[Preparation of Modified Combinatorial DNA Libraries via Emulsion PCR with Subsequent Strand Separation].

A modification of the enzymatic method for the preparation of combinatorial random DNA libraries, which combines amplification in isolated microvolumes with the simultaneous incorporation of modified nucleotides and subsequent separation of DNA strands, was developed. Deoxyuridine triphosphate with hydrophobic substituents such as structural analogues of amino acid side chains in the C5 position of the pyrimidine ring was used to introduce modifications into DNA. To prevent competitive amplification, which reduces the representativeness of combinatorial libraries, PCR in inverse emulsion was used. The separation of the strands of PCR products was carried out. There were six single-stranded DNA libraries with complete substitution of deoxythymidine via modified analogues with various functional groups. These DNA libraries are suitable for generating aptamers to protein targets through additional hydrophobic interactions from the introductions of appropriate modifications, and are completely compatible with the SELEX aptamer selection methodology.

[Enzymatic synthesis of high-modified DNA].

Here, we describe the synthesis and purification of six deoxyuridine triphosphate derivatives that contain protein-like functional groups and alkene linkers of various lengths. Using KOD XL and Deep Vent polymerases, these derivatives have been incorporated into single-stranded DNA, achieving a high degree of DNA modification. These polymerases are able to utilize highly modified DNA strands as templates for synthesizing unmodified DNA. The synthesized deoxyuridine triphosphate derivatives are promising as substrates for producing modified aptamers to various target proteins using, e.g., the systematic evolution of ligands by exponential enrichment (SELEX) methodology.

Targeting duplex DNA with chimeric α,ß-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,ß-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,ß-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,ß-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.

[Oligonucleotides with peptide internucleotide linkage. A novel class of modified oligonucleotides].

Oligonucleotide analogues with replacement of one or more internucleotide phosphodiester linkages with glycine, L- and D-alanine residues have been synthesized (C3'-NH-C(O)-CH(X)-NH-C(O)-C4', X = H, (S)-CH3 and (R)-CH3). The stability of the duplexes formed by the modified oligonucleotides and their wild-type complements have been studied. Incorporation of glycine and L-alanine residues have been shown to substantially decrease the stability the modified duplexes in comparison with that of the wild-type ones (DeltaT(m) approximately -2 degrees C per modification), while the analogs with D-alanine-containing linkages appeared to form duplexes with increased stability (DeltaT(m), approximately +0.2 degrees C per modification).

[Oligonucleotide analogues containing a C3'-NH-C(O)-CH(2)-C5' amide internucleotide bond].

A dinucleoside containing a C3'-NH-C(O)-CH(2)-C5' amide internucleoside bond was synthesized by the interaction of 3'-deoxy-3'-amino-5'-O-(tert-butyldimethylsilyl)thymidine with 3'-O-benzoyl-2'-O-tert-butyldimethylsilyl-5'-deoxy-5'-carboxymethylribosylthymine, which was obtained from 2'-O-acetyl-3'-O-benzyl-5'-deoxy-5'-ethoxycarbonylmethylribosylthymine through the methanolysis of the acetyl group followed by silylation and ester saponification. After standard manipulation with protecting groups, the dinucleoside was converted into 3'-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite), which was used for the synthesis of modified oligonucleotides on an automated synthesizer. The melting curves of the duplexes formed by modified and complementary natural oligonucleotides were registered, and the melting temperatures and thermodynamic parameters of the duplex formation were calculated. The introduction of a single modified bond into the oligonucleotide led to an insignificant decrease in the melting temperature of these duplexes as compared to unmodified ones.

[Synthesis of 5'-deoxy-5'-ethoxycarbonylmethyl nucleosides, the precursors of oligonucleotides with the amide internucleoside bond C3'-NH-C(O)-CH2-C5'].

A method for the synthesis of 5'-deoxy-5'-ethoxycarbonylmethyl nucleosides has been developed. 3-O-Benzyloxymethyl-1,2-O-isopropylidene-alpha-D-allofuranose was oxidized by sodium periodate to form a 5-aldo derivative, which was converted by the reaction with triethylphosphonoacetate in the presence of sodium hydride into a 5-deoxy-5-ethoxycarbonylmethylene derivative. The hydration of the unsaturated compound gave 5-deoxy-5-ethoxycarbonylmethyl-l,2-O-isopropylidene-alpha-D-ribofuranose. After the benzylation of 3-hydroxyl, the removal of the isopropylidene group by heating with acetic acid, and the subsequent acetylation, l,2-di-O-acetyl-3-O-benzyl-5-deoxy-5-ethoxycarbonylmethyl-D-ribofuranose was obtained, which reacted with persilylated nucleic acid bases to form 5'-deoxy-5'-ethoxycarbonylmethyl nucleosides.

[Oligonucleotide analogues containing internucleotide C3'-CH2-C(O)-NH-C5' bonds].

A dinucleoside bearing an amide internucleotide C3'-CH2-C(O)-NH-C5' bond was synthesized by the interaction of 3'-deoxy-3'-carboxylmethylribothymidine-2',3'-lactone obtained by hydrolysis of 2'-O-acetyl-5'-O-benzoyl-3'-deoxy-3'-ethoxycarboxylmethylribothymidine with 5'-deoxy-5'-amino-3'-O-(tert-butyldimethylsilyl)thymidine. After standard manipulations with protective groups, the dinucleoside was converted into 3'-O-(2-cyanoethyl-N,N'-diisopropylphosphoroamidite), which was used for the synthesis of modified oligonucleotides on an automatic synthesizer. Duplex melting curves formed by modified and complementary natural oligonucleotides were measured and the melting temperatures and thermodynamic parameters of duplex formation were calculated. The introduction of one modified bond into oligonucleotides caused only an insignificant decrease in the duplex melting temperatures compared with the nonmodified ones.

[Synthesis of 3'-deoxy-3'-carboxymethylnucleosides, precursors of oligonucleotides with an amide internucleoside bond].

An improved method for the synthesis of 3-deoxy-3-carboxymethylnucleosides was suggested. Oxidation of 5-O-benzoyl-l,2-O-isopropylidene-alpha-D-xylofuranose resulted in the 3-keto derivative, which was treated with triethylphosphonoacetate in the presence of sodium hydride to obtain the 3-deoxy-3-ethoxycarbonylmethylene derivative. Hydrogenation of the unsaturated compound proceeded strictly stereospecifically and gave the product with the ribo configuration. Acetolysis of the resulting compound with AcOH-Ac2O-CH3SO3H led to 1,2-di-O-acetyl-5-O-benzoyl-3-deoxy-3-ethoxycarbonylmethyl-D-ribofuranose, whose interaction with persilylated nucleic bases gave 3-deoxy-3-ethoxycarbonylmethylnucleosides in a total yield of 42-49% from the starting compound.

[Oligonucleotide analogues containing the internucleotide linker C3'-NH-CO-CH2-N(CH3)-C5'].

Oligonucleotide analogues were synthesized whose internucleoside linker contains an amide bond and a methylamino group (C3'-NH-CO-CH2-N(CH3)-C5'). Melting curves for duplexes formed by modified oligonucleotides and natural oligonucleotides complementary to them were measured, and the melting temperatures and thermodynamic parameters of duplex formation were calculated. The introduction of one modified dinucleoside linker into the oligonucleotide only slightly decreases the melting temperatures of these duplexes compared with unmodified ones. The CD spectra of modified duplexes were studied, and their spatial structures are discussed.

[Oligonucleotide analogues bearing an acyclonucleoside linked by an internucleotide amide bond].

Oligonucleotide analogues bearing an acyclocytidine linked to thymidine by an amide (3'-O-CH2-CO-N-5') bond were synthesized. Melting curves of duplexes formed by modified oligonucleotides and complementary natural oligomers were obtained and thermodynamic parameters of their formation were measured. Replacement of dCpT by a modified dinucleotide only moderately decreased the melting temperature of these modified duplexes in comparison with unmodified duplexes containing complementary natural bases. CD spectra of modified duplexes were studied, and the duplex spatial structures are discussed. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.

[Oligodeoxynucleotides containing substituted 4-nitroindoles: synthesis and study of their DNA duplexes].

The synthesis of oligonucleotides containing 1-(2-deoxy-beta-D-ribofuranosyl)-2-methyl-4-nitroindole and 1-(2-deoxy-beta-D-ribofuranosyl)-2-phenyl-4-nitroindole is described. The synthesized modified oligonucleotides were used for studying the stability of intermolecular DNA duplexes with one unnatural strand and for evaluation of discriminating potential of 2-methyl- and 2-phenyl-4-nitroindoles toward nucleic bases. For comparison, an unmodified oligonucleotide and oligonucleotides bearing 5-nitroindole were used. It was shown that 2-methyl-4-nitroindole was only insignificantly inferior in stability to 5-nitroindole and characterized by a similar discriminating potential. 2-Phenyl-4-nitroindole provided a more pronounced duplex destabilization, the discrimination toward natural bases being decreased. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http:// www.maik.ru.

[Binding of unnatural alpha,beta-oligocytidylates with DNA duplexes]. / Issledovanie sviazyvaniia neprirodnykh alpha,beta-oligotsitidilatov s dupleksami DNK.

Binding of short fluorescently labeled AT-containing DNA duplexes with modified oligocytidylates is studied. The latter are modified to contain unnatural alpha-anomers along with natural beta-nucleotides; the nucleotide composition is selected according to putative pattern of unconventional triplex formation between duplex and oligomer bases. Nondenaturing gel electrophoresis is used to study complexation of fluorescent duplexes with cytidyl oligomers and oligocytidylate self-association at low temperatures. A DNA duplex of random AT composition is shown to bind with an excess of the corresponding oligocytidylate in 0.1 M Tris-HCl in the presence of Mg2+. Binding is observed at neutral pH values, while more basic pH (8.0) prevents complexation of the AT duplex and oligocytidylate. Contrary to oligonucleotides of irregular composition, a regular dA30:dT30 duplex does not bind with the dC strand. It is also shown that alternating self-complementary duplex d(AT)16 and oligocytidylate d(CbetaCalpha)15 do not form complexes, and poly-dC self-associates are formed instead. The effect of 2'-O-methylation of the third strand on complex formation and self-association is also analyzed. The results suggest that a modified oligocytidylate binds with a random-composition duplex, albeit with lower efficiency.

[Enhancing the stability of oligonucleotide duplexes. The effect of adding 1-(2'-deoxy-beta-d-ribofuranosyl)-5-nitroindole]. / Povyshenie stabil'nosti dupleksov oligonukleotidov. Effekt prisoedineniia 1-(2'-dezoksi-beta-d-ribofuranozil)-5-nitroindola.

We studied the properties of DNA duplexes containing 5-nitroindole (N) in one of the chains. We synthesized 8-membered oligos with N at the 5' or at the 3' end: 5'-d(NXGACCGTC)-3' or 5'-d(GACCGTCXN)-3', where X is one of the four natural bases, making all four kinds of oligos with and without N. We also prepared 11-membered oligos complementary to the above octanucleotides: 5'-d(TGACGGTCYZT)-3' and 5'-d(TZYGACGGTCT)-3', where Y and Z are A, G, C, or T. The stability of duplexes obtained with these oligos was assessed by melting, and the thermodynamic parameters delta H, delta S, and Tm were calculated. Comparison of the melting curves for modified and nonmodified duplexes demonstrated that the presence of N at the 5' end of one chain raises the Tm by 6.6 degrees C on average; if N is at the 3' end of the same chain, the Tm increases by about 3 degrees C.

Protein-free parallel triple-stranded DNA complex formation.

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.

Structural polymorphism of oligo(dC) with mixed alpha,beta-anomeric backbone.

Oligonucleotides with mixed alpha,beta-anomeric backbone have been proposed recently for the recognition of random DNA sequence via new triplex motif (Doronina and Behr, Chem. Soc. Reviews 26, 63-71 (1997)). In the present work we examined alpha- and beta- anomers of cytidine as possible candidates to recognize AT and TA base pairs of the double stranded DNA. The binding properties of beta-oligo(dC) were studied on a series of synthetic oligodeoxynucleotides by UV absorbtion spectroscopy, measurements of bound EtBr fluorescence polarization, circular dichroism (CD) and non-denaturing gel electrophoresis. The UV thermal denaturation, polarization studies and CD experiments with three stranded oligonucleotide 5'-((dCalpha) (dCbeta))5-L-(dAT)5-L-(dAT)5 (L = triethyleneglycol linker) and other oligonucleotide models showed that the formation of semiprotonated oligocytidilic complexes takes place at low temperatures and neutral pH, rather than folding of the clip into intramolecular triplex. The low-temperature transition was observed in denaturation profiles of any oligonucleotide containing beta- or mixed alpha,beta- cytidine stretches at the concentration of 1 microM. Self-association of alpha,beta-oligo(dC) was additionally confirmed by the appearance of two CD bands (at 290 and 265 nm) characteristic of CC+ base pairs. Despite the effective ability of alpha,beta-oligo(dC) to form self-associates, we succeeded in targeting 30-bp AT containing random DNA duplex by a 30-nt alpha,beta-oligocytidilate as evidenced by non-denaturing gel electrophoresis. A complete binding of the duplex was observed at a 5-fold excess of the third strand at 15 degrees C. Along with the formation of the three-stranded complex, self-association of mixed backbone oligo(dC) strands occurred.

Advanced method for oligonucleotide deprotection.

A new procedure for rapid deprotection of synthetic oligodeoxynucleotides has been developed. While all known deprotection methods require purification to remove the residual protective groups (e.g. benzamide) and insoluble silicates, the new procedure based on the use of an ammonia-free reagent mixture allows one to avoid the additional purification steps. The method can be applied to deprotect the oligodeoxynucleotides synthesized by using the standard protected nucleoside phosphoramidites dG(iBu), dC(Bz)and dA(Bz).

Fabrication of microarray of gel-immobilized compounds on a chip by copolymerization.

The manufacturing of microchips containing oligonucleotides and proteins immobilized within gel pads, ranging in size from 10 x 10 to 100 x 100 microns, is described. The microchips are produced by photo- or persulfate-induced copolymerization of unsaturated derivatives of biomolecules with acrylamide-bisacrylamide mixture. Oligonucleotides containing 5'-allyl or 5'-butenediol units were synthesized using standard phosphoramidite chemistry. Acryloyl residues were attached to a protein by a two-step procedure. Photopolymerization was induced by illumination of the monomer solution containing initiator with UV light through the mask. The mask was applied directly over the monomer solution or projected through a microscope. Alternatively, copolymerization was carried out in drops of aqueous solution of monomers containing ammonium persulfate. Drops with different allyl-oligonucleotides were distributed on a glass slide, and the polymerization was induced by diffusion of N,N,N',N'-tetramethylethylenediamine (TEMED) from a hexane solution that covered the aqueous drops.

Stabilization of parallel (recombinant) triplex with propidium iodide.

Earlier we have shown that the oligonucleotide 5'-d(CATGCTAACT)-L-d(AGTTAGCATG)-L-d(CATGCTAACT)-3' [L = pO(CH2CH2O)3p] is able to fold back forming intramolecular RecA-independent triplex with identical strands oriented parallel to each other (parallel triplex) [A.K. Shchyolkina, E.N. Timofeev, O.F. Borisova, I.A. Il'icheva, E.E. Minyat, E.V. Khomyakova, V.L. Florentiev, FEBS Letters 339, 113-118 (1994) (1)]. In this study the propidium iodide (PI) was found to intercalate into the parallel triplex and increase its stability significantly (Tm increased from 21.4 up to 44.4 degrees C in 0.01 M Na phosphate buffer, pH 7, 0.1 M NaCl, when three PI molecules per triplex were bound). Fluorescence excitation and emission spectra, the quantum yield of fluorescence (q = 0.16) and the fluorescence lifetime of PI (tau = 24.5 ns at 3 degrees C) for the parallel triplex studied were shown to be similar to those for DNA. Scatchard binding plots indicated an anticooperative mode of PI binding to the parallel triplex. The association constant is close to that of PI binding to DNA. The fluorescence experiments revealed the maximum number of binding sites to be five PI molecules per one triplex molecule. Molecular mechanics calculation of possible structures for the parallel triplex-PI complex were performed.