Eugenia M. Ivanova

Antisense activity of an anti-HIV oligonucleotide conjugated to linear and branched high molecular weight polyethylene glycols☆

An anti-HIV 12mer oligonucleotide (ODN) conjugated to two different high molecular weight monomethoxy polyethylene glycols (MPEGs) has been tested for its antisense activity. The capacity of these conjugates to protect the MT-4 cells against HIV infection has been compared to the unmodified, native ODN, and the effect of the different structures of the supporting polymer has been discussed. It was found that only the ODN conjugated to the linear MPEG shows an anti-HIV activity in the investigated conditions. The same 12mer, when conjugated to a branched (MPEG)2, is fully inactive, as well as the native, unmodified ODN.

Hybridization of the Bridged Oligonucleotides with DNA: Thermodynamic and Kinetic Studies

Abstract Hybridization properties of oligonucleotides containing non-nucleotide inserts designed on the basis of synthetic abasic sites, oligomethylene diols or oligoethylene glycols have been characterized. The influence of the inserts which generate extrahelical anucleotidic bulges on thermodynamics, kinetics of hybridization of bridged oligonucleotide with DNA has been studied by UV-melting and stopped-flow techniques. Circular dichroism spectrometry data show that anucleotidic bulges in the middle of the duplex does not alter the B-form helix conformation. Nevertheless, the insert induces destabilization of the duplex structure, caused mostly by the considerable enhancement of the dissociation rates. Free energy increments for the extrahelical anucleotidic bulges can be described in the nearest-neighbor approximation. The thermodynamic effect of the insert lengthening obeys a simple Jacob- son-Stockmayer entropy extrapolation. Independently of the insert type, the free energy term is directly proportional to the logarithm of the number of bonds between the oligo- nucleotide fragments. The behavior of hydrophobic inserts formed by 10-hydroxydecyl-1-phospate units is an exception to the rule.

The Influence of Nearest Neighbours on the Efficiency of Coaxial Stacking at Contiguous Stacking Hybridization of Oligodeoxyribonucleotides

Contiguous stacking hybridization of oligodeoxyribonucleotides with a stem of preformed minihairpin structure of a DNA template was studied with the use of UV‐melting technique. It was shown that the free‐energy of the coaxial stacking interaction (ΔG°ST at 37°C, 1 M NaCl, pH 7.4) at the complementary interface XA*pTY/ZATV (an asterisk stands for a nick) strongly depends on the type of nearest neighbor bases X and Y flanking the nicked dinucleotide step. The maximum efficiency of the coaxial stacking was observed for the PuA*pTPy/PuATPy interface, whereas the minimum efficiency was obtained for the PyA*pTPu/PyATPu interface. A 5′‐phosphate residue in the nick enhances the coaxial stacking. In dependence on duplex structure the observed efficiency of A*T/AT coaxial stacking varied from (− 0.97 kcal/mol) for unphosphorylated TA*TA/TATA interface to three‐fold higher value (− 2.78 kcal/mol) for GA*pTT/AATC interface.

BIOLOGICAL PROPERTIES OF ANTISENSE OLIGONUCLEOTIDES CONJUGATED TO DIFFERENT HIGH-MOLECULAR MASS POLY(ETHYLEN GLYCOLS)

Abstract The effect of the different structures of high-molecular weight poly(ethylen glycol) chains on the biological properties of the conjugated antisense oligonucleotide has been investigated and compared.

Thermodynamic Analysis of Stacking Hybridization of Oligonucleotides with DNA Template

Abstract Contiguous stacking hybridization of oligodeoxyribonucleotides with DNA as template was investigated using three types of complexes: oligonucleotide contiguously stacked with the stem of the preformed minihairpin (complexes I), oligonucleotide tandems containing two (complexes II) or three (complexes III) short oligomers with a common DNA template. Enthalpy ΔH° and entropy ΔS° of the coaxial stacking of adjacent duplexes were determined for GC/G*pC, GT/A*pC, AC/G*pT, AT/A*pT, CT/A*pG, AG/C*pT, AA/T*pT and TT/A*pA nicked (*) dinucleotide base pairs. The maximal efficiency of co-operative interaction was found for the GC/G*pC interface (ΔG°NN/N*pN=-2.7 kcal/mol) and the minimal one for the AA/T*pT interface (ΔG°/NN/N*pN=-1.2 kcal/mol) at 37 °C. As a whole, the efficiency of the base pairs interaction ΔG°NN/N*pN in the nick is not lower than that within the intact DNA helix ΔG°NN/NN). These observed the ΔG°NN/N*pN values are proposed may include the effect of the partial removal of fraying at the adjacent helix ends additionally to the effect of the direct stacking of the terminal base pairs in the duplex junction (ΔG°NN/NN. The thermodynamic parameters have been found to describe adequately the formation of all tandem complexes of the II and III types with oligonucleotides of various length and hybridization properties. The performed thermodynamic analysis reveals features of stacking oligonucleotide hybridization which allow one to predict the temperature dependence of association of oligonucleotides and the DNA template within tandem complexes as well as to determine optimal concentration for formation of these complexes characterized by high co-operativity level.

The Influence of the Non‐Nucleotide Insert on the Hybridization Properties of Oligonucleotides

The effect of different non‐nucleotide inserts incorporated into oligonucleotide chains on their hybridization properties was studied by the method of thermal denaturation. Various types of alkyldiols and oligoethylene glycols were used as inserts modifying oligonucleotide backbone. Such modification of oligonucleotides caused the destabilization of their complementary complexes. It was shown that the hybridization properties of the modified oligonucleotides depend on several features of inserts: the type, number, length of insertions, and positions of interrupted dinucleotide steps in oligonucleotide chain.

Efficiency of coaxial stacking depends on the DNA duplex structure

Abstract Thermodynamic parameters of coaxial stacking at complementary helix-helix interfaces GX*pYG/CZVC (X, Y = A, C, T, G; *—nick) created by contiguous oligonucleotide hybridization were determined. The data obtained were compared to the thermodynamic parameters of coaxial stacking at the interfaces CX*pYC/GZVG. Multiple linear regression analysis has revealed that the free-energy increments of interaction for the contacts GX*pYG/CZVC and CX*pYC/GZVG can be described by a set of uniform ΔG°X*pY/zv values. The difference in the observed free-energy of the coaxial stacking between the two sets is defined by the contribution from the factors reflecting structural differences between compared DNA duplexes.

G-specific RNA-cleaving Conjugates of Short Peptides and Oligodeoxyribonucleotides

Abstract Artificial ribonucleases, conjugates of short oligodeoxyribonucleotides and peptides built of arginine, leucine, proline, and serine, were synthesized and assessed in terms of ribonuclease activity and specificity of RNA cleavage. A specific group of the conjugates was identified that display T1-ribonuclease-like activity and cleave RNA predominantly at G-X sequences. Circular dichroism study of the structures of the most active conjugates, free peptide (LR)4G, and oligonucleotides revealed that conjugation of oligonucleotide to the peptide results in a specific peptide folding that possibly provides ribonuclease activity to the conjugate.

Short Oligonucleotide Tandem Ligation Assay for Genotyping of Single-Nucleotide Polymorphisms in Y Chromosome

We propose a novel universal methodology, Short Oligonucleotide Tandem Ligation Assay (SOTLA), for SNP genotyping. SOTLA is based on using a tandem of short oligonucleotide (TSO) probes consisting of three fragments: the core oligonucleotide and two flanking oligomers, one of which is immobilized onto a solid support and another one contains the biotin label. TSO is self-associated on a complementary DNA template, forms the complex containing two nicks, which are efficiently ligated with DNA ligase giving biotinylated oligonucleotide covalently bound to polymer beads. No ligation of TSO on an imperfect DNA template bearing the base substitution in the core binding site is occurred. We used SOTLA for the highly selective SNP analysis in different DNA fragments of human Y chromosome. Comparison of SOTLA results with those of PCR-RFLP and allele-specific PCR techniques demonstrates that SOTLA ensures the univocal reliable SNP analysis in different PCR fragments varying in length and base composition. The fundamental difference between SOTLA and well known OLA approaches while using T4 DNA ligase is that the accuracy of SNP analysis in OLA is ensured only by the specificity of ligase while that in SOTLA is provided by the specificity of both ligation and hybridization of TSO probes.

Steroid and N-(2-Hydroxyethyl)phenazinium Oligodeoxynucleotides

Abstract Oligonucleotide derivatives carrying steroid and N-(2-hydroxyethyl)phenazinium (Phn) residues were synthesized, and tested for the ability to form complexes.