Vladimir L. Florentiev

An oligonucleotide hybridization approach to DNA sequencing

DNA sequencing; Oligonucleotide hybridization, immobilized

Improved Chips for Sequencing by Hybridization

The SHOM method (Sequencing by Hybridization with Oligonucleotide Matrix) developed in 1988 is a new approach to nucleic acid sequencing by hybridization to an oligonucleotide matrix composed of an array of immobilized oligonucleotides. The original matrix proposed for sequencing by SHOM had to contain at least 65,536 octanucleotides. The present work describes a new family of matrices, which allows one to reduce the number of synthesized oligonucleotides 5-15 times without essentially decreasing the resolving power of the method.

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.

Parallel stranded DNA with AT base pairing

The concentration and temperature dependences of the UV and CD spectra of the oligonucleotide 3′‐d(ApTpApTpApTpApTpApTp)‐O(CH2)6O‐5′‐d(pApTpApTpApTpApTpApT) (eicosamer) in aqueous solution at pH 7 in the presence of 0.5 M NACl were studied. At less than 10−6 M, the eicosamer was shown to form in solution a hairpin with parallel orientation of chains (parallel hairpin). From thermal denaturation profiles [A 260(T)] the thermodynamic parameters, ΔH°, ΔS° and T m for parallel hairpin formation were calculated to be −90±8 kJ/mol. −300±20 J · mol−1 · K−1 and 40.5°C, respectively. The CD spectra of the parallel double helix differed from those of B‐form DNA and had characteristic features: decreasing magnitude of the positive maximum at 265 nm and a negative peak at 285 nm.

Dissociation of duplexes formed by hybridization of DNA with gel-immobilized oligonucleotides.

The method of DNA sequencing by hybridization with oligonucleotides matrix (SHOM) developed in this laboratory (1.2) uses the matrix of oligonucleotides immobilized within polyacrylamide gel. The particular feature of this matrix is that the apparent thermostability of the duplexes depends on the concentration of gel-immobilized oligonucleotides. This dependence is specific for oligonucleotides immobilized in the gel volume (3-D-immobilization) rather than on a flat surface of a filter or glass (2-D-immobilization). The theory has been developed that provides a quantitative description of temperature-dependent duplex dissociation within gel. The theory takes into account that the diffusion of dissociated DNA out of the gel is retarded by multiple acts of association-dissociation of DNA with immobilized oligonucleotides. It allows to calculate the apparent dissociation temperature of duplexes and describes quantitatively its growth upon increase in the enthalpy of duplex dissociation, concentration of immobilized oligonucleotides, gel thickness and decrease of dissociation entropy and washing time. Concentration of gel-immobilized oligonucleotides can be calculated for a normalized matrix in which GC-rich and AT-rich duplexes exhibit the same apparent thermostabilities and are washed off at the same temperature. This simplifies identification of perfect duplexes formed on the matrix which can be carried out for all duplexes at the same temperature. The gel-immobilized oligonucleotide matrix provides also a higher capacity for immobilization and therefore a higher sensitivity of measurements, resulting in a higher discrimination power for identification of perfect duplexes as compared with matrixes of oligonucleotides immobilized on a surface.

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.

Synthesis and hybridization data of oligonucleotide analogs with triazole internucleotide linkages, potential antiviral and antitumor agents

Triazolyl-functionalized oligonucleotide (ON) analogs have received much attention as potential antitumor and antiviral agents. The most promising of such analogs are those exhibiting high binding affinity toward native DNA/RNA, since they may prove to be efficient antisense or siRNA agents. To date, relatively few ON analogs with triazole internucleotide linkages have been described. In this paper, we report an improved synthesis of a modified dinucleoside phosphoramidite and hybridization data of ON analogs with four-bond triazole internucleotide linkages. We believe these data are essential for comprehensive analysis of the relation between the length of triazole internucleotide linkages and duplex stability.

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.