Edward N. Timofeev

Synthesis, characterization and in vitro activity of thrombin-binding DNA aptamers with triazole internucleotide linkages

A series of DNA aptamers bearing triazole internucleotide linkages that bind to thrombin was synthesized. The novel aptamers are structurally analogous to the well-known thrombin-inhibiting G-quadruplexes TBA15 and TBA31. The secondary structure stability, binding affinity for thrombin and anticoagulant effects of the triazole-modified aptamers were measured. A modification in the central loop of the aptamer quadruplex resulted in increased nuclease resistance and an inhibition efficiency similar to that of TBA15. The likely aptamer-thrombin binding mode was determined by molecular dynamics simulations. Due to their relatively high activity and the increased resistance to nuclease digestion imparted by the triazole internucleotide linkages, the novel aptamers are a promising alternative to known DNA-based anticoagulant agents.

Comparison of the 'chemical' and 'structural' approaches to the optimization of the thrombin-binding aptamer.

Noncanonically structured DNA aptamers to thrombin were examined. Two different approaches were used to improve stability, binding affinity and biological activity of a known thrombin-binding aptamer. These approaches are chemical modification and the addition of a duplex module to the aptamer core structure. Several chemically modified aptamers and the duplex-bearing ones were all studied under the same conditions by a set of widely known and some relatively new methods. A number of the thrombin-binding aptamer analogs have demonstrated improved characteristics. Most importantly, the study allowed us to compare directly the two approaches to aptamer optimization and to analyze their relative advantages and disadvantages as well as their potential in drug design and fundamental studies.

Evidence for the tetraplex structure of the d(GT)n repetitive sequences in solution

The ability of oligonucleotides 3′‐d(GT)5pO(CH2)6Opd(GT)5‐5′(anti[d(GT)]) and 3′‐d(GT)5pO(CH2)6Opd(GT)5‐3′(par[d(GT)]) to form tertiary structures has been studied. Circular dichroism (CD) as well as the fluorescence of the ethidium bromide (E1Br) complexes with oligonucleotides and hydrodynamic volume measurements in solutions containing 0.01 M phosphate buffer, pH 7 and NACl in concentrations from 0.1 M to 1 M, have been used. The data obtained in the temperature interval from 30°C to 10°C are in good agreement with the structure suggested earlier [1] where the par[d(GT)] and anti[d(GT)] form structures with four parallel strands in which layers of four G‐residues alternate with unpaired bulged‐out T‐residues. Ethidium bromide interacts with the structure in a cooperative manner. Two ethidium bromide molecules intercalate between two layers of four G‐residues.

Parallel purine-pyrimidine-purine triplex: experimental evidence for existence

Oligonucleotides 5′‐d(CT)5‐L‐d(AG)5‐L‐d(GA)5‐3′ and 5′‐d(GA)5‐L‐d(TC)5‐L‐d(GA)5‐3′ [L = pO(CH2CH2O)3p] were studied by thermal denaturation, chemical modification and binding of fluorescent dyes. Both oligonucleotides are shown to fold back on itself twice forming at pH 7 a sufficiently stable triplex ether with antiparallel‐oriented oligopurine strands (the first compound) or parallel‐oriented oligopurine strands (the second compounds). The parallel triplex is significantly less stable than the antiparallel one. On the basis of conformational modeling, possible types of base tripling in the triplets are proposed. Thus our data provide the first convincingly evidence for the existence of a purine‐pyrimidine‐purine triplex with parallel orientation of identical strands.

Partial thermodynamic parameters for prediction stability and washing behavior of DNA duplexes immobilized on gel matrix.

Earlier we showed that reported in literature nearest-neighbor thermodynamic parameters describe poorly the thermal-induced behavior of DNA duplexes immobilized in gel. Here we present a complete set of partial thermodynamic parameters for all 10 nearest-neighbor interactions specially developed for duplexes immobilized in gel. This thermodynamic library allows to predict dissociation enthalpy and free energy of DNA duplex immobilized in gel matrix from its base sequence. The predicted values are in good agreement with the experimental ones. Dissociation enthalpy and free energy are needed for such application as (i) predicting relative stability of duplexes formed by DNA with oligonucleotides immobilized in cells of gel matrix; (ii) selecting optimal conditions for hybridization experiment; (iii) predicting washing curves and washing temperatures at irreversible temperature-stepped wash of DNA out of oligonucleotide gel matrix; (iv) selecting optimal conditions for washing gel matrix.

Targeting duplex DNA with chimeric α,β-triplex-forming oligonucleotides

Triplex-directed DNA recognition is strictly limited by polypurine sequences. In an attempt to address this problem with synthetic biology tools, we designed a panel of short chimeric α,β-triplex-forming oligonucleotides (TFOs) and studied their interaction with fluorescently labelled duplex hairpins using various techniques. The hybridization of hairpin with an array of chimeric probes suggests that recognition of double-stranded DNA follows complicated rules combining reversed Hoogsteen and non-canonical homologous hydrogen bonding. In the presence of magnesium ions, chimeric TFOs are able to form highly stable α,β-triplexes, as indicated by native gel-electrophoresis, on-array thermal denaturation and fluorescence-quenching experiments. CD spectra of chimeric triplexes exhibited features typically observed for anti-parallel purine triplexes with a GA or GT third strand. The high potential of chimeric α,β-TFOs in targeting double-stranded DNA was demonstrated in the EcoRI endonuclease protection assay. In this paper, we report, for the first time, the recognition of base pair inversions in a duplex by chimeric TFOs containing α-thymidine and α-deoxyguanosine.

A Universal Base in a Specific Role: Tuning up a Thrombin Aptamer with 5-Nitroindole

In this study we describe new modified analogs of the thrombin binding aptamer (TBA) containing 5-nitroindole residues. It has been shown that all modified TBAs form an anti-parallel G-quadruplex structure and retain the ability to inhibit thrombin. The most advanced TBA variant (TBA-N8) has a substantially increased clotting time and two-fold lower IC50 value compared to the unmodified prototype. Molecular modelling studies suggest that the improved anticoagulant properties of TBA-N8 result from changes in the binding mode of the analog. A modified central loop in TBA-N8 is presumed to participate in the binding of the target protein. Studies of FAM labelled TBA and TBA-N8 showed an improved binding affinity of the modified aptamer and provided evidence of a direct interaction between the modified central loop and thrombin. Our findings have implications for the design of new aptamers with improved binding affinities.

Simple and Stereoselective Preparation of an 4‐(Aminomethyl)‐1,2,3‐triazolyl Nucleoside Phosphoramidite

A simple and stereoselective synthesis of a protected 4‐(aminomethyl)‐1‐(2‐deoxy‐β‐D‐ribofuranosyl)‐1,2,3‐triazole cyanoethyl phosphoramidite was developed for the modification of synthetic oligonucleotides. The configuration of the 1,2,3‐triazolyl moiety with respect to the deoxyribose was unambiguously determined in ROESY experiments. The aminomethyl group of the triazolyl nucleotide was fully functional in labelling reactions. Furthermore, the hybridization behavior of 5′ triazole‐terminated oligonucleotide was similar to that of 5′ aminohexyl‐terminated oligomer with the same sequence. Internal modifications of the oligonucleotide strands resulted in significant decrease of duplex stability.

Anomeric DNA quadruplexes

Thrombin-binding aptamer (TBA) is a 15-nt DNA oligomer that efficiently inhibits thrombin. It has been shown that TBA folds into an anti-parallel unimolecular G-quadruplex. Its three-dimensional chair-like structure consists of two G-tetrads connected by TT and TGT loops. TBA undergoes fast degradation by nucleases in vivo. To improve the nuclease resistance of TBA, a number of modified analogs have been proposed. Here, we describe anomeric modifications of TBA. Non-natural α anomers were used to replace selected nucleotides in the loops and core. Significant stabilization of the quadruplex was observed for the anomeric modification of TT loops at T4 and T13. Replacement of the core guanines either prevents quadruplex assembly or induces rearrangement in G-tetrads. It was found that the anticoagulant properties of chimeric aptamers could be retained only with intact TT loops. On the contrary, modification of the TGT loop was shown to substantially increase nuclease resistance of the chimeric aptamer without a notable disturbance of its anticoagulant activity.

Oligodeoxynucleotides with extended zwitter-ionic internucleotide linkage

Two non-natural nucleoside analogues, N-(2-hydroxyethyl)-2′,5′-dideoxy-5′-aminothymidine (dTNH) and N-(2-hydroxyethyl)-N-methyl-2′,5′-dideoxy-5′-aminothymidine (dTNMe), have been prepared and used in the synthesis of oligodeoxythymidilates and mixed-sequence oligodeoxynucleotides, modified at internucleotide linkages. Both modified oligodeoxythymidilates and mixed-sequence oligodeoxynucleotides have been shown to form zwitter-ionic phosphate–amine pairs as evidenced by their decreased electrophoretic mobility in denaturing polyacrylamide gel.