Solution structure of a highly stable DNA duplex conjugated to a minor groove binder.

The tripeptide 1,2-dihydro-(3 H )-pyrrolo[3,2- e ]indole-7-carboxylate (CDPI3) binds to the minor groove of DNA with high affinity. When this minor groove binder is conjugated to the 5'-end of short oligonucleotides the conjugates form unusually stable hybrids with complementary DNA and thus may have useful diagnostic and/or therapeutic applications. In order to gain an understanding of the structural interactions between the CDPI3minor groove binding moiety and the DNA, we have determined and compared the solution structure of a duplex consisting of oligodeoxyribonucleotide 5'-TGATTATCTG-3' conjugated at the 5'-end to CDPI3 and its complementary strand to an unmodified control duplex of the same sequence using nuclear magnetic resonance techniques. Thermal denaturation studies indicated that the hybrid of this conjugate with its complementary strand had a melting temperature that was 30 degrees C higher compared with the unmodified control duplex. Following restrained molecular dynamics and relaxation matrix refinement, the solution structure of the CDPI3-conjugated DNA duplex demonstrated that the overall shape of the duplex was that of a straight B-type helix and that the CDPI3moiety was bound snugly in the minor groove, where it was stabilized by extensive van der Waal's interactions.     

Oligodeoxyribonucleotide length and sequence effects on intermolecular purine-purine-pyrimidine triple-helix formation.

The binding of guanosine/thymidine-rich oligodeoxyribonucleotides containing various deletions, extensions, and point mutations to polypurine DNA targets was investigated by DNase I footprinting. Intermolecular purine-purine-pyrimidine triple-helical DNA formation was best achieved using oligonucleotides 12 nucleotides in length. Longer oligonucleotides were slightly weaker in binding affinity, whereas shorter oligonucleotides were considerably weaker. Oligonucleotide extensions had a slight effect on triplex formation, while single point mutations located near the oligonucleotide ends had a greater effect. In the cases of extensions and point mutations, changes to the 3' end of the oligonucleotide had a consistently greater effect on triplex formation than changes to the 5' end. Such differences in triplex-forming ability were not caused by an intrinsic property of these oligonucleotides, since the same point mutated oligonucleotides could bind with high affinity to duplex DNAs containing complementary sites. Taken together, our data suggest that there may be an asymmetry involved in the process of purine-motif triplex formation, with interactions between the 3' end of the oligonucleotide and complementary sequences on the target duplex DNA being dominant.     

Strand invasion by DNA-peptide conjugates and peptide nucleic acids.

Peptide nucleic acids (PNAs) and conjugates between oligonucleotides and cationic peptides possess superior potential for strand invasion at complementary sequences. We discovered that oligonucleotide-peptide conjugates and PNAs fall into three classes based on their hybridization efficiency; i) those complementary to inverted repeats within AT-rich region hybridize with highest efficiency; ii) those complementary to areas adjacent to inverted repeats or near AT-rich regions hybridize with moderate efficiency; and iii) those complementary to other regions do not detectably hybridize. The correlations between oligomer chemistry, DNA target sequence, and hybridization efficiency that we report here have important implications for the recognition of duplex DNA.     

CD and melting curves structural studies of the tandem DNA complex formed with oligonucleotides carrying photoactive and sensitizing groups in the nick region.

Photoactive derivatives of oligonucleotides are widely used as affinity reagents for the study of structures and functions of nucleic acids and proteins. Between them the binary reagents are the more attractive in the last time. They represent the tandem of two oligonucleotide derivatives complementary to a target sequence and carrying photoactive and sensitizing groups. The efficiency of target modification in this case depends on the mutual arrangement in the nick region of photoactive and sensitizing groups, attached to the oligonucleotides. The use of binary reagents in affinity modification permits to reach the high selectivity of the process. In this work we report our studies on the thermodynamic and structural peculiarities of complementary tandem complex between DNA target and binary oligonucleotide reagent. The complex consisted of the target d(TTGAAGGGGACCGC)and two 7-mer oligonucleotide conjugates,one of which was modified on its 3'-phosphate with a photoreactive p-azidote-trafluorobenzaldehydehydrazone-group,and the other one was linked through its 5'-phosphate to a sensitizing perylene-group. Optical melting curves and thermal changes in circular dichroism (CD)spectra were detected for all possible oligonucleotide and/or conjugate combinations.In addition,molecular modeling simulation of the complex structure was carried out.It was found that CD spectra did not show serious changes in the B-helix structure of the duplex.The interaction between perylene-and azido-groups at the oligonucleotide junction led to considerable increase in duplex stability. CD and molecular modeling data clearly indicated that perylene-group interacted with the duplex in an intercalative manner,but azido-group located on the side of DNA chain minor groove.     

5′-BIS-PYRENYLATED OLIGONUCLEOTIDES DISPLAY ENHANCED EXCIMER FLUORESCENCE UPON HYBRIDIZATION WITH DNA AND RNA

A simple one-step procedure was applied for synthesis of oligonucleotide conjugates bearing two pyrene residues at the 5′-phosphate of oligonucleotide. Excimer fluorescence intensity of the conjugates is highly sensitive to duplex formation: binding of the bis-pyrenylated oligonucleotides to their DNA and RNA targets leads 10-fold increase of fluorescence. The data show that excimer fluorescence intensity of the conjugates depends linearly on the concentration of target DNA and permits quantification of DNA in solution.     

LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA

Locked nucleic acid (LNA) is a nucleic acid analogue containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA mimicking sugar conformation. LNA oligonucleotides display unprecedented hybridization affinity toward complementary single-stranded RNA and complementary single- or double-stranded DNA. Structural studies have shown that LNA oligonucleotides induce A-type (RNA-like) duplex conformations. The wide applicability of LNA oligonucleotides for gene silencing and their use for research and diagnostic purposes are documented in a number of recent reports, some of which are described herein.     

Synthesis and nuclease stability of trilysyl dendrimer-oligodeoxyribonucleotide hybrids.

Hybrids of oligonucleotides and trilysyl-dendrimers with terminal acyl groups were prepared via solid-phase synthesis, including a DNA hexamer bearing an additional 3'-appendage. These were shown to be degraded more slowly by nuclease S1 than control strands, particularly at low pH, and, in one case, to form a duplex with a complementary strand whose melting point at pH 7 was higher than that of the control duplex.     

Specific oligonucleotide invasion into the end of DNA duplex

Phenomenon of the interaction of a double-stranded DNA fragment with an oligonucleotide complementary to the end of the duplex strand was demonstrated to occur via formation of three-stranded DNA structure with an oligonucleotide invasion. It was shown that oligonucleotides complementary to the duplex ends inhibit Holliday junction formation in solutions of homologous linear DNA fragments. This effect depends on the oligonucleotide concentration, sequence and their complementarity to the duplex ends. Formation of three-stranded complexes was demonstrated using radiolabeled oligonucleotides by agarose gel-electrophoresis followed by autoradiography. Analysis of three-stranded DNA structures by chemical cleavage of non-canonical base pairs revealed that oligonucleotide invades into duplex ends via a sequential displacement mechanism and that the level of the invasion may vary considerably.     

Site-specific cleavage of RNA and DNA by complementary DNA--bleomycin A5 conjugates

Bleomycin displays clinical chemotherapeutic activity, but is so nonspecifically toxic that it is rarely administered. It was therefore of interest to determine whether bleomycin could be directed to cleave RNA or DNA at a specific site by conjugation to a complementary oligonucleotide. A 15 nt MYC complementary oligodeoxynucleotide (HMYC55) bearing a 5' bleomycin A5 (Blm) residue was designed to base-pair with nt 7047-7061 of human MYC mRNA. Reactivity of the Blm-HMYC55 conjugate (and mismatch controls) with a MYC mRNA 30-mer, a MYC DNA 30-mer, and a MYC 2'-O-methyl RNA 30-mer, nt 7041-7070, was analyzed in 100 microM FeNH(4)SO(4), 50 mM beta-mercaptoethanol, 200 mM LiCl, 10 mM Tris-HCl, pH 7.5, at 37 degrees C. Cleavage of the substrate RNA or DNA occurred primarily at the junction of the complementary DNA-target RNA duplex, 18-22 nt from the 5' end of the RNA. Reaction products with lower mobility than the target RNA or DNA also formed. Little or no reaction was observed with more than three mismatches in a Blm-oligodeoxynucleotide conjugate. Neither the short RNA or DNA cleavage fragments nor the low mobility products were observed in the absence of Fe(II), or the presence of excess EDTA. The target RNA was also cleaved efficiently by bleomycin within a hybrid duplex with a preformed single-nucleotide bulge in the RNA strand. New Blm-oligodeoxynucleotide conjugates containing long hexaethylene glycol phosphate based linkers between oligodeoxynucleotide and bleomycin were designed to target this bulge region. These conjugates achieved 8-18% cleavage of the target RNA, depending on the length of the linker. Blm-oligodeoxynucleotide conjugates thus demonstrated sequence specificity and site specificity against RNA and DNA targets.     

Specific oligonucleotide invasion into an end of a DNA duplex

A new phenomenon was described: a double-stranded DNA fragment interacted with a single-stranded oligonucleotide complementary to the terminal region of one strand of the duplex to yield a complex with oligonucleotide invasion. Generation of Holliday junctions by homologous linear DNA fragments was less efficient in the presence of single-stranded oligonucleotides complementary to duplex ends. The effect depended on the oligonucleotide concentration, size, and complementarity to a duplex strand. Sequence-specific complexes with single strand invasion were detected in mixtures containing radiolabeled oligonucleotides and duplexes. A single-stranded oligonucleotide invaded a duplex even when its concentration was far lower than the duplex concentration. Complexes with single strand invasion were analyzed by chemical cleavage of noncanonical base pairs. Analysis showed that an oligonucleotide interacts with the complementary region of one strand of the duplex, gradually displacing the other strand. The extent of oligonucleotide invasion into the duplex considerably varied. Oligonucleotide invasion into duplexes became more efficient with increasing oligonucleotide size.     

Cleavage of double-stranded DNA by manganese tris(methylpyridiniumyl)porphyrin linked to 3′-spermine oligonucleotides

 Cleavage of double-stranded DNA was performed with cationic manganese porphyrin complexes linked via a spermine tether to the 3′- or 5′-side of triple-helix-forming oligonucleotides (cleaver-TFO conjugates). The targeted sequence was a 15-polypurine sequence present in the env gene of HIV-1 (positions 7301–7315). The presently used TFOs contain only thymine and 5-methylcytosine residues and one adenine at the 3′-end in order to be able to easily introduce a 3′-polyamine linker by reductive amination of the corresponding 3′-apurinic polypyrimidine oligonucleotides. With this method we prepared these TFO-cleaver conjugates in 45% yield with only two equivalents of the Mn-TrisMPyP-COOH precursor. These new metalloporphyrin-TFO conjugates were able to cleave a complementary 45-mer duplex at 10 nM concentration with only ten equivalents of TFO-cleaver. Conjugates without spermine, without 5-methylcytosine, with a random sequence or with the managanese porphyrin-spermine entity on the 5′-end of TFOs were synthesized for comparative studies. Received: 6 December 1995 / Accepted: 5 February 1996     

Carboranyl oligonucleotides: 4. synthesis and physicochemical studies of oligonucleotides containing 2'-O-(o-carboran-1-yl)methyl group

Boronated oligonucleotides are potential candidates for antisense oligonucleotide technology (AOT), boron neutron capture therapy (BNCT), and as tools in molecular biology. A method was developed for the solid phase synthesis of oligonucleotides containing 2'-O-(o-carboran-1-yl-methyl) (2'-CBM) group. Synthesis was performed using a standard beta-cyanoethyl cycle and automated DNA synthesizer. Manual steps were performed for the insertion of a modified monomer bearing the 2'-CBM group. Several tetradecanucleotides complementary to DNA-HCMV, and bearing 2'-CBM modification near the 3'-end or 5'-end or in the middle of the oligonucleotide chain were synthesized. The resulting oligomers were characterized by polyacrylamide gel electrophoresis (PAGE), reverse phase high-performance liquid chromatography (RP-HPLC), matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and ultraviolet spectroscopy (UV), circular dichroism (CD), and melting temperature (Tm) measurements. Tm of duplexes formed between 2'-CBM-modified tetradecanucleotides and complementary DNA and RNA template were compared with those formed by the unmodified oligonucleotide and complementary sequence. The stability of 2'-CBM oligonucleotides in the presence of phosphodiesterase I from Crotalus atrox venom and in human serum was studied. Oligonucleotides bearing the 2'-CBM group are characterized by increased resistance to enzymatic digestion, increased lipophilicity, and the ability to form stable duplexes with complementary templates.     

Head-to-head bis-hairpin polyamide minor groove binders and their conjugates with triplex-forming oligonucleotides: studies of interaction with target double-stranded DNA

Two hairpin hexa(N-methylpyrrole)carboxamide DNA minor groove binders (MGB) were linked together via their N-termini in head-to-head orientation. Complex formation between these bis-MGB conjugates and target DNA has been studied using DNase I footprinting, circular dichroism, thermal dissociation, and molecular modeling. DNase I footprint revealed binding of these conjugates to all the sites of 492 b.p. DNA fragment containing (A/T)(n)X(m)(A/T)(p) sequences, where n>3, p>3; m=1,2; X = A,T,G, or C. Binding affinity depended on the sequence context of the target. CD experiments and molecular modeling showed that oligo(N-methylpyrrole)carboxamide moieties in the complex form two short antiparallel hairpins rather than a long parallel head-to-head hairpin. Binding of bis-MGB also stabilized a target duplex thermodynamically. Sequence specificity of bis-MGB/DNA binding was validated using bis-conjugates of sequence-specific hairpin (N-methylpyrrole)/(N-methylimidazole) carboxamides. In order to increase the size of recognition sequence, the conjugates of bis-MGB with triplex-forming oligonucleotides (TFO) were synthesized and compared to TFO conjugated with single MGB hairpin unit. Bis-MGB-oligonucleotide conjugates also bind to two blocks of three and more A.T/T.A pairs similarly to bis-MGB alone, independently of the oligonucleotide moiety, but with lower affinity. However, the role of TFO in DNA recognition was demonstrated for mono-MGB-TFO conjugate where the binding was detected mainly in the area of the target sequence consisting of both MGB and TFO recognition sites. Basing on the molecular modeling, three-dimensional models of both target DNA/bis-MGB and target DNA/TFO-bis-MGB complexes were built, where bis-MGB forms two antiparallel hairpins. According to the second model, one MGB hairpin is in the minor groove of 5'-adjacent A/T sequence next to the triplex-forming region, whereas the other one occupies the minor groove of the TFO binding polypurine tract. All these data together give a key information for the construction of MGB-MGB and MGB-oligonucleotide conjugates possessing high specificity and affinity for the target double-stranded DNA.     

Synthesis of metallointercalator-DNA conjugates on a solid support

Metallointercalator-DNA conjugates were prepared by amide bond formation between active esters on the nonintercalating ligands of transition metal complexes and primary amines presented at the 5' or the 3' termini of oligonucleotides attached to solid supports. The conjugates were liberated from the support by aminolysis and purified by HPLC on C18 or C4 stationary phases, which separates the two diastereomeric forms of the conjugates containing either the Lambda or the Delta enantiomer of the octahedral metal complex. The coupling reaction proceeds with approximately 75% conversion of the amino-terminated oligonucleotide into the conjugate; the isolated yield is approximately 200 nmol for syntheses initiated on DNA-synthesis columns with a loading of 2 micromol. The conjugates were characterized by ultraviolet-visible and circular dichorism absorption spectroscopy, electrospray ionization mass spectrometry, enzymatic digestion, and polyacrylamide gel electrophoresis (PAGE). Oligonucleotides bearing [Rh(phi)(2)(bpy')](3+) (phi = 9, 10-phenanthrene quinone diimine; bpy' = 4-butyric acid-4'-methyl bipyridyl) form 1:1 duplexes with the complementary strand, and the electrophoretic mobility under nondenaturating PAGE of duplexes containing Delta-Rh is notably different from duplexes containing Lambda-Rh. High-resolution PAGE of DNA photocleavage reactions initiated by irradiation of the tethered Rh complexes reveal intercalation of the complex only near the tethered end of the duplex. Analogous DNA-binding properties were observed with [Rh(phi)(2)(bpy')](3+) tethered to the 3' terminus. By combining the 3' and 5' modification strategies, a mixed-metal DNA conjugate containing both [Os(phen)(bpy')(Me(2)-dppz)](2+) (Me(2)-dppz = 7, 8-dimethyldipyridophenazine) on the 3' terminus and [Rh(phi)(2)(bpy')](3+) on the 5' terminus was prepared and isolated. Taken together, these strategies for preparing metallointercalator-DNA conjugates offer a useful approach to generate chemical assemblies to probe long-range DNA-mediated charge transfer where the redox initiator is confined to and intercalated in a well-defined binding site.     

Thermoprecipitation of streptavidin via oligonucleotide-mediated self-assembly with poly(N-isopropylacrylamide).

A versatile strategy has been developed for selectively and sequentially isolating targets in a liquid-phase affinity separation environment. The strategy uses a recently developed approach for joining together molecules in linkages that are defined by the complementary pairing of oligonucleotides conjugated to the different molecules [Niemeyer, C. M., Sano, T., Smith, C. L., and Cantor, C. R. (1994) Nucleic Acids Res. 22, 5530-9]. In the work presented here, streptavidin was noncovalently coupled with the temperature-responsive poly(N-isopropylacrylamide) [poly(NIPAAM)] through the sequence-specific hybridization of oligonucleotides conjugated to the protein and polymer. A 20-mer oligonucleotide was covalently linked through a heterobifunctional linker to a genetically engineered streptavidin variant that contained a unique cysteine residue at the solvent-accessible site Glu 116. The complementary DNA sequence was conjugated to the end of a linear ester-activated poly(NIPAAM). The two conjugates were allowed to self-assemble in solution via hybridization of their complementary DNA sequences. The streptavidin-poly(NIPAAM) complex could be used to affinity-precipitate radiolabeled biotin or biotinylated alkaline phosphatase above 32 degrees C through the thermally induced phase separation activity of the poly(NIPAAM). The streptavidin-oligo species could then be reversibly separated from the precipitated polymer-oligo conjugate and recycled by lowering the salt concentration, which results in denaturation of the short double-stranded DNA connection. The use of oligonucleotides to couple polymer to streptavidin allows for selective precipitation of different polymers and streptavidin complexes based on the sequence-specific hybridization of their oligonucleotide appendages.     

Chemical functionalization of oligodeoxynucleotides with multiple boronic acids for the polyvalent binding of saccharides

A novel saccharide host containing four boronic acid recognition units on a single DNA duplex terminus was constructed. This construct allowed boronic acid sugar recognition in the context of double-stranded DNA to be established while highlighting the benefits of multivalency. Following the solid-phase synthesis of a bis-boronic acid tag, two end-functionalized oligonucleotides with complementary sequences were functionalized through amide ligation. By annealing the boronic acid-DNA conjugates, a tetra-boronic acid DNA duplex was assembled. The saccharide binding ability of this tetra-boronic acid host was revealed through cellulose paper chromatography in the absence and presence of various saccharides. While no appreciable saccharide binding was seen in the case of a bis-boronic DNA conjugate, the increased migration of the tetra-boronic acid host relative to the control sequences in the presence of selected monosaccharides underscored the importance of multivalent effects. We thus identified a requirement for multiple recognition sites in these conjugate systems and expect the results to facilitate future efforts toward applying synthetic recognition systems to the realm of macromolecules.     

Synthesis of oligonucleotide derivatives using ChemMatrix supports

The synthesis of oligonucleotides on poly(ethylene glycol)-based (ChemMatrix) supports was studied. Results show that oligonucleotides can be indeed prepared in good yields using slightly modified synthesis cycles and automated DNA synthesizers. The use of these supports for the synthesis of oligonucleotide-peptide conjugates and for the ligation of oligonucleotides using Cu(+)-catalyzed cycloadition reactions is reported. Moreover, these supports can be used for the preparation of oligonucleotides in anhydrous solvents, followed by hybridization of the complementary sequences in aqueous buffers.     

Intercalating nucleic acids containing insertions of 1-O-(1-pyrenylmethyl)glycerol: stabilisation of dsDNA and discrimination of DNA over RNA

We have studied hybridisation affinities and fluorescence behaviour of intercalator-modified oligonucleotides. The phosphoramidite of (S)-1-O-(4, 4′-dimethoxytriphenylmethyl)-3-O-(1-pyrenylmethyl)glycerol, an intercalating pseudo-nucleotide (IPN), was synthesised and by standard methods inserted into 7mer and 13mer oligodeoxyribonucleotides (ODNs) to generate intercalating nucleic acids (INAs). INAs showed greatly increased affinity for complementary single-stranded DNA (ssDNA), as determined by a thermal stabilisation of the formed DNA/INA duplex of up to 10.9°C per modification when the IPN was added as a dangling end and up to 6.7°C per modification when the IPN was inserted as a bulge. There was a positive stabilisation effect of the formed DNA/INA duplex on introducing a second IPN in the INA strand, when the two IPNs were separated by at least 1 bp. The effect is more pronounced the larger the separation of the two IPNs. Contrary to the enhanced affinity for ssDNA, the IPNs lower the affinity for complementary single-stranded RNA (ssRNA), giving rise to a difference in melting temperature of up to 25.8°C for two IPN insertions in an RNA/INA duplex when compared with the corresponding DNA/INA duplex. In this way INA is able to discriminate ssDNA over ssRNA with identical sequences. Fluorescence measurements show a stronger interaction of the pyrene moiety with DNA than with RNA, indicating intercalation as the stabilising factor in DNA/INA duplexes.