A simplified and efficient route to 2'-O, 4'-C-methylene-linked bicyclic ribonucleosides (locked nucleic acid).

A novel efficient method for the synthesis of locked nucleic acid (LNA) monomers is described. The LNA 5',3'-diols containing thymine, 4-N-acetyl- and 4-N-benzoylcytosine, 6-N-benzoyladenine, and 2-N-isobutyrylguanine as nucleobases were prepared via convergent syntheses. The method is based on the use of the common sugar intermediate 1,2-di-O-acetyl-3-O-benzyl-4-C-methanesulfonoxymethyl-5-O-methanesulfonyl-D-erythro-pentofuranose (8) that easily can be prepared from D-glucose in multigram scale. Four different nucleobases were stereoselectively coupled to 8 using a modified Vorbrüggen procedure to give the corresponding 4'-C-branched nucleoside derivatives. Subsequent ring closing furnished the protected LNA nucleosides. The 5'-O-mesyl groups were efficiently displaced by nucleophilic substitution using sodium benzoate. Saponification of the 5'-benzoates followed by catalytic removal of the 3'-O-benzyl groups afforded the free LNA diols. The exocyclic amino groups of adenosine and cytidine were selectively acylated to give 4-N-acetyl- or 4-N-benzoyl-LNA-C and 6-N-benzoyl-LNA-A. The isobutyryl group of guanine was retained during the preparation of 2-N-isobutyryl-LNA-G. The LNA-T diol and base-protected LNA diols can be directly converted into LNA-phosphoramidites for automated chemical synthesis of LNA containing oligonucleotides.

Interaction of LNA oligonucleotides with mdr1 promoter.

LNA oligonucleotides [1] can be used for targeting to double stranded DNA by the "strand invasion" mechanism. We used affinity modification by reactive oligonucleotide conjugates for investigation of oligonucleotides interaction with structured DNA. The tested LNAs and oligonucleotides of the same sequence were assayed as anti-mdr1 drugs in different cell cultures. One of the oligos, LNA79 strongly inhibited mdr1 induction in Hela cells and totally prevented activation of mdr1 in K-562.

The conformations of locked nucleic acids (LNA).

We have used 2D NMR spectroscopy to study the sugar conformations of oligonucleotides containing a conformationally restricted nucleotide (LNA) with a 2'-O, 4'-C-methylene bridge. We have investigated a modified 9-mer single stranded oligonucleotide as well as three 9- and 10-mer modified oligonucleotides hybridized to unmodified DNA. The single-stranded LNA contained three modifications whereas the duplexes contained one, three and four modifications, respectively. The LNA:DNA duplexes have normal Watson-Crick base-pairing with all the nucleotides in anti-conformation. By use of selective DQF-COSY spectra we determined the ratio between the N-type (C3'-endo) and S-type (C2'-endo) sugar conformations of the nucleotides. In contrast to the corresponding single-stranded DNA (ssDNA), we found that the sugar conformations of the single-stranded LNA oligonucleotide (ssLNA) cannot be described by a major S-type conformer of all the nucleotides. The nucleotides flanking an LNA nucleotide have sugar conformations with a significant population of the N-type conformer. Similarly, the sugar conformations of the nucleotides in the LNA:DNA duplexes flanking a modification were also shown to have significant contributions from the N-type conformation. In all cases, the sugar conformations of the nucleotides in the complementary DNA strand in the duplex remain in the S-type conformation. We found that the locked conformation of the LNA nucleotides both in ssLNA and in the duplexes organize the phosphate backbone in such a way as to introduce higher population of the N-type conformation. These conformational changes are associated with an improved stacking of the nucleobases. Based on the results reported herein, we propose that the exceptional stability of the LNA modified duplexes is caused by a quenching of concerted local backbone motions (preorganization) by the LNA nucleotides in ssLNA so as to decrease the entropy loss on duplex formation combined with a more efficient stacking of the nucleobases.

Evaluation of oligonucleotides containing two novel 2'-O-methyl modified nucleotide monomers: a 3'-C-allyl and a 2'-O,3'-C-linked bicyclic derivative.

The two ribo-configured nucleosides 1-(3-C-allyl-2-O-methyl-beta-D-ribo-pentofuranosyl)thymine 3 and (1S,5R,6R,8R)-5-hydroxy-6-(hydroxymethyl)-1-methoxy-8-(thymin-1-yl )- 2,7-dioxabicyclo[3.3.0]octane 6 have been transformed into their corresponding phosphoramidites, 5 and 8 respectively, and used as building blocks for the synthesis of modified oligonucleotides. The oligonucleotides were shown to hybridize with decreased binding affinity towards complementary single stranded DNA and RNA.

The first analogues of LNA (locked nucleic acids): phosphorothioate-LNA and 2'-thio-LNA.

LNA (Locked Nucleic Acids, 1, X = O, Y = O) is a novel oligonucleotide analogue capable of recognizing complementary DNA and RNA with unprecedented thermal affinities. Synthesis of the first chemically modified LNA analogues is reported. A 9-mer phosphorothioate-LNA containing three LNA thymine monomers (1, X = O, Y = S, Base = thymin-1-yl) and 9-mer LNAs containing one, three or five 2'-thio-LNA monomers (1, X = S, Y = O, Base = uracil-1-yl) were able to recognize both complementary DNA and RNA with thermal affinities comparable to those of parent LNA.

Sensitized photomodification of single-stranded DNA by a binary system of oligonucleotide conjugates.

A photoactivatable binary system of oligonucleotide conjugates that form reactive species when assembling on a target nucleotide sequence has been developed. The binary system consists of two oligonucleotides. One contains a photosensitizing group, and the second contains a photoreactive group. Binding of the oligonucleotides to adjacent sequences in the target nucleic acid brings the groups in contact, which allows transfer of the absorbed energy from the sensitizer to the reagent and triggers crosslinking of the reagent to the target. One advantage of the binary system is the improved specificity, which is determined by independent binding of two oligonucleotides to the target sequence. Another advantage is the very high efficiency of the reaction achieved because each molecule of the target-bound sensitizing conjugate can activate many photoreactive oligonucleotide conjugate molecules bound to the target sequence.

[Sensitized photomodification of DNA with binary systems. II. Spectral photosensitivity. One- and two quantum sensitization]. / Sensibilizirovannaya fotomodifikatsiya DNK binarnymi sistemami. II. Spektral'naia svetochuvstvitel'nost'. Odno- i dvukhkvantovaia sensibilizatsiia.

The efficiency of the photomodification of target single-stranded DNA with a decanucleotide derivative of p-azidotetrafluorobenzamide (direct photomodification) and with its complexes with decanucleotide derivatives of pyrene complementary to the adjacent segment of the target (sensitized photomodification) was studied as a function of the wavelength of long-wave UV light. The sensitized photomodification occurs mainly by singlet-singlet energy transfer from pyrene to azide in their complementary complex, which allows a significant increase in the rate and level of photomodification. When irradiation occurred simultaneously in the UV and visible regions (365-580 nm), two-photon triplet-triplet sensitization was revealed for the first time, which leads to a still greater acceleration of the target modification and a change of its site-direction from the G11 to T13 residue. The change of the mode of sensitization depending on the irradiation conditions allows the regulation of the reactivity of the binary system of oligonucleotide derivatives without altering their composition.

[Sensitized photomodification by binary systems. I. Synthesis of oligonucleotide reagents, and the effect of their structure on the efficacy of target modification]. / Sensibilizirovannaia fotomodifikatsiia DNK binarnymi sistemami. I. Sintez oligonukleotidnykh reagentov, vliianie ikh stroeniia na éffektivnost' modifikatsii mishenei.

A highly effective sensitized photomodification of the target DNA by a binary system of oligonucleotide reagents complementary to adjacent regions of the target was accomplished. One of the oligonucleotides carries a photoregent p-azidotetrafluorobenzamide, and the other carries a pyrene sensitizer. Synthesis of the oligonucleotide derivatives was described. The rate and efficacy of the direct and sensitized target photomodifications depending on the location of the photoreagent and sensitizer at the 3'- and 5'-terminal phosphates and on the length of the linker between the sensitizer and addressed nucleotide were studied. The oligonucleotide derivatives with the photoreagent at the 3' terminus proved to be more effective (yield of the covalent adducts 70%). The rate of photomodification sensitized by UV light (365-390 nm) is 100-1500-fold higher than that of the direct site-specific modification and decreases with an increasing length of the linker. In all cases, modification occurs at the guanosine residue located near the photoreagent.

[Effect of the intercalating dyes ethidium and phenazine, covalently jointed to the 5'- or 3'-end of the pentanucleotide d(pGAAAG), on the thermodynamics of complementary and cooperative interaction]. / Vliianie interkaliruiushchikh krasitelei étidiia i fenaziniia, kovalentno prisoedinennykh k 5'- ili 3'-kontsu pentanukleotida d(pGAAAG), na termodinamiku komplementarnogo i kooperativnogo vzaimodeistviia.

Thermal stability was studied and thermodynamic parameters of complex formation were calculated for pentanucleotide complexes [formula: see text] and corresponding complexes of pentanucleotide derivatives carrying at their 5'- or 3'-ends covalently bound residues of intercalating dyes: N-(2-hydroxyethyl)-phenazine (Phn) or 2-N-(3-aminopropionyl)-ethidium. Pentanucleotide derivatives were shown to form more stable complementary complexes. The best stabilizing effect was observed when the dye was oriented towards the long single-stranded fragment of tetradecanucleotide, melting temperature of the complexes being by 22.5 (Phn) and 31.2 degrees C (Etd) greater than that of unmodified complexes in the case of 5'-derivatives and by 21.6 (Phn) and 27.2 degrees C (Etd) for 3'-derivatives. Cooperativity constant of pentanucleotide derivatives in "tandem" complexes IV-VII was higher than that of unmodified pentanucleotide. For complex IV at 37 degrees C the constant values were 33 (unmodified), 35 (5'Phn), 57 (3'Phn), 190 (5'Etd), 100 (3'Etd). With n = 3 in complex VII cooperativity constants approached 1 in any case.

Inhibition of the respiratory burst in mouse macrophages by ultra-low doses of an opioid peptide is consistent with a possible adaptation mechanism.

The respiratory burst induced by phorbol myristate acetate in mouse macrophages was inhibited by ultra-low doses (10(-15)-10(-13) M) of an opioid peptide [D-Ala2]methionine enkephalinamide. The effect disappeared at concentrations above and below this range. The inhibition approached 50% and was statistically significant (P < 0.001). Increasing the time of the opioid incubation with cells brought about a shift in the maximal effect to lower concentrations of the opioid (from 10(-13) to 5 x 10(-15) M) and led to a decrease in the value of the effect, fully in accord with the previously proposed adaptation mechanism of the action of ultra-low doses.

Ethidium and azidoethidium oligonucleotide derivatives: synthesis, complementary complex formation and sequence-specific photomodification of the single-stranded and double-stranded target oligo- and polynucleotides.

Oligodeoxyribonucleotide derivatives containing ethidium or azidoethidium residues attached to 3' and/or 5' end were prepared. These derivatives formed tight specific complexes with complementary oligodeoxyribonucleotides where each attached ethidium residue led to an increase of complex Tm by 20-30 degrees C. Tandem complexes of two oligodeoxyribonucleotides containing ethidium residues with an oligodeoxyribonucleotide having two adjacent complementary sequences for these oligonucleotides were investigated. Photoinduced reactions of a number of ethidium and azidoethidium oligodeoxyribonucleotide derivatives with target complementary single-stranded and double-stranded oligo- and polydeoxyribonucleotides were investigated. The irradiation led to direct photocleavage of the target oligo- or polynucleotide, to formation of hidden (piperidine cleavable) modifications of the target and to formation of covalent adducts between ethidium oligodeoxyribonucleotide derivative and the target. In a number of experiments, azidoethidium dyes were demonstrated to be considerably stronger photosensitizers than ethidium ones. Depending on the nature of the target (single- or double-stranded DNA) and on the irradiation conditions, the total damages to the target oligo- or polydeoxyribonucleotides ranged from 10-70% (for ethidium dyes) to 30-80% (for azidoethidium dyes).

Fluorescence energy transfer as a probe for nucleic acid structures and sequences.

The primary or secondary structure of single-stranded nucleic acids has been investigated with fluorescent oligonucleotides, i.e., oligonucleotides covalently linked to a fluorescent dye. Five different chromophores were used: 2-methoxy-6-chloro-9-amino-acridine, coumarin 500, fluorescein, rhodamine and ethidium. The chemical synthesis of derivatized oligonucleotides is described. Hybridization of two fluorescent oligonucleotides to adjacent nucleic acid sequences led to fluorescence excitation energy transfer between the donor and the acceptor dyes. This phenomenon was used to probe primary and secondary structures of DNA fragments and the orientation of oligodeoxynucleotides synthesized with the alpha-anomers of nucleoside units. Fluorescence energy transfer can be used to reveal the formation of hairpin structures and the translocation of genes between two chromosomes.

[The effect of various substituents, joined through the 5'- and 3'-ends of the primer, initiating properties during the polymerization reaction, catalyzed by AMV-revertase]. / Vliianie razlichnykh zamestitelei, prisoedinennykh po 5'- i 3'-kontsampraimera, na ego initsiiruiushchie svoistva v reaktsii polimerizatsii, kataliziruemoi AMV-revertazoi.

We investigated the interaction of AMV reverse transcriptase and Klenow fragment with oligonucleotide derivatives carrying different 3'- or 5'-terminal reactive groups. It was shown that the attachment of phenazinium, ethidium, and daunomycin residues to the 5'-terminal phosphate stabilized the enzyme template primer complexes, while cholesterol and hemin residues generally decreased their stability. The increased stability in solution correlated to a certain extent with the increase in affinity of the modified primers to the enzyme template complex. Coupling of bulky R residues to the primers had a weak effect on the maximal rate of primer conversion, which is likely to be a result of the lack of strong contacts between the substituents and the enzyme, and steric obstacles hindering translocation of the primer enzyme complex. We analyzed the inhibitory effect of 23 oligonucleotide derivatives (both complementary and noncomplementary to the template) with modified 3'- and 5'-ends, and revealed several analogs inhibiting polymerization catalyzed by AMV reverse transcriptase by 70-100% at 0.1-1 microM concentrations of the reagents.

Comparison of interactions of 5'-derivatives of deoxyoctathymidylate with human DNA polymerize alpha and HIV reverse transcriptase.

Km and Vmax values for d(pT8) and its derivatives containing various 5'-end groups were estimated in the reaction of DNA polymerization alpha catalyzed by DNA polymerase alpha and HIV-RT. The effect of 5'-end modification of primer is more pronounced in the case of HIV-RT. Strong influence is observed for an intercalating (ethidium) group. The affinity of EtpT8 is 200-fold higher than that of d(pT8). Attachment of Phn-, Dnm- and Hem-groups results in the increase of affinity of modified primer from 10 up to 20 times. For DNA polymerase alpha the influence of modifiers on primer affinity is much weaker. The effect of 5'-end residues on the Vmax values is also more pronounced for HIV RT. The way to improve selective interaction of oligonucleotide derivatives with the primer site of HIV RT is suggested.

[Photoactive ethidium and azidoethidium dyes. Synthesis, properties, and covalent bonding to oligodeoxynucleotides]. / Fotoaktivnye étidievye i azidoétidievye krasiteli. Sintez, svoistvai kovalentnoe prisoedinenie k oligodezoksinukleotidam.

Methods of synthesis of ethidium and azidoethidium dyes, containing the primary aliphatic amino group of the 3-aminopropinoic acid residue were developed. The derivatives were characterized by spectral methods (NMR, UV- and fluorescent spectroscopy). Complex formation of ethidium dyes with DNA was studied by fluorescent technique. The stoichiometry of the dye--DNA complex was 1:4 (dye to base pair) for all the derivatives. Photoreactivity of azidoethidium dyes was 10(3) times higher and the quantum yield of the photoproducts was 10(5) times higher than those for ethidium dyes. A method of preparation of 5'-phosphoramide photoreactive oligodeoxynucleotide derivatives was suggested, with ethidium derivative of heptanucleotide pd(CCAAACA) as an example.

[Suppression of transcription of influenza virus RNA by oligonucleotide derivatives]. / Podavlenie transkriptsii RNK virusa grippa proizvodnymi oligonukleotidov.

The inhibitory effects of oligonucleotide derivatives on the transcription of virus RNA in an in vitro system and synthesis of virus proteins was studied. Oligonucleotide derivatives d(T)3, d(T)4, d(T)8, d(T)10, d(CCAAACA), d(TCACCCTC), d(TTCCCATT), d(AATACTCT) and d(TGACCCTCTTCCCATT), that bear residues of ethidium, deuteroporphyrin and its complexes with Fe3+, hemin, cholesterol, deuterocholesterol, estrone and naphthoquinone at the 5'-end phosphate and/or at the 3'-end phosphate were studied. Unmodified oligonucleotides and their derivatives had a negligible effect on the synthesis of cellular proteins, but did inhibit the synthesis of influenza virus proteins. The majority of structural modifications increased the inhibitory effect of oligonucleotides. It was shown that the oligonucleotide derivatives carrying residues of porphyrin, quinone, ethidium, cholesterol, deuterotestosterone and estrone at concentrations near 10 mM inhibit virus development to 50-80%. A clear inhibitory effect (20-25%) of deuteroporphyrin, cholesterol and ethidium derivatives was revealed even at concentration 0.1 mM. The obtained results testified that the inhibition of influenza virus development is dependent on the interaction of oligonucleotide derivatives with the transcription complex proteins.