as model compound for NMR studies of the Topoisomerase I- DNA complex. Special attention was made on the synthetic aspects for the preparation of this challenging compound especially solid supports and protecting groups. The desired peptide was obtained although we did not achieve the amount of the conjugate needed for NMR studies. Most probably the low yield is due to the intrinsic sensitive to hydrolysis of the phosphate bond between oligonucleotide and tyrosine. We have started the synthesis and the structural characterization of oligonucleotides carrying intercalating compounds. At the present state we have obtained model duplex and quadruplex sequences modified with acridine and NMR studies are underway. In addition to this project we have successfully resolved the structure of a fusion peptide derived from hepatitis C virus envelope synthesized by the group of Dr. Haro and we have synthesized and started the characterization of a modified G-quadruplex.

A minimal i-motif stabilized by minor groove G:T:G:T tetrads

The repetitive DNA sequences found at telomeres and centromeres play a crucial role in the structure and function of eukaryotic chromosomes. This role may be related to the tendency observed in many repetitive DNAs to adopt non-canonical structures. Although there is an increasing recognition of the importance of DNA quadruplexes in chromosome biology, the co-existence of different quadruplex-forming elements in the same DNA structure is still a matter of debate. Here we report the structural study of the oligonucleotide d(TCGTTTCGT) and its cyclic analog d. Both sequences form dimeric quadruplex structures consisting of a minimal i-motif capped, at both ends, by a slipped minor groove-aligned G:T:G:T tetrad. These mini i-motifs, which do not exhibit the characteristic CD spectra of other i-motif structures, can be observed at neutral pH, although they are more stable under acidic conditions. This finding is particularly relevant since these oligonucleotide sequences do not contain contiguous cytosines. Importantly, these structures resemble the loop moiety adopted by an 11-nucleotide fragment of the conserved centromeric protein B (CENP-B) box motif, which is the binding site for the CENP-B.

A minimal i-motif stabilized by minor groove G:T:G:T tetrads

The repetitive DNA sequences found at telomeres and centromeres play a crucial role in the structure and function of eukaryotic chromosomes. This role may be related to the tendency observed in many repetitive DNAs to adopt non-canonical structures. Although there is an increasing recognition of the importance of DNA quadruplexes in chromosome biology, the co-existence of different quadruplex-forming elements in the same DNA structure is still a matter of debate. Here we report the structural study of the oligonucleotide d(TCGTTTCGT) and its cyclic analog d. Both sequences form dimeric quadruplex structures consisting of a minimal i-motif capped, at both ends, by a slipped minor groove-aligned G:T:G:T tetrad. These mini i-motifs, which do not exhibit the characteristic CD spectra of other i-motif structures, can be observed at neutral pH, although they are more stable under acidic conditions. This finding is particularly relevant since these oligonucleotide sequences do not contain contiguous cytosines. Importantly, these structures resemble the loop moiety adopted by an 11-nucleotide fragment of the conserved centromeric protein B (CENP-B) box motif, which is the binding site for the CENP-B.

Solution equilibria of cytosine- and guanine-rich sequences near the promoter region of the n-myc gene that contain stable hairpins within lateral loops

Cytosine-and guanine-rich regions of DNA are capable of forming complex structures named i-motifs and G-quadruplexes, respectively. In the present study the solution equilibria at nearly physiological conditions of a 34 -bases long cytosine-rich sequence and its complementary guanin e-rich strand corresponding to the first intron of the n-mycgene were studied. Both sequences , not yet studied, contain a 12 - base tract capable of forming stable hairpins inside the i-motif and G-quadruplex structures, respectively ...