Tethered naphthalene diimide intercalators enhance DNA triplex stability.

Naphthalene diimides function as effective intercalators and when tethered to the 5'-terminus of a pyrimidine-rich oligonucleotide can contribute significantly to the overall stabilization of DNA triplexes. This stabilization can be further enhanced by alterations to the linker tethering the DNA sequence and the intercalator. Less flexible linkers, and particularly one with a phenyl ring present, appear to permit the stabilization afforded by the bound intercalator to be transferred more effectively to the three-stranded complex. The conjugate containing the phenyl linker exhibits a T(M) value that is increased by 28 degrees C relative to the unconjugated triplex. That the linker itself contributes to the observed stabilization is clear since introduction of the phenyl linker increases the observed T(M) by 11 degrees C relative to a simple flexible linker.

Minor groove functional groups are critical for the B-form conformation of duplex DNA.

Two analogue bases are described: 3-deazaadenine is a derivative of adenine from which N3 has been deleted and 3-methyl-2-pyridone is a C-nucleoside that mimics thymine but lacks the O2 carbonyl. The dc(3)A-dm(3)2P base pair is similar to dA-dT but eliminates the polar functional groups in the minor groove. The presence of this base pair in dA-dT rich sequences results in destabilized duplexes or conformational preferences for monomolecular hairpins rather than bimolecular duplexes. When present in dG-dC rich sequences, no significant differences in helix stability are observed. These differences are explained on the basis of hydration effects, most notably, the elimination of the minor groove spine of hydration normally present in dA-dT rich sequences. CD spectra suggest that sequences with a fully modified core (four analogue base pairs) are more A-like helices than B-like helices. Sequences containing two analogue base pairs can be transformed to A-like helices under conditions of high salt, or 65% trifluoroethanol. These conformational changes are also explained in terms of a loss of hydration in the minor groove that normally stabilizes the B-form conformation. In the absence of such hydration, the helices are conformationally mobile and adopt a more A-like helix form.

Engineering specific cross-links in nucleic acids using glycol linkers.

One of the most convenient methods for generating oligonucleotides possessing intra- or interstrand cross-links is through incorporation of oligoethylene glycol bridges by solid-phase synthesis. The reagents are commercially available or can be synthesized in a few easy synthetic steps. Unlike many other DNA and RNA cross-links, aspects of the structural and thermodynamic impact of modifying nucleic acids with oligoethylene glycols have been studied. This unit covers protection, phosphitylation, and preparation of the glycol linker for oligonucleotide synthesis.

Synthesis of a dA-dT base pair analogue and its effects on DNA-ligand binding.

Two nucleoside derivatives containing the base analogues 3-deazaadenine and 3-methyl-2-pyridone have been prepared as analogues of dA and dT, respectively. After conversion into the appropriately protected phosphoramidites, DNA sequences were prepared with site-specifically placed analogues. When present in a duplex DNA sequence, the analogues result in the deletion of one or both of the hydrogen bonding functional groups (the N3-nitrogen of dA and the O2-carbonyl of dT) present in the minor groove. Binding by two ligands, 4',6-diamidine-2-phenyl indole (DAPI) and Hoechst 33258 in the minor groove has been probed using a variety of DNA sequences. These sequences contain a d(GAATTC)2 core with analogue nucleosides substituted for one or more of the dA and dT residues. DAPI bound strongly to any sequence that contained both O2-carbonyls of the central two dT residues. The presence of a dc3A residue did in some cases enhance binding. With one of the central O2-carbonyls deleted, the binding was noticeably reduced, and with both absent, no significant binding could be detected. Similar although less dramatic results were observed with Hoechst 33258 binding to analogue sequences.

Use of pK(a) differences to enhance the formation of base triplets involving C-G and G-C base pairs.

Two C-nucleosides are employed for the recognition of dC-dG base pairs. Both derivatives are related to dC but lack the O2-carbonyl. The absence of the carbonyl should eliminate any unfavorable steric interactions at this site. One of the derivatives contains a 2-aminopyridine heterocycle (d2APy) while the second contains a 2-aminopyrimidine heterocycle (d2APm). The former with a pK(a) of 6. 8 functions better for the recognition of dG-dC base pairs than it does in the binding to dC-dG base pairs. The d2APm derivative with a pK(a) of 3.3 functions better to form base triplets with dC-dG base pairs than with dG-dC targets. Triplex T(m)'s in both cases are compared with the sequence containing the native dC residue. The dC analogues appear to make two hydrogen bonds to a target dG base residue, one of which requires protonation of the ring nitrogen. Recognition of a target dC residue appears to require the formation of a single hydrogen bond to the C-nucleoside and having that nitrogen largely in the unprotonated state facilitates its formation.

Expanding the structural and functional diversity of RNA: analog uridine triphosphates as candidates for in vitro selection of nucleic acids.

Two analog uridine triphosphates tethering additional functionality, one a primary amino group and the second a mercapto group, were prepared and tested for their compatibility with in vitro RNA selection procedures. 5-(3-Aminopropyl)uridine triphosphate (UNH(2)) as a uridine substitute was a more effective substrate for T7 RNA polymerase than 5-(2-mercaptoethyl)uridine triphosphate (USH). However, both functioned in transcription assays of 100 nt templates to generate RNA transcripts in quantities sufficient to initiate RNA selection procedures. Transcription of RNA pools with T7 RNA polymerase and UNH(2) or USH occurred with efficiencies of 43 and 29%, respectively, of the values obtained for native UTP transcription. In addition, the transcribed RNA containing roughly 25% UNH(2) residues exhibited better substrate properties for SuperScript(TM) II RNase H reverse transcriptase than did RNA transcripts containing approximately 25% of the USH analog. With either analog, both transcription and reverse transcription proceeded with high fidelity for insertion of the analog residue.

Long-range guanine oxidation in DNA restriction fragments by a triplex-directed naphthalene diimide intercalator.

Naphthalene diimide (NDI), a powerful oxidant that binds avidly to DNA by intercalation, is seen to damage the 5' guanine of 5'-GG-3' sites by photoactivated charge transport through DNA. When covalently tethered to the center of a triplex-forming oligonucleotide and delivered by triplex formation within a pyrimidine.purine-pyrimidine motif to a specific site on a restriction fragment, NDI can photooxidize guanine over at least 25-38 bp in each direction from the site of binding. Charge migration occurs in both directions from the NDI intercalator and on both DNA strands of the target, but the oxidation is significantly more efficient to the 3' side of the triplex. NDI and octahedral rhodium intercalators, when tethered directly to the 5' terminus of the triplex-forming strand as opposed to the center, generate significant amounts of oxidative damage only in the immediate vicinity of the intercalation site. Given that long-range charge transport depends on DNA stacking, these results suggest that the base stack is distorted at the 5' end of the triplex region in the duplex-triplex junction. Targeting of photooxidative damage by triplex formation extends our previous studies of long-range charge transport to significantly longer DNA sequences through a strategy that does not require covalent attachment of the photooxidant to the DNA being probed. Moreover, triplex targeting of oxidative damage provides for the first time a typical distance distribution for genomic charge transport of approximately 200 A around the oxidant.

Tethered naphthalene diimide-based intercalators for DNA triplex stabilization.

The synthesis and triplex stabilizing properties of oligodeoxyribonucleotides functionalized at the 5'- and/or 3'-termini with a naphthalene diimide-based (NDI) intercalator is described. The NDI intercalator was prepared in a single step from the corresponding dianhydride and was attached to the 5'-terminus of an oligodeoxyribonucleotide following a reverse coupling procedure. The DMT protecting group was removed and the sequence phosphitylated to generate the phosphoramidite derivative on the 5'-terminus of the support-bound oligodeoxyribonucleotide. The NDI intercalator with a free hydroxyl was then added in the presence of tetrazole. Attachment of the NDI to the 3'-terminus relied upon a tethered amino group that could be functionalized first with the naphthalene dianhydride, which was subsequently converted to the diimide. Using both procedures, an oligonucleo-tide conjugate was prepared having the NDI intercalator at both the 5'- and 3'-termini. Thermal denaturation studies were used to determine the remarkable gain in stability for triplexes formed when the NDI-conjugated oligonucleotide was present as the third strand in the complex.

Nucleoside analogue substitutions in the trinucleotide DNA template recognition sequence 3'-(CTG)-5' and their effects on the activity of bacteriophage T7 primase.

Bacteriophage T7 primase catalyzes the synthesis of the oligoribonucleotides pppACC(C/A) and pppACAC from the single-stranded DNA template sites 3'-d[CTGG(G/T)]-5' and 3'-(CTGTG)-5', respectively. The 3'-terminal deoxycytidine residue is conserved but noncoding. A series of nucleoside analogues have been prepared and incorporated into the conserved 3'-d(CTG)-5' site, and the effects of these analogue templates on T7 primase activity have been examined. The nucleosides employed include a novel pyrimidine derivative, 2-amino-5-(beta-2-deoxy-D-erythro-pentofuranosyl)pyridine (d2APy), whose synthesis is described. Template sites containing d2APy in place of the cryptic dC support oligoribonucleotide synthesis whereas those containing 3-deaza-2'-deoxycytidine (dc(3)C) and 5-methyl-6-oxo-2'-deoxycytidine (dm(5ox)C) substitutions do not, suggesting that the N3 nitrogen of cytidine is used for a critical interaction by the enzyme. Recognition sites containing 4-amino-1-(beta-2-deoxy-D-erythro-pentofuranosyl)-5-methyl-2,6[1H, 3H]-pyrimidione (dm(3)2P) or 2'-deoxyuridine (dU) substitutions for dT support oligoribonucleotide synthesis whereas those containing 5-methyl-4-pyrimidinone 2'-deoxyriboside (d(2H)T) substitutions do not, suggesting the importance of Watson-Crick interactions at this template residue. Template sites containing 7-deaza-2'-deoxyguanosine (dc(7)G) or 2'-deoxyinosine (dI) in place of dG support oligoribonucleotide synthesis. The reduced extent to which dc(7)G is successful within the template suggests a primase-DNA interaction. Inhibition studies suggest that the primase enzyme binds "null" substrates but cannot initiate RNA synthesis.

Anomalous Migration of Short Sequences of DNA: Comparison of Theory and Experiment.

Abstract A new model to replace the Ogstron and tube reptation models for gel retardation of DNA is proposed that explicitly takes into account screening of the hydrodynamic interactions and polyelectrolyte effects. At short DNA sequence lengths, significant anomalous migration is predicted whose onset is dependent on the size of polyacrylamide gel pores. Thus, a 2-residue fragment has the same electrophoretic mobility as a 12-residue fragment for a polyacrylamide gel with a mesh size of 60Å. The oligonucleotide length at which anomalous migration is observed also depends on pore size. Experimental measurement of gel mobility for DNA fragments of the form N(pN)(n), where n = 1-11, 14 and 19 substantiate this phenomenon.

Synthesis and triplex forming properties of pyrimidine derivative containing extended functionality.

Two pyrimidine nucleosides have been synthesized containing extended hydrogen bonding functionality. In one case the side chain is based upon semicarbazide and in the second monoacetylated carbohydrazide was employed. DNA sequences could be prepared using both analogue nucleosides in a reverse coupling protocol, and provided that the normal capping step was eliminated and that the iodine-based oxidizing solution was replaced with one based upon 10-camphorsulfonyl oxaziridine. Both derivatives exhibited moderate effects in targeting selectively C-G base pairs embedded within a polypurine target sequence.

Critical nature of a specific uridine O2-carbonyl for cleavage by the hammerhead ribozyme.

Three modified hammerhead ribozyme/substrate complexes have been prepared in which individual uridine O2-carbonyls have been eliminated. The modified complexes were chemically synthesized with the substitution of a single 2-pyridone (2P) base analogue for residues U4, U7, and U16.1. Steady-state kinetic analyses indicate that the cleavage efficiencies for the U7 and U16.1 complexes were not significantly reduced relative to the native complex as measured by kcat/KM. The cleavage efficiency for the 2P4 complex, with the analogue present within the uridine loop, was reduced by greater than 2 orders of magnitude. This significant reduction in catalytic efficiency was due primarily to a decrease in kcat. The pH vs cleavage rate profile suggests that the O2-carbonyl of the U4 residue of the hammerhead complex is critical for transition state stabilization and efficient cleavage activity. The results of a Mg2+ rescue assay do not implicate the O2-carbonyl of U4 in an interaction with a divalent metal ion. In addition, the results of a ribozyme folding assay suggest that the presence of the 2P4 within the uridine loop does not alter the folding pathway (relative to the native sequence) both in the absence and in the presence of Mg2+. The O2-carbonyl of U4 appears oriented toward the interior of the catalytic pocket where it may be involved in a critical hydrogen bonding interaction necessary for transition state stabilization.

A 2,2"-bipyridine ligand for incorporation into oligodeoxynucleotides: synthesis, stability and fluorescence properties of ruthenium-DNA complexes.

A non-nucleoside linker based upon the ligand 2,2'-bipyridine and ethylene glycol is prepared and placed into the backbone of a number of oligonucleo-tides. The bipyridine ligand is reacted with cis -dichloro bis(2,2'-bipyridyl) Ru(II) to generate the relatively substitutionally inert complex based upon the well-characterized tris -2,2'-bipyridyl Ru(II). The ruthenium-containing DNA complexes exhibited UV and fluorescence characteristics that are consistent with those previously observed for simple tris -2,2'-bipyridyl Ru(II) complexes. Oligonucleotides containing the ruthenium complex will form both DNA duplexes and triplexes with stabilities that are slightly better than those formed from simple tethered oligonucleotide probes in which the two hybridizing sequences are tethered by simple tri(ethylene glycol) or hexa(ethylene glycol) linkers.

Synthesis and triplex forming properties of an acyclic N7-glycosylated guanine nucleoside.

A chiral acyclic nucleoside, one in which the ribose carbohydrate has been replaced with a glycerol-based linker, is prepared by glycosylating guanine at the N7-nitrogen. The stereochemically pure derivative is converted to a DMT-protected phosphoramidite for incorporation into DNA sequences. Sequence containing the acyclic N7-dG nucleoside are capable of forming DNA triplexes in which it is likely that the N1-H and N2-amino groups of the N7-dG are involved in recognition of the guanine base in G-C base pairs.

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.

Functional group substitutions of the branchpoint adenosine in a nuclear pre-mRNA and a group II intron.

Splicing of nuclear mRNA precursors (pre-mRNAs) takes place in the spliceosome, a large and complex ribonucleoprotein. Nuclear pre-mRNA splicing and group II intron self-splicing occur by a chemically identical pathway involving recognition of a specific branchpoint adenosine and nucleophilic activation of its 2'-hydroxyl group. The chemical similarity between these two splicing reactions, as well as other considerations, have suggested that the catalytic core of the spliceosome and group II introns may be related. Here we test this hypothesis by analyzing splicing and RNA branch formation of a pre-mRNA and a group II intron in which the branchpoint adenosine was substituted with purine base analogues. We find that replacement of the branchpoint adenosine with either of two modified adenosine analogues or guanosine leads to remarkably similar patterns of splicing and RNA branch formation in the two systems.

Application of a 5'-bridging phosphorothioate to probe divalent metal and hammerhead ribozyme mediated RNA cleavage.

This paper describes the preparation and application of a chimeric DNA/RNA oligonucleotide that contains a single 5'-bridging phosphorothioate linkage adjacent to a ribonucleotide and embedded in an otherwise all-DNA sequence. The influence of pH, divalent metal cation, hybridization, and secondary structure on the susceptibility of the thio linkage towards transesterification is investigated in an effort to better understand the metal-phosphorothioate interactions and the basis for catalysis. In addition to the chemical cleavage, we have examined the hammerhead ribozyme mediated cleavage of the 5'-bridging phosphorothioate linkage specifically to test the hypothesis that the ribozyme employs a second metal cofactor, which functions as a Lewis acid, to catalyze transesterification. The results of our kinetics experiments do not support this double-metal model.

Probing contacts to the DNA backbone in the trp repressor-operator sequence-specific protein-nucleic acid complex using diastereomeric methylphosphonate analogues.

Fourteen analogue DNA sequences containing the trp operator sequence and a single diastereomeric methylphosphonate linkage are each prepared from the stereochemically pure nucleoside methylphosphonate dimer building block, prepared as a phosphoramidite. The analogue sequences are shown to be single diastereomers on the basis of HPLC analysis of the digestion mixture; in each case, only a single diastereomeric dimer is present. These analogue sequences can be used effectively to probe for interactions to either of the prochiral phosphate oxygens as illustrated by their use to identify critical interactions in the trp repressor-operator complex. In a number of cases, the pairs of diastereomeric analogue sequences exhibit variable binding affinities that can be used to identify one of the prochiral phosphate oxygens as a critical site for complex-stabilizing interactions. Upon the basis of dissociation constants, apparent incremental binding energies are assigned to specific interactions. In all but one example, these identified sites for interactions to the phosphate backbone can be correlated with contacts implicated by the crystal structure analysis of the trp repressor-operator complex.

Oligonucleotides tethering Hoechst 33258 derivatives: effect of the conjugation site on duplex stabilization and fluorescence properties.

A series of DNA conjugates have been prepared in which two different derivatives of Hoechst 33258 have been tethered to a sequence containing a 5'-GAATTC-3' target site. The two derivatives differ only in the length of the tether between the DNA and the Hoechst fluorophore. By using a DNA backbone labeling protocol, one in which the Hoechst dye is tethered to an internucleotide phosphoramidate residue, it was possible to easily vary the site of attachment with respect to the A-T rich binding site. When tethered outside the GAATTC sequence, little if any helix stabilization results upon hybridization of the conjugate to its complementary sequence. As the site of conjugation is moved to one end of the target sequence and finally within the AATT sequence, more effective helix stabilization results. When tethered between the two A residues, or between the A and T residue, a delta Tm of at least +20 degrees C is observed. Upon hybridization and formation of the B-form DNA, binding by the tethered Hoechst dye results, and the bound dye becomes brightly fluorescent. Upon a simple titration of the single-stranded conjugate with the complementary target sequence the quantum yield enhancement for hybridization only appears to be 5-7-fold at best. These fluorescence effects, generally less dramatic than those observed with other sequences, result from an increase in quantum yield for the single-stranded conjugate relative to the free Hoechst 33258. Heating the single-stranded conjugate reduces the inherent fluorescence of the single-stranded conjugate to a level comparable with that of the free Hoechst dye. In experiments monitoring absorbance vs temperature, a cooperative transition is observed for the single-stranded conjugate. Both the high quantum yield observed for the single-stranded conjugate and the observed thermally induced transition suggest that the single-stranded conjugate can dimerize (at the GAATTC site), mediated by the groove-binding fluorophore.