Pyrovanadolysis, a pyrophosphorolysis-like reaction mediated by pyrovanadate, Mn2+, and DNA polymerase of bacteriophage T7.

DNA polymerases catalyze the 3'-5'-pyrophosphorolysis of a DNA primer annealed to a DNA template in the presence of pyrophosphate (PP(i)). In this reversal of the polymerization reaction, deoxynucleotides in DNA are converted to deoxynucleoside 5'-triphosphates. Based on the charge, size, and geometry of the oxygen connecting the two phosphorus atoms of PP(i), a variety of compounds was examined for their ability to carry out a reaction similar to pyrophosphorolysis. We describe a manganese-mediated pyrophosphorolysis-like activity using pyrovanadate (VV) catalyzed by the DNA polymerase of bacteriophage T7. We designate this reaction pyrovanadolysis. X-ray absorption spectroscopy reveals a shorter Mn-V distance of the polymerase-VV complex than the Mn-P distance of the polymerase-PP(i) complex. This structural arrangement at the active site accounts for the enzymatic activation by Mn-VV. We propose that the Mn(2+), larger than Mg(2+), fits the polymerase active site to mediate binding of VV into the active site of the polymerase. Our results may be the first documentation that vanadium can substitute for phosphorus in biological processes.

Stability of DNA containing a structural water mimic in an A-T rich sequence.

We describe here the synthesis and properties of A-T rich DNA containing covalently bound water mimics located in the DNA minor groove.

Preparation of the 2'-deoxynucleosides of 2,6-diaminopurine and isoguanine by direct glycosylation.

The purine nucleoside 2,6-diaminopurine-2'-deoxyriboside is prepared by the direct glycosylation of the 2,6-bis(tetramethylsuccinimide) derivative of the parent purine heterocycle 4 with 2-deoxy-3,5-di-O-(p-toluoyl)-alpha-D-erythro-pentofuranosyl chloride 5 using the sodium salt method. 2'-Deoxyisoguanosine is prepared from 2,6-diaminopurine by a five-step procedure. The purine heterocycle isoguanine is prepared by selective diazotization of 2,6-diaminopurine and then converted to the N9-trityl derivative to increase solubility. After silylation of the O(2)-carbonyl with TMSCl, the N(6)-amino group is protected as the tetramethylsuccinimide (M(4)SI). The O(2)-carbonyl is protected as the DPC derivative, and the trityl group is removed. The resulting product is glycosylated in good yield to generate fully protected 2'-deoxyisoguanosine.

A Janus-Wedge DNA triplex with A-W1-T and G-W2-C base triplets.

A new type of double-stranded DNA targeting format by formation of a Janus-Wedge (J-W) triple helix is described. The "wedge" residue W1 is used for A-T and T-A base pairs while W2 is used for G-C and C-G base pairs. Both wedge residues are attached to a PNA backbone that is designed to insert the probe strand into double-stranded DNA and base pair with both Watson-Crick faces. To study the stability of such an assembly, we have examined the formation of the J-W triplex with various sequences.

2',3'-Dideoxy-3'-thionucleoside triphosphates: syntheses and polymerase substrate activities.

All four 2',3'-dideoxy-3'-thio-nucleosides (ddtNTPs) function as substrates for the Y410F mutant of Deep Vent (exo-) DNA polymerase. Not only are the ddtNTPs incorporated to form the N + 1 product, but further elongations are observed in which the key step is attack of the 3'-thiol on the 5'-triphosphate. Although other polymerases are likely to differ in their use of the ddtNTPs, there does not appear to be a fundamental prohibition against using a thiol nucleophile on a phosphate anhydride electrophile. The syntheses of four ddtNTPs (C, T, A, G) are described. [structure: see text]

High fidelity TNA synthesis by Therminator polymerase.

Therminator DNA polymerase is an efficient DNA-dependent TNA polymerase capable of polymerizing TNA oligomers of at least 80 nt in length. In order for Therminator to be useful for the in vitro selection of functional TNA sequences, its TNA synthesis fidelity must be high enough to preserve successful sequences. We used sequencing to examine the fidelity of Therminator-catalyzed TNA synthesis at different temperatures, incubation times, tNTP ratios and primer/template combinations. TNA synthesis by Therminator exhibits high fidelity under optimal conditions; the observed fidelity is sufficient to allow in vitro selection with TNA libraries of at least 200 nt in length.

Glycerol nucleoside triphosphates: synthesis and polymerase substrate activities.

[Structure: see text] The synthesis of (S)-glycerol nucleoside triphosphates (gNTPs) and the analysis of their substrate activities for enzymatic polymerization is described. NTPs with simplified carbohydrate backbones such as the tNTPs (alpha-L-threose-NTPs) are polymerase substrates and offer the potential to create non-natural aptamer sequences with simplified backbones through enzymatic means. The acyclic (S)-GNA was modeled after the shortened alpha-threofuranosyl backbone. Here we describe the synthesis of (S)-glycerol NTPs and initial enzymatic testing of this further simplified nucleic acid backbone.

The role of minor groove functional groups in DNA hydration.

Here we describe the crystal structure of modified [d(CGCGAATTCGCG)]2 refined to 2.04 A. The modification, which affects only the two thymines at the central ApT step, involves isosteric removal of the 2-keto oxygen atoms and substitution of the N1 nitrogen with carbon. The crystal structure reveals the ability of this modified thymine to effectively base pair with adenine in [d(CGCGAAtTCGCG)]2. The structure also suggests that the minor groove 'spine of hydration' is destabilized but essentially intact.

Effect of terminal 3'-hydroxymethyl modification of an RNA primer on nonenzymatic primer extension.

The significance of the precise position of the hydroxyl at the 3'-end of an RNA primer for nonenzymatic template-directed primer extension is not well understood. We show that an RNA primer terminating in 3'-hydroxymethyl-2',3'-dideoxy-guanosine has greatly diminished activity, suggesting that the spatial preorganization of the terminal sugar contributes significantly to the efficiency of primer extension.

Synthesis of alpha-L-threofuranosyl nucleoside triphosphates (tNTPs).

[structure: see text] The alpha-l-threofuranosyl nucleoside triphosphates of T, G, and D (tTTP, tGTP, and tDTP) were synthesized from the described 2'-O-DMT-protected derivatives using the Eckstein method, while the corresponding C derivative (tCTP) was prepared from the 2'-O-acetyl derivative. The prepared alpha-l-threofuranosyl nucleoside triphosphates, despite being one carbon shorter than the native 2'-deoxyfuranosyl nucleoside triphosphates, are effective substrates for selected DNA polymerases.

Design and synthesis of DNA-tethered ruthenium complexes that self-assemble into linear arrays.

Ruthenium(II) bis(terpyridine) complexes have been prepared with two triethylene glycol linkers to which DNA sequences have been attached; hybridization at various complex ratios results in linear arrays of varying lengths.

Hoechst 33258 Tethered by a Hexa(ethylene glycol) Linker to the 5'-Termini of Oligodeoxynucleotide 15-Mers: Duplex Stabilization and Fluorescence Properties.

A fluorescent Hoechst 33258 derivative has been prepared in which a hexa(ethylene glycol) linker is attached to the terminal phenol residue. Conjugation of this derivative to DNA sequences is accomplished by a reversed coupling protocol, one in which the 5'-terminal nucleoside residue of a fully protected DNA sequence is converted to a terminal phosphoramidite. In the presence of the Hoechst derivative and tetrazole the final coupling reaction is achieved to generate the conjugated nucleic acid. After deprotection and cleavage of the conjugate from the support, HPLC analysis indicates that the conjugation reaction proceeds with yields as high as 75%. The presence of the conjugated Hoechst derivative increases the stability of DNA duplexes typically by 10-16 degrees C. A variety of sequence variants indicate that the tether length is sufficient to reach beyond the terminus of the DNA duplex and bind to internal A-T rich target sequences as far away as four base pairs from the site of attachment. A four base pair binding site appears to be necessary for effective helix stabilization by the conjugate, but in some cases can include a G-C base pair, which is consistent with a previous X-ray diffraction study regarding the binding of Hoechst 33258 to duplex DNA. When A-T base pairs alternate with G-C base pairs, a small but discernible increase is T(m) is observed (3.6 degrees C), indicating that binding to this sequence still occurs, but not in the same manner as to A-T rich sequences. Upon formation of the conjugated duplex, an enhanced quantum yield for the fluorescence emission spectrum of the tethered Hoechst derivative is observed. When an A-T rich binding site is present, the enhanced quantum yield increases by at least 16- and in some cases to nearly 30-fold relative to the value obtained for the single-stranded DNA-Hoechst conjugate.

Triplex mediated delivery of a platinum complex to a specific DNA target site.

Tethering an ethylene diamine linker to the 5' terminus of an oligothymidine sequence provides a site for complexation with K(2)PtCl(4). Due to the low reactivity of dT toward a platinum source, we chose dT(8) and dT(15) as our initial synthetic targets for platination. Post-synthetic reaction of the platinum reagent with the diamino oligothymidine generates the diamino dichloro platinum-DNA conjugate that can be used for DNA duplex targeting by oligodeoxyncleotide-mediated triplex formation. The dT(8) sequence is not sufficiently long to facilitate triplex formation and Pt-cross-linking, whereas with a dT(15) sequence cross-linking between the third strand and the duplex occurs exclusively with the duplex target strand directly involved in triplex formation. No examples of cross-linking to the complementary target strand, or of cross-linking to both target strands are observed. Most efficient cross-linking occurs when the dinucleotide d(GpG) is present in the target strand and no cross-linking occurs with the corresponding 7-deazaG dinucleotide target. Cross-linking is also observed when dC or dA residues are present in the target strand, or even with a single dG residue, but it is not observed in any cases to dT residues. Triplex formation provides the ability to target specific sequences of double-stranded DNA and the orientational control arising from triplex formation is sufficient to alter the binding preferences of platinum. Conjugates of the type described here offer the potential of delivering a platinum complex to a specific DNA site.

2,3-Dicyclohexylsuccinimide as a directing/protecting group for the regioselective glycosylation or alkylation of purines.

Here we describe the synthesis and application of a novel 2,3-dicyclohexylsuccinimide (Cy2SI) protecting group towards regioselective purine glycosylation and alkylation reactions. This bulky protecting group promotes high regioselectivity during the glycosylation (as well as diastereoselectivity) or alkylation of purines using Hoffer's chlorosugar or tert-butyl bromoacetate, respectively. Cy2SI offers the additional synthetic advantage that other base-labile protecting groups, such as toluoyl esters, can be selectively removed in its presence without affecting the imide.

Synthesis and NMR assignment of the two diastereomers of 8,6'-cyclo-2',6'-dideoxyadenosine.

We herein present the first synthesis and characterization of the two C5' diastereomers of 8,6'-cyclo-2',6'-dideoxyadenosine. Starting from commercially available 2'-deoxyadenosine, the target cyclonucleosides were synthesized in 11 linear steps. Following a zinc-mediated cyclization reaction to form the seven-membered ring, the stereochemistry of the newly formed chiral center was established using two-dimensional NOESY NMR experiments.

An efficient synthetic approach to 6,5'-(S)- and 6,5'-(R)-cyclouridine.

Here we present new routes for the efficient syntheses of 6,5'-(S)- and 6,5'-(R)-cyclouridine. The syntheses utilize readily accessible uridine as a starting material. This route to the R diastereomer is significantly more efficient than previous synthetic efforts, allowing us to obtain large amounts of pure material for future biological testing.

Synthesis and properties of DNA containing cyclonucleosides.

Here, we present efficient syntheses of the R and S diastereomers of 8,5'-cyclo-2'-deoxyadenosine and 6,5'-cyclo-2'-deoxyuridine. We incorporated these interesting nucleosides into DNA to study how the cyclo linkage affects the stability of duplex formation.

Tetramethylsuccinimide as a directing/protecting group in purine glycosylations.

Tetramethylsuccinic anhydride can be used to protect the exocyclic amine of 6-aminopurine derivatives by forming the corresponding tetramethysuccinimide. X-ray crystallography confirms that the imide carbonyl and the methyl groups are positioned to sterically block the N7 nitrogen so that glycosylations occur with very high regiochemical control at N9. This approach is particularly effective for 3-substituted purines where the substituent tends to block access to N9 and inhibit glycosylation at that site.