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'--TBDMS-ribonucleosides (N-OTP and N-OTP, respectively; nucleobase protected) were synthesized and separated into pure P-diastereomers. X-ray analysis showed the absolute configuration of the phosphorus atom in the -eluting diastereomer of A-OTP. The - and -eluting P-diastereomers of N-OTP and N-OTP were used in the solid-phase synthesis of phosphorothioate dinucleotides (NT and NT, respectively), which were subsequently hydrolyzed with -selective phosphodiesterase svPDE and -selective nuclease P1 to determine the absolute configuration of the phosphorus atoms.

Boron clusters as a platform for new materials: composites of nucleic acids and oligofunctionalized carboranes (CBH) 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.

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.

Homopurine R-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 role of the Hint1 protein in the metabolism of phosphorothioate oligonucleotides drugs and prodrugs, and the release of HS 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.

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

Enantiomerically pure, protected acyclic nucleosides of the GNA type (glycol nucleic acids) (N'), obtained from ()-(+)- and ()-(-)-glycidols and the four canonical DNA nucleobases (Ade, Cyt, Gua and Thy), were transformed into 3'--DMT-protected 2-thio-4,4-pentamethylene-1,3,2-oxathiaphospholane derivatives (OTP-N') containing a second stereogenic center at the phosphorus atom. These monomers were chromatographically separated into -diastereoisomers, which were further used for the synthesis of -stereodefined "dinucleoside" phosphorothioates NT (N = A, C, G, T). The absolute configuration at the phosphorus atom for all eight NT was established enzymatically and verified chemically, and correlated with chromatographic mobility of the OTP-N' monomers on silica gel.

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.

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.

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.

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.

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.

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.

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.

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.

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.