Synthesis and hybridizing properties of P-stereodefined chimeric [PS]-{DNA:RNA} and [PS]-{DNA:(2'-OMe)-RNA} oligomers.

Oxathiaphospholane derivatives of 2'-OMe-ribonucleosides and 2'-O-TBDMS-ribonucleosides (MN-OTP and TN-OTP, respectively; nucleobase protected) were synthesized and separated into pure P-diastereomers. X-ray analysis showed the R P absolute configuration of the phosphorus atom in the fast-eluting diastereomer of TA-OTP. The fast- and slow-eluting P-diastereomers of MN-OTP and TN-OTP were used in the solid-phase synthesis of phosphorothioate dinucleotides (MNPST and NPST, respectively), which were subsequently hydrolyzed with R P-selective phosphodiesterase svPDE and S P-selective nuclease P1 to determine the absolute configuration of the phosphorus atoms. P-Stereodefined phosphorothioate ([PS]) 10-mer chimeric oligomers [PS]-{DNA:(2'-OMe)-RNA} and isosequential [PS]-{DNA:RNA} containing two MNPS or NPS units were synthesized. Melting experiments performed for their complexes with Watson-Crick paired DNA matrix showed that MNPS or NPS units decrease the thermal stability of the duplexes (ΔT m = -0.5 ÷ -5.5 °C per modification) regardless of the absolute configuration of the P-atoms. When the (2'-OMe)-RNA matrix was used an increase in T m was noted in all cases (ΔT m = +1 ÷ +7 °C per modification). The changes in thermal stability of the duplexes formed by [PS]-chimeras with DNA and (2'-OMe)-RNA matrices do not correlate with the absolute configuration of the phosphorus atoms.

Boron clusters as a platform for new materials: composites of nucleic acids and oligofunctionalized carboranes (C2B10H12) and their assembly into functional nanoparticles.

Nucleic acids are key biomolecules in all life forms. These biomolecules can encode and transfer information via Watson-Crick base-pairing interactions and can form double-stranded structures between complementary sequences with high precision. These properties make nucleic acids extremely successful in applications in materials science as nanoconstruction materials. Herein, we describe a method for the automated synthesis of "oligopeds", which are building blocks based on the boron cluster structure equipped with short DNA adapters; these building blocks assemble into functional nanoparticles. The obtained, well defined, torus-like structures are the first DNA nanoconstructs based on a boron cluster scaffold. The results indicate the potential of boron clusters in DNA nanoconstruction and open the way for the design of entirely new types of buildings blocks based on polyhedral heteroborane geometry and its unique properties. The use of antisense oligonucleotides as DNA adapters illustrates one of the possible applications of the obtained nanoconstructs as vectors for therapeutic nucleic acids.

A Flp-SUMO hybrid recombinase reveals multi-layered copy number control of a selfish DNA element through post-translational modification.

Mechanisms for highly efficient chromosome-associated equal segregation, and for maintenance of steady state copy number, are at the heart of the evolutionary success of the 2-micron plasmid as a stable multi-copy extra-chromosomal selfish DNA element present in the yeast nucleus. The Flp site-specific recombination system housed by the plasmid, which is central to plasmid copy number maintenance, is regulated at multiple levels. Transcription of the FLP gene is fine-tuned by the repressor function of the plasmid-coded partitioning proteins Rep1 and Rep2 and their antagonist Raf1, which is also plasmid-coded. In addition, the Flp protein is regulated by the host's post-translational modification machinery. Utilizing a Flp-SUMO fusion protein, which functionally mimics naturally sumoylated Flp, we demonstrate that the modification signals ubiquitination of Flp, followed by its proteasome-mediated degradation. Furthermore, reduced binding affinity and cooperativity of the modified Flp decrease its association with the plasmid FRT (Flp recombination target) sites, and/or increase its dissociation from them. The resulting attenuation of strand cleavage and recombination events safeguards against runaway increase in plasmid copy number, which is deleterious to the host-and indirectly-to the plasmid. These results have broader relevance to potential mechanisms by which selfish genomes minimize fitness conflicts with host genomes by holding in check the extra genetic load they pose.

Homopurine RP-stereodefined phosphorothioate analogs of DNA with hampered Watson-Crick base pairings form Hoogsteen paired parallel duplexes with (2'-OMe)-RNAs.

3'-O-(2-Thio-4,4-pentamethylene-1,3,2-oxathiaphospholane) derivatives of 5'-O-DMT-N6-methyl-deoxyadenosine and 5'-O-DMT-N2,N2-dimethyl-O6-diphenylcarbamoyl-deoxyguanosine (OTP-NY, NY = DMT-m6dA or DMT-m,m2dGDPC) were synthesized, resolved onto pure P-diastereomers, and used in P-stereocontrolled synthesis of dinucleoside 3',5,-phosphorothioates NXPST (NX = m6dA or m,m2dG), in which the absolute configuration of the stereogenic phosphorus atom was established enzymatically. Diastereomerically pure OTP-NY and standard OTP-N (N = DMT-dABz or DMT-dGBz,DPC) were used in the synthesis of chimeric RP-stereodefined phosphorothioate oligomers ((RP-PS)-DN(NX)A) with hampered Watson-Crick base pairings. It was found that the m6dA units slightly reduce the thermodynamic stability of antiparallel duplexes formed with RNA and (2'-OMe)-RNA matrices, whereas m,m2dG units prevent their formation. The m6dA units stabilize (by up to 4.5 °C per modified unit) the parallel duplexes formed by (RP-PS)-DN(NX)A with Hoogsteen-paired (2'-OMe)-RNA templates compared to the analogous reference duplex containing only unmodified nucleobases. In contrast, the m,m2dG units destabilize such duplexes by up to 3 °C per modified unit. Both units prevent the formation of the corresponding parallel triplexes.

The role of the Hint1 protein in the metabolism of phosphorothioate oligonucleotides drugs and prodrugs, and the release of H2S under cellular conditions.

Phosphorothioate oligonucleotides (PS-oligos) containing sulfur atom attached in a nonbridging position to the phosphorus atom at one or more internucleotide bond(s) are often used in medicinal applications. Their hydrolysis in cellular media proceeds mainly from the 3'-end, resulting in the appearance of nucleoside 5'-O-phosphorothioates ((d)NMPS), whose further metabolism is poorly understood. We hypothesize that the enzyme responsible for (d)NMPS catabolism could be Hint1, an enzyme that belongs to the histidine triad (HIT) superfamily and is present in all organisms. We previously found that (d)NMPS were desulfurated in vitro to yield (d)NMP and H2S in a Hint1-assisted reaction. Here, we demonstrate that AMPS/GMPS/dGMPS introduced into HeLa/A549 cells are intracellularly converted into AMP/GMP/dGMP and H2S. The level of the released H2S was relative to the concentration of the compounds used and the reaction time. Using RNAi technology, we have shown decreased levels of AMPS/GMPS desulfuration in HeLa/A549 cells with reduced Hint1 levels. Finally, after transfection of a short Rp-d(APSAPSA) oligomer into HeLa cells, the release of H2S was observed. These results suggest that the metabolic pathway of PS-oligos includes hydrolysis into (d)NMPS (by cellular nucleases) followed by Hint1-promoted conversion of the resulting (d)NMPS into (d)NMP accompanied by H2S elimination. Our observations may be also important for possible medicinal applications of (d)NMPS because H2S is a gasotransmitter involved in many physiological and pathological processes.

P-Stereodefined phosphorothioate analogs of glycol nucleic acids-synthesis and structural properties.

Enantiomerically pure, protected acyclic nucleosides of the GNA type (glycol nucleic acids) (GN'), obtained from (R)-(+)- and (S)-(-)-glycidols and the four canonical DNA nucleobases (Ade, Cyt, Gua and Thy), were transformed into 3'-O-DMT-protected 2-thio-4,4-pentamethylene-1,3,2-oxathiaphospholane derivatives (OTP-GN') containing a second stereogenic center at the phosphorus atom. These monomers were chromatographically separated into P-diastereoisomers, which were further used for the synthesis of P-stereodefined "dinucleoside" phosphorothioates GNPST (GN = GA, GC, GG, GT). The absolute configuration at the phosphorus atom for all eight GNPST was established enzymatically and verified chemically, and correlated with chromatographic mobility of the OTP-GN' monomers on silica gel. The GNPS units (derived from (R)-(+)-glycidol) were introduced into self-complementary PS-(DNA/GNA) octamers of preselected, uniform absolute configuration at P-atoms. Thermal dissociation experiments showed that the thermodynamic stability of the duplexes depends on the stereochemistry of the phosphorus centers and relative arrangement of the GN units in the oligonucleotide strands. These results correlate with the changes of conformation assessed from circular dichroism spectra.

Thermal stability and conformation of antiparallel duplexes formed by P-stereodefined phosphorothioate DNA/LNA chimeric oligomers with DNA and RNA matrices.

3'-O-(2-Thio-4,4-pentamethylene-1,3,2-oxathiaphospholane) derivatives of LNA-type nucleosides (LNA-OTPs, 2a-d; B' = Thy, Ade(Bz), Cyt(Bz), Gua(dmf), respectively) were synthesized and separated into pure P-diastereomers. X-ray analysis allowed for assignment of the absolute configuration of the phosphorus atom in the detritylated, fast-eluting diastereomer 2a. The diastereomerically pure LNA-OTP monomers were used in solid phase synthesis of P-stereodefined chimeric PS-(DNA/LNA) 11-mers containing 2-3 LNA units. Formally, among the phosphorothioate oligomers the biggest enhancement in thermal stability of Watson-Crick paired duplexes was found for [SP-PS]-(DNA/LNA)/RNA duplexes (on average 8.2 °C per LNA nucleotide), followed by [RP-PS]-(DNA/LNA)/RNA (6.3 °C), [RP-PS]-(DNA/LNA)/DNA (3.8 °C) and [SP-PS]-(DNA/LNA)/DNA (2.4 °C per LNA nucleotide). However, detailed analysis of the thermal dissociation data showed that the thermal stability of (PS-LNA)-containing duplexes does not depend on the spatial orientation of the sulfur atoms. This conclusion received support from CD measurements.

Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination.

Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5' to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions.

LNA units present in the (2'-OMe)-RNA strand stabilize parallel duplexes (2'-OMe)-RNA/[All-R(P)-PS]-DNA and parallel triplexes (2'-OMe)-RNA/[All-R(P)-PS]-DNA/RNA. An improved tool for the inhibition of reverse transcription.

Homopurine phosphorothioate analogs of DNA, possessing all phosphorus atoms of RP configuration ([All-RP-PS]-DNA), when interact with appropriate complementary RNA or (2'-OMe)-RNA templates, form parallel triplexes or parallel duplexes of very high thermodynamic stability. The present results show that T-LNA or 5-Me-C-LNA units introduced into the parallel Hoogsteen-paired (2'-OMe)-RNA strands (up to four units in the oligomers of 9 or 12 nt in length) stabilize these parallel complexes. At neutral pH, dodecameric parallel duplexes have Tm values of 62-68 °C, which are by 4-10 °C higher than Tm for the reference duplex (with no LNA units present), while for the corresponding triplexes, Tm values exceeded 85 °C. For nonameric parallel duplexes, melting temperatures of 38-62 °C were found and (2'-OMe)-RNA oligomers containing 5-Me-C-LNA units stabilized the complexes more efficiently than the T-LNA containing congeners. In both series the stability of the parallel complexes increased with an increasing number of LNA units present. The same trend was observed in experiments of reverse transcription RNA→DNA (using AMV RT reverse transcriptase) where the formation of parallel triplexes (consisting of an RNA template, [All-RP-PS]-DNA nonamer and Hoogsteen-paired (2'-OMe)-RNA strands containing the LNA units) led to the efficient inhibition of the process. Under the best conditions checked (four 5-Me-C-LNA units, three-fold excess over the RNA template) the inhibition was 94% effective, compared to 71% inhibition observed in the reference system with the Hoogsteen-paired (2'-OMe)-RNA strand carrying no LNA units. This kind of complexation may "arrest" harmful RNA oligomers (e.g., viral RNA or mRNA of unwanted proteins) and, beneficially, exclude them from enzymatic processes, otherwise leading to viral or genetic diseases.

Transformation of a wobble 2-thiouridine to 2-selenouridine via S-geranyl-2-thiouridine as a possible cellular pathway.

The newly discovered S-geranylated 2-thiouridines (geS2U) (Dumelin et al., 2012) and 2-selenouridines (Se2U) were recently shown to be synthesized by a single enzyme (selenouridine synthase, SelU) through two distinct pathways using the same 2-thiouridine substrate (S2U); however, no clear catalytic mechanism was proposed. We suggest that S-geranyl-2-thiouridine is an intermediate of the SelU-catalyzed conversion of S2U to Se2U. The successful chemical transformation of S2U→geS2U→Se2U is demonstrated here as an initial approximation of the intracellular pathway. The structure of Se2U was confirmed by spectroscopic methods, which included, for the first time, (77)Se NMR data (δ 354ppm).

Stereoselective formation of a P-P bond in the reaction of 2-alkoxy-2-thio-1,3,2-oxathiaphospholanes with O,O-dialkyl H-phosphonates and H-thiophosphonates.

A new method for the formation of organohypophosphates containing a P-P bond under mild conditions, based on the DBU-assisted reaction of 2-alkoxy-2-thio-1,3,2-oxathiaphospholanes with O,O-dialkyl H-phosphonates or H-thiophosphonates, has been elaborated. The resulting triesters of P(1)-thio- and P(1),P(2)-dithiohypophosphoric acids, respectively, having O-methyl or O-ethyl groups, can be selectively dealkylated to form the corresponding di- or monoesters. Appropriately protected 2'-deoxyguanosine-3'-O-(2-thio-1,3,2-oxathiaphospholane) was converted into the corresponding P(1)-thio- and P(1),P(2)-dithiohypophosphate esters in a highly stereoselective manner (98%+ and 90%+, respectively).

Restoration of catalytic functions in Cre recombinase mutants by electrostatic compensation between active site and DNA substrate.

Two conserved catalytic arginines, Arg-173 and Arg-292, of the tyrosine site-specific recombinase Cre are essential for the transesterification steps of strand cleavage and joining in native DNA substrates containing scissile phosphate groups. The active site tyrosine (Tyr-324) provides the nucleophile for the cleavage reaction, and forms a covalent 3'-phosphotyrosyl intermediate. The 5'-hydroxyl group formed during cleavage provides the nucleophile for the joining reaction between DNA partners, yielding strand exchange. Previous work showed that substitution of the scissile phosphate (P) by methylphosphonate (MeP) permits strand cleavage by a Cre variant lacking Arg-292. We now demonstrate that MeP activation and cleavage are not blocked by substitution of Arg-173 or even simultaneous substitutions of Arg-173 and Arg-292 by alanine. Furthermore, Cre(R173A) and Cre(R292A) are competent in strand joining, Cre(R173A) being less efficient. No joining activity is detected with Cre(R173A, R292A). Consistent with their ability to cleave and join strands, Cre(R173A) and Cre(R292A) can promote recombination between two MeP-full-site DNA partners. These findings shed light on the overall contribution of active site electrostatics, and tease apart distinctive contributions of the individual arginines, to the chemical steps of recombination. They have general implications in active site mechanisms that promote important phosphoryl transfer reactions in nucleic acids.

Electrostatic suppression allows tyrosine site-specific recombination in the absence of a conserved catalytic arginine.

The active site of the tyrosine family site-specific recombinase Flp contains a conserved catalytic pentad that includes two arginine residues, Arg-191 and Arg-308. Both arginines are essential for the transesterification steps of strand cleavage and strand joining in DNA substrates containing a phosphate group at the scissile position. During strand cleavage, the active site tyrosine supplies the nucleophile to form a covalent 3'-phosphotyrosyl intermediate. The 5'-hydroxyl group produced by cleavage provides the nucleophile to re-form a 3'-5' phosphodiester bond in a recombinant DNA strand. In previous work we showed that substitution of the scissile phosphate (P) by the charge neutral methylphosphonate (MeP) makes Arg-308 dispensable during the catalytic activation of the MeP diester bond. However, in the Flp(R308A) reaction, water out-competes the tyrosine nucleophile (Tyr-343) to cause direct hydrolysis of the MeP diester bond. We now report that for MeP activation Arg-191 is also not required. In contrast to Flp(R308A), Flp(R191A) primarily mediates normal cleavage by Tyr-343 but also exhibits a weaker direct hydrolytic activity. The cleaved MeP-tyrosyl intermediate formed by Flp(R191A) can be targeted for nucleophilic attack by a 5'-hydroxyl or water and channeled toward strand joining or hydrolysis, respectively. In collaboration with wild type Flp, Flp(R191A) promotes strand exchange between MeP- and P-DNA partners. Loss of a catalytically crucial positively charged side chain can thus be suppressed by a compensatory modification in the DNA substrate that neutralizes the negative charge on the scissile phosphate.

Hoogsteen-paired homopurine [RP-PS]-DNA and homopyrimidine RNA strands form a thermally stable parallel duplex.

Homopurine deoxyribonucleoside phosphorothioates possessing all internucleotide linkages of R(P) configuration form a duplex with an RNA or 2'-OMe-RNA strand with Hoogsteen complementarity. The duplexes formed with RNA templates are thermally stable at pH 5.3, while those formed with a 2'-OMe-RNA are stable at neutrality. Melting temperature and fluorescence quenching experiments indicate that the strands are parallel. Remarkably, these duplexes are thermally more stable than parallel Hoogsteen duplexes and antiparallel Watson-Crick duplexes formed by unmodified homopurine DNA molecules of the same sequence with corresponding RNA templates.

Unusual thermal stability of RNA/[RP-PS]-DNA/RNA triplexes containing a homopurine DNA strand.

Homopurine deoxyribonucleoside phosphorothioates, as short as hexanucleotides and possessing all internucleotide linkages of RP configuration, form a triple helix with two RNA or 2'-OMe-RNA strands, with Watson-Crick and Hoogsteen complementarity. Melting temperature and fluorescence quenching experiments strongly suggest that the Hoogsteen RNA strand is parallel to the homopurine [RP-PS]-oligomer. Remarkably, these triplexes are thermally more stable than complexes formed by unmodified homopurine DNA molecules of the same sequence. The triplexes formed by phosphorothioate DNA dodecamers containing 4-6 dG residues are thermally stable at pH 7.4, although their stability increases significantly at pH 5.3. FTIR measurements suggest participation of the C2-carbonyl group of the pyrimidines in the stabilization of the triplex structure. Formation of triple-helix complexes with exogenously delivered PS-oligos may become useful for the reduction of RNA accessibility in vivo and, hence, selective suppression/inhibition of the translation process.

Oxathiaphospholane approach to the synthesis of oligodeoxyribonucleotides containing stereodefined internucleotide phosphoroselenoate function.

5'-O-DMT-deoxyribonucleoside-3'-O-(2-selena-4,4-pentamethylene-1,3,2-oxathiaphospholane) monomers, derivatives of dA, dC, dG, and T, can be resolved into pure P-diastereomers by silica gel column chromatography. They have been used for DBU-promoted, either solution- or solid-phase synthesis of P-stereodefined phosphoroselenoate analogues of oligodeoxyribonucleotides. Fast- and slow-eluting monomers are precursors of phosphoroselenoate internucleotide linkage of R(P) and S(P) absolute configuration, respectively. [reaction: see text]