X-ray structure of a lectin-bound DNA duplex containing an unnatural phenanthrenyl pair.

DNA duplexes containing unnatural base-pair surrogates are attractive biomolecular nanomaterials with potentially beneficial photophysical or electronic properties. Herein we report the first X-ray structure of a duplex containing a phen-pair in the center of the double helix in a zipper like stacking arrangement.

DNA triple-helix formation at pyrimidine-purine inversion sites.

A systematic investigation of a series of triplex forming oligonucleotides (TFOs) containing alpha-, and beta-thymidine, alpha- and beta-N7-hypoxanthine, and alpha- and beta-N7 and N9 aminopurine nucleosides, designed to bind to T-A inversion sites in DNA target sequences was performed. Data obtained from gel mobility assays indicate that T-A recognition in the antiparallel triple-helical binding motif is possible if the nucleoside alpha N9-aminopurine is used opposite to the inversion site in the TFO.

Conformational diversity versus nucleic acid triplex stability, a combinatorial study.

The stability of a triple helix formed between a DNA duplex and an incoming oligonucleotide strand strongly depends on the solvent conditions and on intrinsic chemical and conformational factors. Attempts to increase triple helix stability in the past included chemical modification of the backbone, sugar ring, and bases in the third strand. However, the predictive power of such modifications is still rather poor. We therefore developed a method that allows for rapid screening of conformationally diverse third strand oligonucleotides for triplex stability in the parallel pairing motif to a given DNA double helix sequence. Combinatorial libraries of oligonucleotides of the requisite (fixed) base composition and length that vary in their sugar unit (ribose or deoxyribose) at each position were generated. After affinity chromatography against their corresponding immobilized DNA target duplex, utilizing a temperature gradient as the selection criterion, the oligonucleotides forming the most stable triple helices were selected and characterized by physicochemical methods. Thus, a series of oligonucleotides were identified that allowed us to define basic rules for triple helix stability in this conformationally diverse system. It was found that ribocytidines in the third strand increase triplex stability relative to deoxyribocytidines independently of the neighboring bases and position along the strand. However, remarkable sequence-dependent differences in stability were found for (deoxy)thymidines and uridines.

Synthesis of pyrrolidine C-nucleosides via Heck reaction.

[figure: see text] A novel method for the synthesis of pyrrolidine C-nucleosides has been developed. The key step of the synthesis is the palladium(0)-mediated coupling of a disubstituted N-protected 2-pyrroline and 5-iodouracil. C-Nucleoside 14 and its N-methyl derivative 15 can easily be converted to the corresponding phosphoramidite building blocks for DNA synthesis.

Triple-helix formation in the antiparallel binding motif of oligodeoxynucleotides containing N(9)- and N(7)-2-aminopurine deoxynucleosides.

Triplex-forming oligodeoxynucleotide 15mers, designed to bind in the antiparallel triple-helical binding motif, containing single substitutions (Z) of the four isomeric alphaN(7)-, betaN(7)-, alphaN(9)- and betaN(9)-2-aminopurine (ap)-deoxyribonucleosides were prepared. Their association with double-stranded DNA targets containing all four natural base pairs (X-Y) opposite the aminopurine residues was determined by quantitative DNase I footprint titration in the absence of monovalent metal cations. The corresponding association constants were found to be in a rather narrow range between 1.0 x 10(6) and 1.3 x 10(8) M(-1). The following relative order in Z x X-Y base-triple stabilities was found: Z = alphaN(7)ap: T-A > A-T> C-G approximately G-C; Z = betaN(7)ap: A-T > C-G > G-C > T-A; Z = alphaN(9)ap: A-T = G-C > T-A > C-G; and Z = betaN(9)ap: G-C > A-T > C-G > T-A.

A stable DNA duplex containing a non-hydrogen-bonding and non-shape-complementary base couple: interstrand stacking as the stability determining factor.

The stabilizing effect of a dG:dC base-pair can also be imparted to a DNA duplex by a non-hydrogen-bonding, non-shape-complementary nucleoside analogue when interstrand stacking interactions come into play. This is the case, for example, with dBP, which has a bipyridyl (BP) residue as a nucleobase surrogate (the picture shows a dBP:dBP pair).

Oligodeoxynucleotides containing conformationally constrained abasic sites: a UV and fluorescence spectroscopic investigation on duplex stability and structure.

The synthesis and incorporation into oligodeoxy-nucleotides of two novel, conformationally restricted abasic (AB) site analogs are described. The stability of oligonucleotide 18mer duplexes containing one such AB site opposite any of the four natural DNA bases was investigated by UV melting curve analysis and compared to that of duplexes containing a conformationally flexible propanediol unit 1 or a tetrahydrofuran unit 2 as an AB site analog. No major differences in the melting temperatures (DeltaT(m) 0-3 degrees C) between the different abasic duplexes were observed. All AB duplexes were found to have T(m)s that were lower by 9-15 degrees C relative to a fully matched 18mer control duplex, and by 4-10 degrees C relative to the corresponding 19mer duplexes in which the AB site is replaced by a mismatched nucleobase. Thus we conclude that the loss of stability of a duplex that is encountered by removal of a nucleobase from the stack cannot be compensated with conformational restriction of the AB site. From the van't Hoff transition enthalpies obtained from the melting curves, it appears that melting cooperativity is higher for the duplexes containing the conformationally rigid AB sites. Fluorescence quenching experiments with duplexes containing the fluorescent base 2-amino-purine (2AP) opposite the AB sites showed a weak tendency towards more efficient stacking of this base in duplexes containing the conformationally constrained AB sites. Thus, such AB sites may structurally stabilize the cavity formed by the removal of a base. Potential applications emerging from the properties of such conformationally constrained AB sites in DNA diagnostics are discussed.

Total synthesis of coraxeniolide-A.

The first total synthesis of optically active coraxeniolide-A (1a) and 4-epi-coraxeniolide-A (1b) is described. The approach is highly stereoselective and flexible in the preparation of a wide variety of members of the xeniolide family. The use of the Grob-fragmentation was pivotal for the stereospecific elaboration of the nine-membered ring. Coraxeniolide-A (1a) was synthesized in 28 steps by using the Hajos-Parrish diketone 2 as starting material which is available enantiomerically pure.

Selective recognition of a C-G base-pair in the parallel DNA triple-helical binding motif.

Selective recognition of a C-G base-pair within the parallel DNA triple-helical binding motif was achieved by a third strand containing the base 5-methyl pyrimidin-2-one. The third strand affinities (K(D)) for a representative 15-mer duplex sequence containing all four Watson-Crick base pairs (X-Y) in the center are C-G (26 nM) >> A-T (270 nM) approximately T-A (350 nM) > G-C (ca 700 nM).

Inhibition of DNA polymerase reactions by pyrimidine nucleotide analogues lacking the 2-keto group.

To investigate the influence of the pyrimidine 2-keto group on selection of nucleotides for incorporation into DNA by polymerases, we have prepared two C nucleoside triphosphates that are analogues of dCTP and dTTP, namely 2-amino-5-(2'-deoxy-beta-d-ribofuranosyl)pyridine-5'-triphosphate (d*CTP) and 5-(2'-deoxy- beta-d-ribofuranosyl)-3-methyl-2-pyridone-5'-triphosphate (d*TTP) respectively. Both proved strongly inhibitory to PCR catalysed by Taq polymerase; d*TTP rather more so than d*CTP. In primer extension experiments conducted with either Taq polymerase or the Klenow fragment of Escherichia coli DNA polymerase I, both nucleotides failed to substitute for their natural pyrimidine counterparts. Neither derivative was incorporated as a chain terminator. Their capacity to inhibit DNA polymerase activity may well result from incompatibility with the correctly folded form of the polymerase enzyme needed to stabilize the transition state and catalyse phosphodiester bond formation.

Strong, specific, monodentate G-C base pair recognition by N7-inosine derivatives in the pyrimidine.purine-pyrimidine triple-helical binding motif.

The nucleoside analogs 7-(2'-deoxy-alpha-D-ribofuranosyl)hypoxanthine (alpha7H,1), 7-(2'-deoxy-beta-D-ribofuranosyl)hypoxanthine (beta7H,2) and 7-7-(2'-O-methyl-beta-D- ribofuranosyl)hypoxanthine (beta7HOMe,3) were prepared and incorporated into triplex forming oligodeoxynucleotides, designed to bind to DNA in the parallel (pyrimidine.purine-pyrimidine) motif. By DNase I footprinting techniques and UV-melting curve analysis it was found that, at pH 7. 0, the 15mer oligonucleotides d(TTTTTMeCTXTMeCTMeCTMeCT) (MeC = 5-methyl-deoxycytidine, X =beta7H,beta7HOMe) bind to a DNA target duplex forming a H.G-C base triple with equal to slightly increased (10-fold) stability compared to a control oligodeoxynucleotide in which the hypoxanthine residue is replaced by MeC. Remarkably, triple-helix formation is specific to G-C base pairs and up to 40 microM third strand concentration, no stable triplex exhibiting H.A-T, H.T-A or H.C-G base arrangements could be found (target duplex concentration approximately 0.1 nM). Multiply substituted sequences containing beta7H residues either in an isolated [d(TTTTTbeta7HTbeta7HTbeta7HTbeta7HTbeta7HT)] or in a contiguous [d(TTTbeta7Hbeta7Hbeta7Hbeta7HTTTTbeta7HTTT)] manner still form triplexes with their targets of comparable stability as the control (MeC-containing) sequences at pH 7.0 and high salt or spermine containing buffers. General considerations lead to a structural model in which the recognition of the G-C base pair by hypoxanthine takes place via only one H-bond of the N-H of hypoxanthine to N7 of guanine. This model is supported by a molecular dynamics simulation. A general comparison of the triplex forming properties of oligonucleotides containing beta7H with those containing MeC or N7-2'-deoxyguanosine (N7G) reveals that monodentate recognition in the former case can energetically compete with bidentate recognition in the latter two cases.

Bicyclo-DNA: a Hoogsteen-selective pairing system.

BACKGROUND: The natural nucleic acids (DNA and RNA) can adopt a variety of structures besides the antiparallel double helix described by Watson and Crick, depending on base sequence and solvent conditions. Specifically base-paired DNA structures with regular backbone units include left-handed and parallel duplexes and triple and quadruple helical arrangements. Given the base-pairing pattern of the natural bases, preferences for how single strands associate are determined by the structure and flexibility of the sugar-phosphate backbone. We set out to determine the role of the backbone in complex formation by designing DNA analogs with well defined modifications in backbone structure. RESULTS: We recently developed a DNA analog (bicyclo-DNA) in which one (gamma) of the six torsion angles (alpha-zeta) describing the DNA-backbone conformation is fixed in an orientation that deviates from that observed in B-DNA duplexes by about + 100 degrees , a shift from the synclinal to the antiperiplanar range. Upon duplex formation between homopurine and homopyrimidine sequences, this analog preferentially selects the Hoogsteen and reversed Hoogsteen mode, forming A-T and G-C+ base pairs. Base-pair formation is highly selective, but degeneracy is observed with respect to strand orientation in the duplex. CONCLUSIONS: The flexibility and orientation of the DNA backbone can influence the preferences of the natural bases for base-pairing modes, and can alter the relative stability of duplexes and triplexes.

A new strategy for the generation of catalytic antibodies.

The high binding affinity and specificity of antibodies for a wide range of ligands has recently been exploited in the generation of catalysts for acyl-transfer reactions, carbon-carbon bond forming and carbon-carbon bond cleaving reactions. In addition, a number of strategies are emerging for the generation of catalytic antibodies including transition state stabilization, catalysis by approximation, and the introduction of catalytic groups or cofactors into antibody combining sites. An important goal in the design of catalytic antibodies is the development of general rules relating hapten structure to the corresponding catalytic groups in the antibody combining site. We report here that electrostatic interactions between a hapten and the complementary antibody can be exploited to generate catalytic amino-acid side chains in an antibody-combining site. The antibody-catalysed reaction, a beta-elimination reaction, exhibits saturation kinetics, substrate specificity, competitive inhibition by hapten, and specific inactivation by a reagent that modifies carboxylate residues.