Novel Method for the Synthesis of 2′ -Phosphorylated Oligonucleotides

We have developed a new method for the preparation of oligodeoxyribonucleotides and oligo(2′-O-methylribonucleotides) that contain a 2′-phosphorylated ribonucleoside residue, and optimized it to avoid 2′ -3′ -isomerization and chain cleavage. Structures of the 2′ -phosphorylated oligonucleotides were confirmed by MALDI-TOF MS and enzymatic digestion, and the stability of their duplexes with DNA and RNA was investigated. 2′-Phosphorylated oligonucleotides may be useful intermediates for the introduction of various chemical groups for a wide range of applications.     

Binary hammerhead ribozymes with high cleavage activity

Binary hammerhead ribozymes consisted of two oligoribonucleotides capable of assembling into hammerhead structure (without loop II) on the RNA target were engineered. Catalytic activities of such ribozymes were investigated in comparison with their full-length analog and ribozyme where two strands were jointed by non-nucleotidic linker. Binary constructs were shown to be significantly more active than the parent full-length hammerhead ribozyme.     

Functionalization of the Oligonucleotides Containing an Internucleotide Phosphoramidate Bond

A new method for functionalization of oligonucleotides by addition of aminoalkyl derivatives to the intermolecular phosphorus atom of the oligonucleotide N3"–P5" phosphoramidate bond in the presence of triphenylphosphine, 4-dimethylaminopyridine, and 2,2"-dipyridyl disulfide was suggested. The reaction proceeded with both low-molecular alkylamines (1,6-diaminohexane or N,N-dimethyl-1,3-diaminopropane) and a minor groove binder containing an aminoalkyl group.     

Head-to-head bis-hairpin polyamide minor groove binders and their conjugates with triplex-forming oligonucleotides: studies of interaction with target double-stranded DNA

Two hairpin hexa(N-methylpyrrole)carboxamide DNA minor groove binders (MGB) were linked together via their N-termini in head-to-head orientation. Complex formation between these bis-MGB conjugates and target DNA has been studied using DNase I footprinting, circular dichroism, thermal dissociation, and molecular modeling. DNase I footprint revealed binding of these conjugates to all the sites of 492 b.p. DNA fragment containing (A/T)(n)X(m)(A/T)(p) sequences, where n>3, p>3; m=1,2; X = A,T,G, or C. Binding affinity depended on the sequence context of the target. CD experiments and molecular modeling showed that oligo(N-methylpyrrole)carboxamide moieties in the complex form two short antiparallel hairpins rather than a long parallel head-to-head hairpin. Binding of bis-MGB also stabilized a target duplex thermodynamically. Sequence specificity of bis-MGB/DNA binding was validated using bis-conjugates of sequence-specific hairpin (N-methylpyrrole)/(N-methylimidazole) carboxamides. In order to increase the size of recognition sequence, the conjugates of bis-MGB with triplex-forming oligonucleotides (TFO) were synthesized and compared to TFO conjugated with single MGB hairpin unit. Bis-MGB-oligonucleotide conjugates also bind to two blocks of three and more A.T/T.A pairs similarly to bis-MGB alone, independently of the oligonucleotide moiety, but with lower affinity. However, the role of TFO in DNA recognition was demonstrated for mono-MGB-TFO conjugate where the binding was detected mainly in the area of the target sequence consisting of both MGB and TFO recognition sites. Basing on the molecular modeling, three-dimensional models of both target DNA/bis-MGB and target DNA/TFO-bis-MGB complexes were built, where bis-MGB forms two antiparallel hairpins. According to the second model, one MGB hairpin is in the minor groove of 5'-adjacent A/T sequence next to the triplex-forming region, whereas the other one occupies the minor groove of the TFO binding polypurine tract. All these data together give a key information for the construction of MGB-MGB and MGB-oligonucleotide conjugates possessing high specificity and affinity for the target double-stranded DNA.     

3'-modified oligo (2'-O-methylribonucleotides) as improved probes for hybridization with RNA

A series of octa (2-O-methylribonucleotides) with an additional 3'-terminal deoxynucleoside (T, dC, dA or dG) linked by the 3'-3' (inverted) bond was synthesized. The exceptional stability of these oligomers to a 3'-exonuclease (SVP) and nucleases in culture medium containing 10% heat-inactivated fetal calf serum was demonstrated. It was shown that the addition of the 3'-dangling inverted deoxynucleoside increases substantially the thermal stability of the duplexes of oligo(2'-O-methylribonucleotides) with complementary RNA and DNA in the case of a relatively weak terminal AmU(T) pair and enhances the mismatch sensitivity.     

Sequence-specific conjugates of oligo(2'-O-methylribonucleotides) and hairpin oligocarboxamide minor-groove binders: design, synthesis, and binding studies with double-stranded DNA

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 (Tm), faster complex formation, and lower dissociation constants (Kd) 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 a 3',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 74 degrees). However, under these conditions, binding was retained only by the MGB part of the conjugate.     

Mutational and Kinetic Analysis of APE1 Endoribonuclease Activity

Molecular Biology - Human apurinic/apyrimidinic endonuclease 1 (APE1) participates in the DNA repair system. It is believed that the main biological function of APE1 is Mg2+-dependent hydrolysis of...     

Photoactivatable CRISPR/Cas9 System

Russian Journal of Bioorganic Chemistry - A photoactivatable CRISPR/Cas9 system consisting of the Cas9 protein, synthetic 102-nt sgRNA or a pair of guide crRNA/tracrRNA, and blocking photocleavable...