A 14 nt DNA sequence 5'-AGAATGTGGCAAAG-3' from the zinc finger repeat of the human KRAB zinc finger protein gene ZNF91 bearing the intercalator 2-methoxy,6-chloro,9-amino acridine (Acr) attached to the sugar-phosphate backbone in various positions has been shown to form a specific triple helix (triplex) with a 16 bp hairpin (intramolecular) or a two-stranded (intermolecular) duplex having the identical sequence in the same (parallel) orientation. Intramolecular targets with the identical sequence in the antiparallel orientation and a non-specific target sequence were tested as controls. Apparent binding constants for formation of the triplex were determined by quantitating electrophoretic band shifts. Binding of the single-stranded oligonucleotide probe sequence to the target led to an increase in the fluorescence anisotropy of acridine. The parallel orientation of the two identical sequence segments was confirmed by measurement of fluorescence resonance energy transfer between the acridine on the 5'-end of the probe strand as donor and BODIPY-Texas Red on the 3'-amino group of either strand of the target duplex as acceptor. There was full protection from OsO(4)-bipyridine modification of thymines in the probe strand of the triplex, in accordance with the presumed triplex formation, which excluded displacement of the homologous duplex strand by the probe-intercalator conjugate. The implications of these results for the existence of protein-independent parallel triplexes are discussed.

The preparation of C'-methylnucleosides and their phosphoric esters as well as their physicochemical and substrate properties in various enzymic reactions are reviewed. The use of functionally competent analogs for the determination of the substrate conformation during enzymic reactions is summarized.

The number of synthetic UTP analogues containing methyl groups in different positions of the ribose moiety were tested as substrates for T7 RNA polymerase (T7 RNAP). Two of these compounds (containing substituents in the 5' position) were shown to be weak substrates of T7 RNAP. 3'Me-UTP was neither substrate nor inhibitor of T7 RNAP while 2'Me-UTP was shown to terminate RNA chain synthesis. Conformational analysis of the analogues and parent nucleotide using the force-field method indicates that the allowed conformation of UTP during its incorporation into the growing RNA chain by T7 RNAP is limited to the chi angle range of 192-256 degrees of N-type conformation.

The aim of this work is to determine the conformation of the nucleobase adjacent to the cleavable phosphodiester bond in the productive enzyme-substrate complex of RNA-depolymerizing enzymes. To this end the kinetic parameters of hydrolysis of UpA, 2'-C-Me- and 3'-C-Me-UpA were determined for RNase A, RNase Pb2, nuclease S1 and snake venom phosphodiesterase. In these derivatives the ranges of the allowed orientation of uridine residues are restricted due to the substitution of methyl groups for the ribose hydrogen atoms. The results described demonstrate that the proposed method is of general value for the estimation of the nucleotide glycoside angles in the productive enzyme-substrate complexes.

DNA sequencing by hybridization on oligonucleotide microchip (SHOM) allows the determination of a spectrum of overlapping oligonucleotides constituting a DNA fragment that hybridizes to form perfect duplexes with an array of immobilized oligonucleotides and, as a result, enables reconstitution of the nucleotide sequence of the fragment. In longer DNA fragments, unambiguous reconstitution of DNA sequence is often impeded by the presence of repetitive regions and simple sequence repeats. Here it is demonstrated that SHOM supplemented by measurement of the distance between certain sites (for example, restriction sites or priming sites for PCR) within the analyzed DNA enables sequencing of much longer DNA fragments, containing repeats of different complexity.

A study was made of the potentialities of DNA sequencing by hybridization with oligonucleotide matrix (SHOM) in the version of contiguous stacking hybridization on gapped matrices. The reconstruction efficacy was assessed for various combinations of matrices and l-oligonucleotide libraries. It was shown that contiguous stacking hybridization on gapped matrices allows the number of additional hybridizations to be cut down by half without loss of reconstruction efficacy.

Temperature dependence of UV and CD spectra of the oligonucleotide 5'-(dT)10-L-(dT)10-L-(dA)10-3' [tripl(ATT)] [L = -pO(CH2CH2O)3p-] in phosphate buffer, pH 7, at various NaCl concentrations and in the presence or absence of 0.01 M MgCl2 has been studied. At low oligonucleotide concentrations (2.2 x 10(-5) M nucleotide concentration) all structural transitions proceed intramolecularly. Tripl(ATT) exists in three forms: as a three-stranded clip (at low temperatures), a double-stranded hairpin (at intermediate temperatures), and as an open strand (at high temperatures). Thermodynamic parameters of the triplex formation depending on the NaCl concentration were calculated. The CD spectra were assigned to the single-, double-, and three-stranded forms. Ethidium bromide (EtBr) binding to the three-stranded clip was studied. Ethidium bromide molecules were shown to intercalate into the triple helix with the stable complex formation (association constant is 10(6)). One molecule of three-stranded clip binds not more than three EtBr molecules. The proposed synthetic model (oligonucleotide blocks coupled by hydroxyalkyl chains) has been shown to be convenient for studies of the physical and chemical properties of the triplex and other multistranded complexes of DNA.

Temperature dependence of UV and CD spectra of the oligonucleotide 5'-(dT)10-L-(dT)10-L-(dA)10-3' [tripl(ATT)] [L = -pO(CH2CH2O)3p-] in phosphate buffer, pH 7, at various NaCl concentrations and in the presence or absence of 0.01 M MgCl2 has been studied. At low oligonucleotide concentrations (2.2 x 10(-5) M nucleotide concentration) all structural transitions proceed intramolecularly. Tripl(ATT) exists in three forms: as a three-stranded clip (at low temperatures), a double-stranded hairpin (at intermediate temperatures), and as an open strand (at high temperatures). Thermodynamic parameters of the triplex formation depending on the NaCl concentration were calculated. The CD spectra were assigned to the single-, double-, and three-stranded forms. Ethidium bromide (EtBr) binding to the three-stranded clip was studied. Ethidium bromide molecules were shown to intercalate into the triple helix with the stable complex formation (association constant is 10(6)). One molecule of three-stranded clip binds not more than three EtBr molecules. The proposed synthetic model (oligonucleotide blocks coupled by hydroxyalkyl chains) has been shown to be convenient for studies of the physical and chemical properties of the triplex and other multistranded complexes of DNA.

DNA sequencing by hybridization with a matrix of immobilized oligonucleotides is not directly is not directly applicable for fragments with long monotonous repeats. Auxiliary information for this purpose can be obtained from the distances between certain segments within the DNA fragment examined, using DNA restriction or PCR with defined primers followed by measuring the length of the resulting segments in gel electrophoresis. Such additional information increases the reconstruction efficiency and in many cases solves the problem of repeating and monotonous segments. The work demonstrates the use and appraises the usefulness of this information.

In this paper we consider the efficiency of additional rounds of "continuous stacking" hybridization in DNA sequence reconstruction by hybridization with oligonucleotide matrix (SHOM). After the initial hybridization of target DNA with the matrix of oligonucleotides of fixed length L some additional hybridizations should be carried out in the presence of fluorescently labeled oligonucleotides of another length l. These additional oligonucleotides can hybridize in tandem with matrix tuples (continuous stacking hybridization) thus forming an extended duplex with the target DNA strand. The additional data obtained allows resolutions of branching points arising in the reconstruction procedure. Multiple rounds of continuous stacking hybridization considerably increase the efficiency of the sequencing method, eventually approaching the power of (L+l)-matrix. We develop here an algorithm that allows us to minimize the number of additional hybridization steps, by assembling sets of l-tuples to be added together in each round of continuous stacking hybridization. For SHOM using a matrix of octanucleotides, continuous stacking hybridization with pentanucleotides increases the length of unambiguously sequenced DNA from 200 to several thousands of base pairs.

DNA sequencing efficacy can be enhanced through extending the duplex length whereby L-oligonucleotides are added in solution to a matrix of immobilized L-oligonucleotides. The reconstruction efficacy has been estimated for fragments up to 30,000 units, considering various L+l combinations. The results obtained allow an assessment of the potentialities of the proposed technique with various matrices, as well as of the experimental labor involved. Contiguous stacking hybridization substantially increases the length of DNA fragments that can be sequenced. An approach is proposed to solve ambiguities at branch points caused by long repeats, using contiguous stacking hybridization to form a concatenation of several added oligonucleotides stabilized by pair stacking interactions.

An open CAN format (Compressed Aminoacids and Nucleotides) is presented for storing genetic information in compressed form in data banks (DB). The data compression principles are considered in detail with EMBL (nucleotide sequences, SWISSPROT (amino acid sequences), and PDB (3D structures) as examples. A unified compressed data format permits integration of EMBL, SWISSPROT, and PDB into a single DB. This approach is intended to be applied for integrating GENBANK and other analogous DBs. Another outcome of the work is a library of DB access and retrieval procedures providing the composers of applied software with a uniform interface to biologically related DBs. The proposed data storage scheme was recommended by the Expert Commission of the Informatics Section of the Human Genome State program as a standard for DB distribution in Russia.

Conformational analysis was carried out for parallel homopolymeric RNA helices poly(A).poly(U) in the framework of the heteronomic model, and for four-stranded poly(U).poly(A).poly(A). poly(U) helices in which the chains with the same bases are parallel and related by a twofold symmetry axis. All possible models of base binding into a symmetrical four-stranded complex and all variants of base orientation relative to the ribose-phosphate backbone were considered. Optimization of potential energy calculated using atom-atom potentitals was performed to determine the dihedral angles and the helix parameters corresponding to minimal conformational energies of parallel two- and four-stranded RNA helices. The possibility of stabilization of four-stranded RNA structures by intramolecular hydrogen bonding was discussed. The results of calculations for parallel double-stranded RNA helices were compared with the spectroscopic and X-ray data on parallel poly(A).poly(U) in solution and in crystalline fibers, as well as with the earlier analogous calculations for DNA helical complexes. Such a comparison allowed a number of conclusions concerning the structural properties of triple-stranded complexes poly(dA).2poly(dT) and poly(A).2poly(U), which contain fragments corresponding to parallel heteronomic helices.

Consideration is given to the effectiveness of additional stacking-hybridization rounds in DNA sequence reconstruction by hybridization with oligonucleotide matrix. First, DNA is hybridized with a matrix of oligonucleotides of length L. Overlapping of the tuples forming perfect duplexes with the DNA permits unambiguous reconstruction of a part of its sequence and suggests reconstruction versions for the remaining part. Then additional hybridization is done in the presence of shorter oligonucleotides (length l) hybridizing to DNA in such a way that they abut on some matrix tuples, forming perfect duplexes of length L + l, whereby the l part of the duplex is additionally stabilized by stacking interaction. The information obtained on the sequence of the L + l regions allows the reconstruction efficacy to be considerably increased, ultimately to reach that of a matrix of (L + l)-long tuples. An algorithm is described for compiling such a set of added l-oligonucleotides as would minimize the number of additional hybridizations. For an octanucleotide matrix, use of additional stacking hybridization with different pentanucleotide sets increases the length of unambiguously sequenced DNA from 200 to several thousand bases.

The ability of the 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 hairpins and higher associates was studied. Optical methods of thermal denaturation and circular dichroism (CD) as well as the fluorescence of the dyes ethidium bromide (EtBr) and acridine orange (AO) bound to the oligonucleotides were used in solutions containing 0.01 M phosphate buffer, pH 7, and NaCl from 0.1 to 1 M. At room temperature formation of hairpin structures with parallel and antiparallel strands is possible. The energy parameters of par[d(GT)] and anti[d(GT)] are similar and are DELTAH almost-equal-to -15 kcal/mole and DELTAS almost-equal-to -50 cal/mole.degree. In the temperature range from approximately 3 to approximately 10-degrees-C par[d(GT)] and anti[d(GT)] form four-stranded structures with parallel strands in which layers of four G residues alternate with unpaired T residues which are easily pushed outward. EtBr and AO form insertion complexes with the four strands and AO forms dimeric complexes with the unpaired nucleotides. Two molecules of EtBr are bound between two layers of four G residues. On the basis of comparing the frequency of appearance in the genome of alternating (GT)n-, (G)n-, and (GC)n residues it can be asserted that the functional characteristics Of (GT)n sequences are connected with the conformational possibilities of four-stranded structures with unpaired T residues.

The results of conformational analysis of homopolymeric double helices of poly(dG) . poly(dG) . poly(dC) DNA with parallel orientation of the sugar-phosphate backbones are presented for all possible types of base pairing. The dihedral angles of sugar-phosphate chains and the parameters corresponding to minimal conformational energy of the DNA helix were determined from the results of optimizing the potential energy calculated by the method of atom-atom potentials. Analysis was carried out for the dependence of the conformational energy on the type of base hydrogen bonding and on the direction of the sugar-phosphate chains. The possible structure of parallel DNA helices with different nucleotide composition is discussed.

Temperature dependences of the UV and CD spectra were studied for linked oligonucleotides 3'-(dA)10-L-(dT)10-5' [anti(AT)], 3'-(dA)10-L-(dT)10-3' [par(AT)], and 3'-(dA)10-L-(dT)10-L-(dT)10-5' [tripl(ATT)] (where L is -pO(CH2CH2O)3P-) in phosphate buffer (pH 7) at different NaCl concentrations with and without 10 mM MgCl2. At low oligonucleotide concentrations (approximately 2.2. 10(-5) M nucleotides) all structural transitions are intramolecular. Par(AT) and anti(AT) exist only in two interconvertible forms: hairpin (at low temperatures) with parallel and antiparallel chain orientation in the stem, or unfolded chain (at elevated temperatures), whereas tripl(ATT) has three forms: triple-stranded "paperclip," double-stranded hairpin, and unfolded chain. For the first time the thermodynamic parameters were calculated for the triplex formation from oligodeoxynucleotides as a function of NaCl concentration. The CD spectra were assigned to single-, double-, and triple-stranded forms. A study was made of ethidium bromide binding to the triple-stranded "paperclip." It was found that ethidium bromide molecules are intercalated into the triple chain to form stable complexes (association constant of 10(6) M-1). One triple-stranded "paperclip" binds maximally three ethidium bromide molecules. The synthetic model proposed (three oligonucleotide blocks linked by oxyalkyl spacers) is shown to be the most convenient to date for studying the physicochemical properties of triplexes.

Thermal denaturation of four oligonucleotides, viz. 3'-d(AT)5pO(CH2)6Opd(AT)5-3' (par(AT)), 3'-d(AT)5pO(CH2)6Opd(AT)5-5'(anti(AT)),3'-d(A)10pO(CH2)6Op(T)10-3' (par(A-T)), and 3'-d(A)10pO(CH2)6Opd(T)10-5'(anti(A-T)), was studied in 0.01 M phosphate buffer, pH7, in the presence of 0.1, 0.25, 0.5 and 1.0 M NaCl. All the oligomers were found to exist at a lower temperature (0 to 20-degrees-C) as complexes composed either of two oligomer molecules (a canonical duplex) or of more oligomer molecules whereas, at a higher temperature (30 to 70-degrees-C), they formed hairpins with a parallel (par(AT) and par(A-T)) or antiparallel (anti(AT) and anti(A-T)) orientation of the chains. Melting curves (A260(T)) were used to calculate thermodynamic parameters for the formation of hairpins and "low-temperature" duplexes. Experiments on ethidium bromide binding to the oligonucleotides have shown that the oligomer anti(A-T) exists, at a low ionic strength, as a four stranded complex ("quadruplex") contains two antiparallel helices, d(A). d(T), which have a parallel orientation and are bound to one another owing to the formation of additional hydrogen bonds between nucleic acid bases. The possible biological function of quadruplexes is discussed.

The SHOM method (Sequencing by Hybridization with Oligonucleotide Matrix) developed in 1988 is a new approach to nucleic acid sequencing by hybridization to a octanucleotide 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 for sequencing, which allows one to reduce the number of synthesized oligonucleotides 5-15 times without essentially decreasing the resolving power of the method.