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.

ZrIV-tetraphenylporphyrinates as nuclease mimics: structural, kinetic and mechanistic studies on phosphate diester transesterification.

The Zr(IV)-tetraphenylpor-phyrinates Zr(TPP)(X,X'), (X,X' = -OAc, -OMe, Cl ) 4-6, 8 were prepared and their complexing properties as well as catalytic properties towards solvolysis of the phosphate diesters hpp (2), dmp (3) and pmp (16) characterised. The diesters 2 and 16, representing model phosphates for RNA and DNA, were substrates for the catalyst Zr(TPP)Cl2 (4), and rate accelerations over background by 6-9 orders of magnitude were measured. These accelerations are comparable to those of dinuclear transition metal catalysts and lanthanide ions. Catalytic turnover was observed. Kinetic studies revealed that the catalytically active species of 4 in the solvolysis of 2 and 16 in methanol-containing solvents are dinuclear complexes containing either one or two phosphate esters depending upon the phosphate concentration. Besides the usual solvolysis pathway of the RNA model hpp (2), which proceeds via the cyclophosphate 20, a second, unusual pathway via direct substitution of the hydroxypropyl substituent was found. X-ray analysis of the Zr(TPP)(dmp) complex 19 revealed a dinuclear structure with two bridging dmp ligands and one monomethyl phosphate unit. In 19 one of the two dmp residues occurs in a very unusual high energy ac,ap conformation. Based on this structure and on the kinetic data, mechanistic models for the two solvolysis reaction pathways were developed. From an extensive CSD search on phosphodiester structures no correlation between P-O ester bond lengths and diester conformations could be found. However, P-O ester bonds decrease in length with increasing formal charge of the complexing metal ions. This underlines the higher importance of electrostatic activation relative to stereoelectronic effects in phosphodiester hydrolysis.

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).

Triple helix formation inhibits DNA gyrase activity.

The goal of this work was to examine the effect of triple helix-forming oligonucleotides on a gyrase target region and on the activity of the enzyme. Using melting temperature measurements and gel mobility shift analysis, it was found that modified oligonucleotides can form a triple helix along the 29-nucleotide region of a 32-bp duplex representing part of the gyrase DNA-target sequence of the 162-bp fragment from pBR322. Triplex formation with this target region has been achieved at pH 7.5 by using a synthetic oligonucleotide in which cytosine was replaced by the C-nucleoside of 2-aminopyridine. The results of the enzymic experiments in vitro with the 162-bp fragment demonstrated that the cleavage reaction mediated by gyrase can be efficiently inhibited by the triplex-forming oligonucleotide modified with 2-aminopyridine. A possible inhibitory mechanism is discussed.

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.

Bicyclo[3.2.1]-DNA, a new DNA analog with a rigid backbone and flexibly linked bases: pairing properties with complementary DNA.

BACKGROUND: The structural and conformational variety in nucleic acid complexes is largely controlled by the sugar-phosphate backbone. In order to modulate specific features such as strength or selectivity of complex formation by designing nucleotide analogs, a deeper understanding of the relationship between mononucleotide structures and the properties of their oligomers is necessary. One approach involves comparing the properties of DNA analogs displaying well defined modifications in their backbone structure with those of natural DNA and RNA. RESULTS: We have designed and synthesized a new DNA analog, 'bicyclo[3.2.1]-DNA', which has a rigid phosphodiester backbone that emulates a B-DNA-type conformation, to which the nucleobases are attached via a flexible open-chain linker. A UV-melting curve analysis shows that bicyclo[3.2.1]-DNA forms stable duplexes with complementary DNA, although generally with lower Tm values than pure DNA duplexes. Duplex formation is strictly constrained to antiparallel complementary sequences, and base-mismatch discrimination is slightly enhanced compared to pure DNA duplexes. In addition, bicyclo[3.2.1]-DNA sequences are resistant to a 3'-exonuclease. CONCLUSIONS: The furanose unit present in natural nucleosides is not necessary for a competent and stable phosphodiester-based pairing system, provided that the backbone is conformationally constrained. The information for the preference of antiparallel strand association in B-DNA is not merely a consequence of bases being attached to a specific side of the furanose unit, but is also encoded in the backbone itself. Furthermore, conformational flexibility in the base-pairing region does not lead to a loss of selectivity in base-pair 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.

Watson-Crick base-pairing properties of bicyclo-DNA.

A series of sequences of the DNA analog bicyclo-DNA, 6-12 nucleotides in length and containing all four natural nucleobases, were prepared and their Watson-Crick pairing properties with complementary RNA and DNA, as well as in its own series, were analyzed by UV-melting curves and CD-spectroscopy. The results can be summarized as follows: bicyclo-DNA forms stable Watson-Crick duplexes with complementary RNA and DNA, the duplexes with RNA generally being more stable than those with DNA. Pyrimidine-rich bicyclo-DNA sequences form duplexes of equal or slightly increased stability with DNA or RNA, whereas purine-rich sequences show decreased affinity to complementary DNA and RNA when compared with wild-type (DNA-DNA, DNA-RNA) duplexes. In its own system, bicyclo-DNA prefers antiparallel strand alignment and strongly discriminates for base mismatches. Duplexes are always inferior in stability compared with the natural ones. A detailed analysis of the thermodynamic properties was performed with the sequence 5'-GGATGGGAG-3'x 5'-CTCCCATCC-3' in both backbone systems. Comparison of the pairing enthalpy and entropy terms shows an enthalpic advantage for DNA association (delta deltaH = -18 kcal x (mol)-1)) and an entropic advantage for bicyclo-DNA association (delta deltaS = 49 cal x K(-1) x mol(-1), leading to a delta deltaG 25 degrees C of -3.4 kcal x mol(-1) in favor of the natural duplex. The salt dependence of Tm for this sequence is more pronounced in the case of bicyclo-DNA due to increased counter ion screening from the solvent. Furthermore bicyclo-DNA sequences are more stable towards snake venom phosphodiesterase by a factor of 10-20, and show increased stability in fetal calf serum by a factor of 8 compared with DNA.

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.