Modification of oligodeoxynucleotides by on-column Suzuki cross-coupling reactions.

The on-column functionalization of oligodeoxynucleotides via base-free Suzuki cross-coupling reactions is reported herein. These cross-coupling reactions were carried out with various boronic acids and either full-length modified oligonucleotides containing one or more 2'-deoxy-5-iodouridine (5IdU) monomer(s) or on oligonucleotide fragments immediately after incorporation of 5IdU. Five different functionalities were coupled to oligonucleotides containing one or three attachment points.

Synergy of Two Highly Specific Biomolecular Recognition Events: Aligning an AT-Hook Peptide in DNA Minor Grooves via Covalent Conjugation to 2'-Amino-LNA.

Two highly specific biomolecular recognition events, nucleic acid duplex hybridization and DNA-peptide recognition in the minor groove, were coalesced in a miniature ensemble for the first time by covalently attaching a natural AT-hook peptide motif to nucleic acid duplexes via a 2'-amino-LNA scaffold. A combination of molecular dynamics simulations and ultraviolet thermal denaturation studies revealed high sequence-specific affinity of the peptide-oligonucleotide conjugates (POCs) when binding to complementary DNA strands, leveraging the bioinformation encrypted in the minor groove of DNA duplexes. The significant cooperative DNA duplex stabilization may pave the way toward further development of POCs with enhanced affinity and selectivity toward target sequences carrying peptide-binding genetic islands.

Folding Topology of a Short Coiled-Coil Peptide Structure Templated by an Oligonucleotide Triplex.

The rational design of a well-defined protein-like tertiary structure formed by small peptide building blocks is still a formidable challenge. By using peptide-oligonucleotide conjugates (POC) as building blocks, we present the self-assembly of miniature coiled-coil α-helical peptides guided by oligonucleotide duplex and triplex formation. POC synthesis was achieved by copper-free alkyne-azide cycloaddition between three oligonucleotides and a 23-mer peptide, which by itself exhibited multiple oligomeric states in solution. The oligonucleotide domain was designed to furnish a stable parallel triplex under physiological pH, and to be capable of templating the three peptide sequences to constitute a small coiled-coil motif displaying remarkable α-helicity. The formed trimeric complex was characterized by ultraviolet thermal denaturation, gel electrophoresis, circular dichroism (CD) spectroscopy, small-angle X-ray scattering (SAXS), and molecular modeling. Stabilizing cooperativity was observed between the trimeric peptide and the oligonucleotide triplex domains, and the overall molecular size (ca. 12 nm) in solution was revealed to be independent of concentration. The topological folding of the peptide moiety differed strongly from those of the individual POC strands and the unconjugated peptide, exclusively adopting the designed triple helical structure.

3'-Pyrene-modified unlocked nucleic acids: synthesis, fluorescence properties and a surprising stabilization effect on duplexes and triplexes.

Efficient synthesis of a new 3'-O-amino-UNA monomer was developed as a scaffold for further functionalization and incorporation into oligonucleotides (ONs). Pyrene-functionalized 3'-O-amino-UNA was incorporated one, two or three times into 21-mer DNA and 2'-O-Me-RNA ONs. Duplex melting temperatures, circular dichroism (CD) spectra, steady-state fluorescence emission spectra, UV/Vis absorption spectra and triplex melting temperatures were measured for the modified duplexes. The presence of the pyrene-modified UNA monomer lead to a surprising and unprecedented thermal stabilization of especially DNA:DNA duplexes when compared to the corresponding unmodified DNA:DNA duplexes. Improved mismatch discrimination was also seen for some of the modified duplexes. CD spectra revealed no major differences between modified and unmodified duplexes. Molecular modeling showed that the pyrene moieties were located in the minor groove of DNA:DNA duplexes as confirmed by CD and UV/Vis absorption studies. Upon multiple incorporations of the monomer in single-stranded ONs, steady-state fluorescence emission studies revealed the formation of a pyrene excimer which in most cases was quenched upon duplex hybridization, and fluorescence-based detection of mismatched hybridization was observed for some modified strand constitutions. Incorporation of the monomer in a triplex-forming oligonucleotide (TFO) strand lead to an increase of triplex melting temperature both at pH 6.0 and pH 7.0 for parallel triplexes - again an effect that has not been reported earlier for UNA-containing ONs. Steady-state fluorescence emission studies revealed significant differences in fluorescence for single-stranded ONs and triplexes.

Bis-pyrene-modified unlocked nucleic acids: synthesis, hybridization studies, and fluorescent properties.

Efficient synthesis of a building block for the incorporation of a bis-pyrene-modified unlocked nucleic acid (UNA) into oligonucleotides (DNA*) was developed. The presence of bis-pyrene-modified UNA within a duplex leads to duplex destabilization that is more profound in DNA*/RNA and less distinct in DNA*/DNA duplexes. Nevertheless, the destabilization effect of bis-pyrene-modified UNA is weaker than that of unmodified UNA. Some oligonucleotides with bis-pyrene-modified UNA incorporations displayed superior mismatch discrimination capabilities. UV/Vis absorption and molecular modeling studies indicate that the pyrene groups of bis-pyrene-modified UNA are located in the major groove of a duplex. Oligonucleotides containing two bis-pyrene-modified UNA monomers showed low pyrene monomer emission in bulge-containing duplexes, high pyrene monomer emission in fully matched duplexes, and 5-(pyrenyl)uracil:pyrene exciplex emission in the single-stranded form. Such fluorescent properties enable the application of bis-pyrene-modified UNA in the development of fluorescence probes for DNA/RNA detection and for detection of deletions at specific positions.