Interactions of derivatives of short oligonucleotides with nucleic acids. VII. Effect of conformation changes in the duplex structure on on the specificity and efficacy of modification of target DNA by alkylating oligonucleotide derivatives]

The modification of a target DNA by alkylating oligonucleotide derivatives possessing various capacities for complex formation was studied. The binding properties of oligonucleotides were changed either by increasing their length (tetra-, octa-, and dodecamers) or by introducing a point substitution and/or an N-(2-hydroxyethylphenazinium) residue. It was found that conformational changes occurring in the structure of the target.reagent complex upon elevating the reaction temperature affect the efficiency and site-specificity of the alkylation. In the case of complete saturation of the target with the reagent, an increase in the hybridization ability of the reagent reduced the efficiency of the target modification. It was found that the modification by the tetranucleotide reagent (in the presence of an effector adjacent to the 3' end) occurs exclusively at an intracomplex target base. In the case of the dodecamer, which forms a stable, highly cooperative complex with the target, several bases of the target undergo alkylation, and an increase in temperature changes the site-specificity of alkylation. In this process, the redistribution of the target modification sites toward stronger nucleophilic centers enhances alkylation at temperatures near the melting temperature of the target.dodecanucleotide complex despite a decrease in the extent of target association.     

Mini-antisense Oligonucleotides

Abstract

A new strategy of selective DNA target modification was proposed. The using of reactive derivatives of short oligonucleotides in the presence of flanking effector pair allows one to modify DNA target only when the perfect complementary complex of DNA target and oligonucleotide tandem is formed.     

A New Strategy of Discrimination of a Point Mutation by Tandem of Short Oligonucleotides

Abstract

A new strategy based on the use of cooperative tandems of short oligonu-cleotide derivatives (TSOD) has been proposed to discriminate a “right” DNA target from a target containing a single nucleotide discrepancy. Modification of a DNA target by oligodeoxyribonucleotide reagents was used to characterize their interaction in the perfect and mismatched complexes. It is possible to detect any nucleotide changes in the binding sites of the target with the short oligonucleotide reagent. In the presence of flanking di-3′,5′-N-(2-hydroxyethyl)phenazinium derivatives of short oligonucleotides (effectors) the tetranucleotide alkylating reagent modifies DNA target efficiently and site-specifically only in the perfect complex and practically does not modify it in the mismatched complex. It has been shown that TSOD is much more sensitive tool for the detection of a point mutation in DNA as compared to a longer oligonucleotides.     

Effective selective modification of a single-stranded fragment of DNA with alkylating derivatives of short oligodeoxyribonucleotides in the presence of reaction effectors N-(2-hydroxyethyl)phenazine derivatives of oligodeoxyribonucleotides

Tri-, tetra-, penta- and hexanucleotides bearing a reactive 4-(N-methylamino-N-2-chloroethyl)benzylamide group can effectively and selectively modify a single-stranded DNA fragment (302 nucleotides) in the presence of effectors, N-(2-hydroxyethyl)phenazinium derivatives of oligonucleotides complementary to DNA sequences adjacent to the binding site of the reagent. The reagents investigated modify not only single-stranded but also secondary-structured DNA regions. The modification extent depends on the length of oligonucleotide parts of the reagent and effector. A gap between the two stretches associated with the target DNA prevents the effector from functioning. The substitution of an octanucleotide effector by two tetranucleotide ones only slightly reduces the modification extent with a hexanucleotide reagent. A very efficient and specific modification can be achieved by using two effectors flanking the reactive oligonucleotide derivative. The approach leads to the modification extent of up to 89% with a hexanucleotide reagent.     

Reactivity of oligonucleotide derivatives, containing methylphosphonate groups. VI. Increase in the effectiveness of directed action of alkylating derivatives of oligonucleotide methylphosphonate analogs on nucleic acids in the presence of effectors--3',5'-bis-N-(2-hydroxyethyl)-phenazine derivatives of oligonucleotides

Effectors for increasing the efficiency of DNA modification with the alkylating methylphosphonate analogues of oligodeoxyribonucleotides (MFAO) were suggested. Oligodeoxyribonucleotide d(pC5A8ACAATG) used as a target DNA treated with alkylating derivatives of octathymidylate having alternating methylphosphonate and phosphodiester internucleotide bonds (both Rp- and Sp-individual diastereoisomers of MFAO were used) and bearing alkylating 4-(N-methyl-N-2-chloroethylamino)benzyl phosphoramide residue at the 3'-end. The reactions were carried out in the presence of an effector, hexadeoxyribonucleotide derivative PhnNH(CH2)2NHpCATTGTpNH(CH2)2NHPhn bearing two N-(2-hydroxyethyl)phenazinium (Phn) residues at the 3'- and 5'-ends and being complementary to the part of the target DNA neighbouring with octaadenylate. It was shown that Tm of the duplex formed by the target DNA, octathymidylate and effector is by 7-13 degrees C higher than in the absence of the effector, thus considerably increasing the efficiency of the intracomplex alkylation of the target (e.g., at 40 degrees C, the increase for the reagent based on the Rp-isomer is sixfold). Specificity of the target DNA modification by the MFAO alkylating derivatives in the presence of effector is same as with reagents based on oligodeoxyribonucleotides with natural internucleotide bonds.     

OLigonucleotide (Cyanomethyl) Phosphonates

Abstract

A dinucleoside (cyanomethyl) phosphonate has been prepared, and its properties have been studied. This compound was converted into an oligonucleotide possessing alternating (cyanomethyl) phosphonate and phosphodiester backbone groups and its hybridization to complementary DNA and RNA sequences was studied versus methylphosphonate and phosphodiester controls.     

Comparative Study of Modification of DNA and RNA by Oligo(2′-O-Methylribonucleotide) Derivatives

Abstract

The results of modification of the model DNA and RNA targets by the alkylating derivatives of 2′-O-methylribo-, ribo-, and deoxyhexanucleotides in the presence and absence of effectors (N-(2-hydroxyethyl)phenazinium derivatives of the same type of octanucleotides) are presented. It has been shown that the alkylating 4(N-methyl-N-2-chloroethyl-)benzylmethylamidophosphate derivatives of oligo(2′-O-methylribonucleotides) are the high effective reagents for the site specific modification of nucleic acids especially RNA.

     

Oligonucleotide Conjugates Designed for Discriminative Hybridization at Physiological Temperature

Abstract

We report the synthesis of oligonucleotide conjugates engineered to allow discriminative hybridization at temperatures around physiological. Two types of structural modifications were introduced: 1) internal oligomethylene and oligoethylene glycol spacers, and 2) terminal phenazinium residues. The thermal denaturation behaviour of the complexes formed by these oligonucleotide conjugates with a target sequence is compared to that of natural duplexes. We observed a lowering of the T_m of the duplexes formed by the internal modified oligonucleotides, whilst the terminal phenazinium residues enhance their stability. The effect of the spacers is modulated by their length and hydrophobic or hydrophilic nature. Alkylating substituents, which modify the target DNA strand on hybridization, were introduced on all conjugates, and the target cleavage obtained after piperidine treatment used as a further indicator of hybridization.     

DNA Sequencing by Hybridization to Oligonucleotide Matrix. Calculation of Continuous Stacking Hybridization Efficiency

Abstract

In this paper we consider the efficiency of additional rounds of “continuous stacking” hybridization in DNA sequence reconstruction by hybridization with oligonucleotide matrix (SHOM). After the initial hybridization of target DNA with the matrix of oligonucleotides of fixed length L some additional hybridizations should be carried out in the presence of fluorescently labeled oligonucleotides of another length l. These additional oligonucleotides can hybridize in tandem with matrix tuples (continuous stacking hybridization) thus forming an extended duplex with the target DNA strand. The additional data obtained allows resolutions of branching points arising in the reconstruction procedure. Multiple rounds of continuous stacking hybridization considerably increase the efficiency of the sequencing method, eventually approaching the power of (L+l)-matrix. We develop here an algorithm that allows us to minimize the number of additional hybridization steps, by assembling sets of l-tuples to be added together in each round of continuous stacking hybridization. For SHOM using a matrix of octanucleotides, continuous stacking hybridization with pen- tanucleotides increases the length of unambiguously sequenced DNA from 200 to several thousands of base pairs.     

Cooperative Interactions of the Oligodeoxyribonucleotides on the Complementary Template. The Influence of Chemical Groups and Mismatched Nucleotides at the 5′- and 3′-ends of Oligonucleotides on the Parameters of Cooperativity

Abstract

Parameters of cooperative interactions of two or three oligodeoxyribonucleotides or their derivatives bound with the adjacent sites of the complementary template were measured using method of “complementary addressed modification titration” (CAMT). Complementary template (target) were modified with the reactive oligonucleotide derivatives (reagents) bearing covalently attached alkylating 4-[N-(2-chloroethyl)-N-methylaminojbenzylamino- group (C1RCH₂NH)- at 5′-terminal phosphate. The targets had only one binding site for the reagent and either no (T10), or one (T'22 and T22) or two sites (T26) for the oligonucleotides (effectors) cooperatively bound with the adjacent sites on the template. Both unmodified oligonucleotides E₁, E₂ and their derivatives E₁ ^phn, E₂ ^phn bearing N- (2-hydroxyethyl)-phenazinium residues Phn- both at 5′- and 3′- ends covalently linked via ethylenediamine linker were used as effectors. Effectors E₁ and E₂ (E₁ ^Phn and E₂ ^Phn) bind, respectively, upstream or downstream from the reagent. Hexameric (X6) or octameric (X8 or X8^m) reagents were used for the target modification. The reagent X8^m formed one TT-mismatch with the target at the end opposite to location of the reactive moiety. The cooperativity parameter values characterizing the mutual interactions between the reagents X6, X8, X8^m and effectors E₁, E₂, E₁ ^phn, E₂ ^Phn have been found as the ratio of the association constants of the reagents in the presence of effectors. The association constants were calculated from the dependencies of the target modification extent on initial concentrations of the reagents. The use of T26 existing both in linear and hairpin conformations permitted us to estimate additionally the role of indirect cooperativity originating from the induction of the target conformational change by the effectors. The following conclusions were done from the quantitative results. The efficiency of direct cooperativity is independent on the length of oligonucleotide for the same nature of the contact. The cooperativity parameter increases by factor about 3 in the presence of Phn-group covalently attached to oligonucleotides and located at the junctions. The presence of either alkylating group CIRCH₂NH- or TT-mismatch at the junctions eliminates cooperative interaction between the bases. In the same time sufficiently effective cooperative interaction takes place in the case of simultaneous presence of both Phn- and either CIRCH₂NH- group or TT-mismatch at the junction.     

Synthesis of alkylating oligonucleotide derivatives containing cholesterol or phenazinium residues at their 3'-terminus and their interaction with DNA within mammalian cells

5'-[32P]-labelled alkylating decathymidylate [4-(N-2-chloroethyl)N-methylaminobenzyl]-5'-phosphamide derivatives containing cholesterol or phenazinium residues at their 3'-termini were synthesized and used for alkylation of DNA within mammalian cells. The uptake of the cholesterol derivative by the cells and the extent of DNA alkylation are about two orders of magnitude higher than those of a similar alkylating derivative lacking the groups at the 3'-termini. The presence of the phenazinium residue at the 3'-terminus of the oligonucleotide reagent does not improve the reagent uptake by the cells but drastically increases the DNA modification efficiency.     

The Derivatives of Phosphorothioate Oligonucleotides

Abstract

Derivatives of phosphorothioate oligonucleotide analogues bearing alkylating N-methyl-4-(N-2-chloroethyl)-N-methylamino)benzylamine or stabilizing complementary complexes N-(2-hydroxyethyl)phenazinium residues at the 3′-terminal phosphate group were synthesized and investigated.     

A new strategy of discrimination of a point mutation by tandem of short oligonucleotides

A new strategy based on the use of cooperative tandems of short oligonucleotide derivatives (TSOD) has been proposed to discriminate a "right" DNA target from a target containing a single nucleotide discrepancy. Modification of a DNA target by oligodeoxyribonucleotide reagents was used to characterize their interaction in the perfect and mismatched complexes. It is possible to detect any nucleotide changes in the binding sites of the target with the short oligonucleotide reagent. In the presence of flanking di-3',5'-N-(2-hydroxyethyl)phenazinium derivatives of short oligonucleotides (effectors) the tetranucleotide alkylating reagent modifies DNA target efficiently and site-specifically only in the perfect complex and practically does not modify it in the mismatched complex. It has been shown that TSOD is much more sensitive tool for the detection of a point mutation in DNA as compared to a longer oligonucleotides.     

Synthesis of a 2-Hydroxy-oxolane Derivative as a New Potential Crosslinking Agent of DNA

Abstract

The design and the synthesis of a new potential crosslinking agent of DNA is reported. It is based on the alkylating properties of a natural toxin (botryodiplodin) and will be further linked to antisense oligonucleotides.     

Stabilization of DNA Double and Triple Helices by Conjugation of Minor Groove Binders to Oligonucleotides

Abstract

New conjugates containing two parallel or antiparallel carboxamide minor groove binders (MGB) attached to the same terminal phosphate of one oligonucleotide strand were synthesized. The conjugates interact with their target DNA stronger than the individual components. Effect of conjugated MGB on DNA duplex and triplex stability and their sequence specificity was demonstrated on the short oligonucleotide duplexes and on the triplex formed by model 16-mer oligonucleotide with HIV polypurine tract.     

Activation by laser irradiation of a "switched on" oligonucleotide reagent for an addressed modification of nucleic acids

Properties of oligonucleotide reagents containing an alkylating group of regulated reactivity (nitrogen mustard residue activatable upon mild borohydride reduction of the aromatic formyl group) have been studied. It was shown that these reagents can also be activated by irradiation with nitrogen laser light (lambda 337 nm). Activation of the reagent in complex with a target polydeoxyribonucleotide resulted in the addressed chemical modification of the target. The positional direction of the modification depended on the way of the activation (borohydride reduction or laser irradiation).     

Synthesis And Binding Study Of Phosphonate Analogues Of Pnas And Their Hybrids With Pnas

Abstract

The preparation of monomers for the synthesis of phosphonate analogues of peptide nucleic acids containing the four natural nucleobases: thymine, cytosine, adenine and guanine, has been ccomplished. The monomers obtained were used for the automated online solid phase synthesis of pure phosphono-PNA oligomers as well as chimeras consisting of phosphono-PNA and PNA resudies. The hybridization properties of these oligonucleotide mimics to complementary DNA and RNA fragments were studied.     

Synthesis and Triplex Binding Properties of Oligonucleotides Containing a Novel Nucleobase

Abstract

The thiazolo-indole compound 1 bearing the complementary donor-acceptor-donor sites (dad) was designed for specific recognition of an AT inverted base pair in pyrimidine triple helix motif. It was successfully incorporated into 14-mer oligonucleotide using a serinol unit as sugar derivative. The triple helix hybridization studies were examined by means of thermal denaturation experiments with a 26-mer DNA duplex containing the AT inverted base pair.     

SNP Detection for Cytochrome P450 Alleles by Target-assembled Tandem Oligonucleotide Systems Based on Exciplexes

Abstract

We report the first use of exciplex-based split-probes for detection of the wild type and *3 mutant alleles of human cytochrome P450 2C9. A tandem 8-mer split DNA oligonucleotide probe system was designed that allows detection of the complementary target DNA sequence. This exciplex-based fluorescence detector system operates by means of a contiguous hybridization of two oligonucleotide exciplex split-probes to a complementary target nucleic acid target. Each probe oligonucleotide is chemically modified at one of its termini by a potential exciplex-forming partner, each of which is fluorescently silent at the wavelength of detection. Under conditions that ensure correct three-dimensional assembly, the chemical moieties on suitable photoexcitation form an exciplex that fluoresces with a large Stokes shift (in this case 130 nm). Preliminary proof-of-concept studies used two 8-mer probe oligonucleotides, but in order to give better specificity for genomic applications, probe length was extended to give coverage of 24 bases. Eight pairs of tandem 12-mer oligonucleotide probes spanning the 2C9*3 region were designed and tested to find the best set of probes. Target sequences tested were in the form of (i) synthetic oligonucleotides, (ii) embedded in short PCR products (150 bp), or (iii) inserted into plasmid DNA (～ 3 Kbp). The exciplex system was able to differentiate wild type and human cytochrome P450 2C9 *3 SNP (1075 A→C) alleles, based on fluorescence emission spectra and DNA melting curves, indicating promise for future applications in genetic testing and molecular diagnostics.     

Selective Attachment of Oligonucleotides to Interleukin-1β and Targeted Delivery to Cells

Abstract

Conjugates between oligodeoxyribonucleotides and an interleukin-1β mutant protein have been constructed using a heterobifunctional cross-linker. These protein-DNA conjugates had conserved binding activity to the interleukin-1 receptor. The oligonucleotide hybridization properties were unchanged.