STUDIES IN OLIGONUCLEOTIDE-BASED ARTIFICIAL NUCLEASE SYSTEMS. INTRAMOLECULAR COPPER (II) COMPLEX FORMATION IN AN OLIGONUCLEOTIDE BIS-PHENANTHROLINE CONJUGATE

We have recently developed oligonucleotide based artificial nuclease (OBAN) systems based on 2′-O-methyloligoribonucleotides carrying a 2,9-dimethylphenanthroline · Zn(II) complex. These hybridize to an RNA molecule with bulge formation in the central region of the target and cleave the RNA target in a catalytic manner. When studying an 11-mer 2′-O-methyloligoribonucleotide carrying two 2,9-dimethylphenanthroline moieties, located 5 base pairs apart from each other, we found that this forms a cyclic structure in the presence of Cu²⁺ ions. This is due to intramolecular Cu(2,9-dimethylphenanthroline)₂ complex formation, i.e., with the two ligands conjugated to the same oligonucleotide.     

Efficient new ribozyme mimics: direct mapping of molecular design principles from small molecules to macromolecular, biomimetic catalysts

Dramatic improvements in ribozyme mimics have been achieved by employing the principles of small molecule catalysis to the design of macromolecular, biomimetic reagents. Ribozyme mimics derived from the ligand 2,9-dimethylphenanthroline (neocuproine) show at least 30-fold improvements in efficiency at sequence-specific RNA cleavage when compared with analogous o-phenanthroline- and terpyridine-derived reagents. The suppression of hydroxide-bridged dimers and the greater activation of coordinated water by Cu(II) neocuproine (compared with the o-phananthroline and terpyridine complexes) better allow Cu(II) to reach its catalytic potential as a biomimetic RNA cleavage agent. This work demonstrates the direct mapping of molecular design principles from small-molecule cleavage to macromolecular cleavage events, generating enhanced biomimetic, sequence-specific RNA cleavage agents.     

A method for synthesis of an artificial ribonuclease

5-Amino-2,9-dimethyl-1,10-phenanthroline-oligonucleotide conjugates have been synthesized. A 2'-O-methyl octaribonucleotide carrying a 2'-aminoethoxymethyl linker in a central position was produced. Reaction of the aminoneocuproine phenyl carbamate with the fully deprotected oligonucleotide in aqueous solution gave virtually quantitative conversion into the conjugate. Preliminary cleavage studies in presence of zinc ions show nuclease activity towards RNA targets.     

A METHOD FOR SYNTHESIS OF AN ARTIFICIAL RIBONUCLEASE

5-Amino-2,9-dimethyl-1,10-phenanthroline-oligonucleotide conjugates have been synthesized. A 2′-O-methyl octaribonucleotide carrying a 2′-aminoethoxymethyl linker in a central position was produced. Reaction of the aminoneocuproine phenyl carbamate with the fully deprotected oligonucleotide in aqueous solution gave virtually quantitative conversion into the conjugate. Preliminary cleavage studies in presence of zinc ions show nuclease activity towards RNA targets.     

Synthesis of 3'-3'-linked oligonucleotides branched by a pentaerythritol linker and the thermal stabilities of the triplexes with single-stranded DNA or RNA

Synthesis of 3'-3'-linked oligonucleotides branched by a pentaerythritol linker is described. The branched oligonucleotides were synthesized on a DNA/RNA synthesizer using a controlled pore glass (CPG) with a pentaerythritol linker carrying 4,4'-dimethoxytrityl (DMTr) and levulinyl (Lev) groups. The stability of the triplexes between the branched oligonucleotides and the target single-stranded DNA or RNA was studied by thermal denaturation. The oligonucleotides with the pentaerythritol linker formed thermally stable triplexes with the single-stranded DNA and RNA. Furthermore, the branched oligonucleotides containing 2'-O-methylribonucleosides, especially the oligonucleotide composed of 2'-deoxyribonucleosides and 2'-O-methylribonucleosides, stabilized the triplexes with the single-stranded DNA or RNA. Thus, the branched oligonucleotide containing 2'-O-methylribonucleosides may be a candidate for a novel antisense molecule by the triplex formation.     

Further evidence supporting an inner sphere mechanism in the NO reduction of the copper(II) complex Cu(dmp)2 (dmp = 2,9-dimethyl-1,10-phenanthroline)

Described are further studies directed towards elucidating the mechanism of the nitric oxide reduction of the copper(II) model system, Cu(dmp)2(2+) (I, dmp=2,9-dimethyl-1,10-phenanthroline). The reaction of I with NO in methanol results in the formation of Cu(dmp)2+ (II) and methyl nitrite (CH3ONO), with a second order rate constant kNO=38.1 M-1 s-1 (298K). The activation parameters for this reaction in buffered aqueous medium were measured to be DeltaH(double dagger)=41.6 kJ/mol and DeltaS(double dagger)=-82.7 kJ/mol deg. The addition of azide ion (N3-) as a competing nucleophile results in a marked acceleration in the rate of the copper(II) reduction. Analysis of the kinetics for the NO reduction of the bulkier Cu(dpp)(2)2+ (IV, dpp=2,9-diphenyl-1,10-phenanthroline) and the stronger oxidant, Cu(NO2-dmp)2(2+) (V, NO2-dmp=5-nitro-2,9-dimethyl-1,10-phenanthroline), gave the second order rate constants kNO=21.2 and 29.3 M-1 s-1, respectively. These results argue against an outer sphere electron transfer pathway and support a mechanism where the first step involves the formation of a copper-nitrosyl (Cu(II)-NO or Cu(I)-NO+) adduct. This would be followed by the nucleophilic attack on the bound NO and the labilization of RONO to form the nitrite products and the cuprous complex.     

Some Properties of Site-Specific Nickase BspD6I and the Possibility of Its Use in Hybridization Analysis of DNA

A new method for hybridization analysis of nucleic acids is proposed on the basis of the ability of site-specific nickases to cleave only one DNA strand. The method is based on the use of a labeled oligonucleotide with the recognition site of the nickase hybridized with the target (DNA or RNA) at an optimal temperature of the enzyme (55°C). The two shorter oligonucleotides formed after the cleavage with the nickase do not complex with the target. Thus, a multiple cleavage of the labeled oligonucleotide takes place on one target molecule. The cleavage of the nucleotide is recorded either by polyacrylamide gel electrophoresis (when a radioactive labeled oligonucleotide is used) or by fluorescence measurements (if the oligonucleotide has the structure of a molecular beacon). The new method was tested on nickase BspD6I and a radioactive oligonucleotide complementary to the polylinker region of the viral DNA strand in bacteriophage M13mp19. Unfortunately, nickase BspD6I does not cleave DNA in the RNA–DNA duplexes and therefore cannot be used for detection of RNA targets.     

Triple helix-forming oligonucleotides conjugated to indolocarbazole poisons direct topoisomerase I-mediated DNA cleavage to a specific site.

Topoisomerase I is an ubiquitous DNA-cleaving enzyme and an important therapeutic target in cancer chemotherapy for camptothecins as well as for indolocarbazole antibiotics such as rebeccamycin. To achieve a sequence-specific cleavage of DNA by topoisomerase I, a triple helix-forming oligonucleotide was covalently linked to indolocarbazole-type topoisomerase I poisons. The three indolocarbazole-oligonucleotide conjugates investigated were able to direct topoisomerase I cleavage at a specific site based upon sequence recognition by triplex formation. The efficacy of topoisomerase I-mediated DNA cleavage depends markedly on the intrinsic potency of the drug. We show that DNA cleavage depends also upon the length of the linker arm between the triplex-forming oligonucleotide and the drug. Based on a known structure of the DNA-topoisomerase I complex, a molecular model of the oligonucleotide conjugates bound to the DNA-topoisomerase I complex was elaborated to facilitate the design of a potent topoisomerase I inhibitor-oligonucleotide conjugate with an optimized linker between the two moieties. The resulting oligonucleotide-indolocarbazole conjugate at 10 nM induced cleavage at the triple helix site 2-fold more efficiently than 5 microM of free indolocarbazole, while the other drug-sensitive sites were not cleaved. The rational design of drug-oligonucleotide conjugates carrying a DNA topoisomerase poison may be exploited to improve the efficacy and selectivity of chemotherapeutic cancer treatments by targeting specific genes and reducing drug toxicity.     

Specific DNA duplex formation at an artificial lipid bilayer: towards a new DNA biosensor technology

A novel technique is described which comprises a base-specific DNA duplex formation at a lipid bilayer-H(2) O-phase boundary layer. Two different probes of oligonucleotides both carrying a double-tailed lipid at the 5'-terminus were incorporated into stable artificial lipid bilayers separating two compartments (cis/trans-channel) of an optically transparent microfluidic sample carrier with perfusion capabilities. Both the cis- and trans-channels are filled with saline buffer. Injection of a cyanine-5-labeled target DNA sequence, which is complementary to only one of the oligonucleotide probes, into the cis-channel, followed by a thorough perfusion, leads to an immobilization of the labeled complementary oligonucleotide on the membrane as detected by single-molecule fluorescence spectroscopy and microscopy. In the case of fluorescent but non-complementary DNA sequences, no immobilized fluorescent oligonucleotide duplex could be detected on the membrane. This clearly verifies a specific duplex formation at the membrane interface.     

Electrochemical properties of copper complexes with unsubstituted and substituted 1,10-o-phenanthrolines in N,N-dimethylformamide solvent

The electrochemical reduction of a series of copper(II) complexes with 1,10-o-phenanthrolines, namely the 1:1 and 1:2 metal:ligand complexes with 2,9-dimethylphenanthroline, 4,7-dimethylphenanthroline and unsubstituted phenanthroline, respectively, has been studied in N,N-dimethylformamide using platinum electrodes. As to the 1:2 complexes, the effect of the presence of substituents with different electronic and steric effects on the phenanthroline ligands has been studied with the aim of rationalizing the different values of the standard potentials which have been measured. Furthermore, the possibility of electrogenerating neutral species, with a formally zerovalent copper centre, exhibiting different stability depending on the nature of the ligands, has been ascertained. In out solvent medium, 1:1 complexes have been found to be in equilibrium with the corresponding 1:2 complexes. A scheme for the reduction of solutions of these compounds, including the different equilibria associated to the electrode charge transfers, has been outlined.     

Closed-tube human leukocyte antigen DQA1∗05 genotyping assay based on switchable lanthanide luminescence probes

Genotyping in closed tube is commonly performed using polymerase chain reaction (PCR) amplification and allele-specific oligonucleotide probes using fluorescence resonance energy transfer (FRET). Here we introduce a homogeneous human leukocyte antigen (HLA)–DQA1∗05 end-point PCR assay based on switchable lanthanide luminescence probe technology and a simple dried blood sample preparation. The switchable probe technology is based on two non-luminescent oligonucleotide probes: one carrying a non-luminescent lanthanide chelate and the other carrying a light-absorbing antenna ligand. Hybridization of the probes in adjacent positions to the target DNA leads to the formation of a highly luminescent lanthanide chelate complex by self-assembly of the reporter molecules. Performance of the HLA–DQA1∗05 assay was evaluated by testing blood samples collected on sample collection cards and was prepared by lysing the punched samples (3-mm discs) using alkaline reaction conditions and high temperature. Testing of 147 blood samples yielded 100% correlation to the heterogeneous DELFIA technology-based reference assay. Genotyping requires carefully designed probe sequences able to discriminate matched and mismatched target sequences by hybridization. Furthermore, definite genotype discrimination was achieved because inherently non-luminescent switchable probes together with time-resolved measurement mode led to very low background signal level and, therefore, very high signal differences averaging 54-fold between DQA1∗05 and other alleles.     

Study of the structural-energy aspects of complementary-addressed modifications of nucleic acid with reactive 4-]N-methyl-N-(2-chloroethyl)-amino]benzyl-3'- or 5'-phosphamide oligonucleotide derivatives by a molecular mechanics method

Calculations of probabilities of the complementary addressed modification of target NA by 3'- or 5'-reactive derivatives of oligonucleotides carrying a 4-[N-(2-chloroethyl)-N-methyl]aminobenzyl group attached to the 3'- or 5'-terminal phosphates through a phosphoroamide linkage have been made. It is shown that the structural basis of the high efficiency and positional specificity depending on the NA target base sequence is the extent of structural correspondence of the energetically optimal conformation of the active group in the complex to the mutual arrangement of the active group and nucleophilic site needed for the chemical reaction. The 3'-derivative has the highest dependence of efficiency and positional specificity of the alkylation on the target NA base sequence. The maximal positional specificity of the alkylation is found for the modification of the cytidine at the first position from the terminal complementary base pair at the 5'-end of the target NA. For the 5'-derivative, the alkylating ability was determined to depend on the insertion of additional methylene bridges into the standard phosphoroamide linker: two methylene groups provide for the maximal increase of the modification ability of the nucleophilic site of the target NA in the double-stranded part of the complex. The efficiency of alkylation of the target NA in a three component complex with oligonucleotide-effector also complementary to the target NA have been studied. It was found that formation of the three-component complex lead to an additional stabilization of the conformation needed for the reaction of the active group, in comparison with two-component complex, by means of the intercalation of the phenyl group of the reagent in the gap between the oligonucleotide derivative and the oligonucleotide effector.     

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.     

Cleavage of RNA in hybrid duplexes by ribonuclease H ofE. coli: I. Substrate properties of complexes formed by RNA and a tandem of short oligodeoxyribonucleotides

We studied theE. coli RNase H cleavage of a 5′-labeled RNA fragment within two hybrid duplexes with identical sequences, one of which is formed by RNA and a 20-mer oligodeoxyribonucleotide (RNA/p20) whereas the second, by RNA and a tandem of short oligodeoxyribonucleotides (octanucleotide : tetranucleotide : octanucleotide) (RNA/tandem). It was shown that RNA in the RNA/p20 complex is hydrolyzed from the 3′-end to yield consecutively the 17-, 14-, 11-, 8-, and 5-mer 5′-labeled fragments. On hydrolysis of RNA in complex RNA/tandem, the same products were registered but their accumulation rates in this case differed. Thus, the initial rates of accumulation of the 17- and 8-mer were close. Moreover, the accumulation of the final 5-mer differed considerably: in the RNA/tandem complex it appeared within first minutes of the reaction but only after a considerable lag period in complex RNA/p20. These data testify that the tandem is involved not only in the consecutive accumulation of the shortened products (which is characteristic of complexes including extended oligonucleotides) but also in the parallel accumulation. This results from hydrolysis of each duplex segment formed by RNA and the short oligonucleotide of the tandem. Although the order of recognition and cleavage of RNA target by ribonuclease H at certain bonds depends on the type of the hybrid duplex, the destruction of RNA target within complex RNA/tandem and in complex with the full-size oligonucleotide occurs with a close effectiveness.     

Carbohydrate Modifications in Antisense Oligonucleotide Therapy: New Kids on the Block

Abstract

Chemical modifications to improve the efficacy of an antisense oligonucleotide are designed to increase the binding affinity to target RNA, to enhance the nuclease resistance, and to improve cellular delivery. Among the different sites available for chemical modification in a nucleoside building block, the 2′-position of the carbohydrate moiety¹ has proven to be the most valuable for various reasons: (1) 2′-modification can confer an RNA-like 3′-endo conformation to the antisense oligonucleotide. Such a preorganization for an RNA like conformation^2,3,4,5 greatly improves the binding affinity to the target RNA; (2) 2′-modification provides nuclease resistance to oligonucleotides; (3) 2′-modification provides chemical stability against potential depurination conditions pharmacology evaluations and correlation with pharmacokinetic changes are emerging from these novel chemical modifications. Analytical chemistry of modified oligonucleotides before and after biological administration of antisense oligonucleotides with techniques such as capillary gel electrophoresis (CGE) and mass spectrometry help to determine the purity as well as the in vivo fate of these complex molecules. Large-scale synthesis is becoming a tangible reality for antisense oligonucleotides. Nucleic acid chemists and biologists alike are beginning to understand the structure-biological activity in terms of basic physical-organic parameters such as the gauche effect, the charge effect and conformational constraints. Synthesis of chimeric designer oligonucleotides bringing the attractive features of different modifications to a given antisense oligonucleotide sequence to generate synergistic interactions is forthcoming³⁰. These advances along with the potential availability of complete human genome sequence information promise a bright future for the widespread use of nucleic acid based therapeutics.     

Inhibition of in vitro and ex vivo translation by a transplatin-modified oligo(2'-O-methylribonucleotide) directed against the HIV-1 gag-pol frameshift signal

A 2'-O-methylribooligonucleotide containing a G1.U.G3 triad modified by trans-diamminedichloro-platinum(II) was targeted to the RNA region responsible for the gag-pol frameshifting during translation of the HIV-1 mRNA. The binding of the platinated oligonucleotide to its target RNA induced a rearrangement of the (G1, G3)-intrastrand crosslink, leading to the formation of an intermolecular oligonucleotide-RNA G-A crosslink. This resulted in the selective arrest of translation of a luciferase gene placed downstream of the HIV-1 frameshift signal both in a cell-free extract (rabbit reticulocyte lysate) and in RNA-transfected cells. A specific inhibition of luciferase activity was still observed when the oligonucleotide-RNA complex was not pre-formed prior to either translation or transfection. Moreover, a selective inhibition was also observed when the oligonucleotide and the plasmid DNA encoding the luciferase and bearing the RNA gag- pol frameshifting signal were co-transfected in NIH 3T3 cultured cells. Therefore the intra-strand-->interstrand conversion of the platinum crosslink kinetically competes with the translation machinery and blocks the polypeptide elongation. These transplatin-modified oligonucleotides which operate within a live cell on a 'real-time' basis and do not need an external triggering signal constitute a promising new class of selective reactive probes.     

Cleavage of RNA in hybrid duplexes by ribonuclease H from E. coli. I. Substrate properties of complexes formed by RNA and tandem of short oligodeoxyribonucleotides

We studied the E. coli RNase H cleavage of a 5'-labeled RNA fragment within two hybrid duplexes with identical sequences, one of which is formed by RNA and a 20-mer oligodeoxyribonucleotide (RNA/p20), whereas the second, by RNA and a tandem of short oligodeoxyribonucleotides (octanucleotide: (RNA/tandem). It was shown that RNA in the RNA/p20 complex is hydrolyzed from the 3'-end to yield consecutively the 17-, 14-, 11-, 8-, and 5-mer 5'-labeled fragments. On hydrolysis of RNA in complex RNA/tandem, the same products were registered, but their accumulation rates in this case differed. Thus, the initial rates of accumulation of the 17- and 8-mer were close. Moreover, the accumulation of the final 5-mer differed considerably: in the RNA/tandem complex it appeared within first minutes of the reaction, but only after a considerable lag period in complex RNA/p20. These data testify that the tandem is involved not only in the consecutive accumulation of the shortened products (which is characteristic of complexes including extended oligonucleotides) but also in the parallel accumulation. This results from hydrolysis of each duplex segment formed by RNA and the short oligonucleotide of the tandem. Although the order of recognition and cleavage of RNA target by ribonuclease H depends on the type of the hybrid duplex, the destruction of RNA target within complex RNA/tandem and in complex with the full-size oligonucleotide occurs with a close effectiveness.