Special Properties of Modification of SsDNA Target by Alkylating Derivatives of Oligonucleotides in Tandem Complexes

Abstract

The influence of an effector (di-N-(2-hydroxyethyl)-phenazinium derivative of oligonucleotide) on modification of the DNA target by alkylating derivatives of oligonucleotides having various hybridization properties was studied. Being adjacent to the alkylating group of the reagent, the effector enhances the target modification if the oligonucleotide reagent has low hybridization properties and suppresses the modification if the reagent can form the stable complex with the DNA target at the used conditions.

     

Complementary addressed modification of plasmid DNA

The possibility to accomplish the sequence-specific chemical modification of superhelical DNA with reactive oligonucleotide derivatives was demonstrated. Plasmids containing fragments of the immunoglobulin gene were modified with alkylating derivatives of oligonucleotides complementary to a nucleotide sequence in the immunoglobulin gene. In contrast to the relaxed plasmid DNAs, superhelical DNAs (sigma = -0.1) were found to be attacked by the derivatives at the target nucleotide sequence. The efficiency of the reaction increases with the increase of the plasmids negative superhelicity. It was found also that the denatured derivatives. The sequence-specific modification of plasmid DNAs with the reactive oligonucleotide derivatives can be used for the site-directed mutagenesis and the investigation of the repair processes.     

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.     

Interaction of cholesterol-conjugated alkylating oligonucleotide derivatives with cellular biopolymers.

Interactions of oligonucleotide derivatives with mammalian cells and cellular biopolymers have been investigated. The derivatives were oligonucleotides bearing an alkylating 2-chloroethylamino group at the 3'-end and a cholesterol residue at the 5'-terminal phosphate. These compounds are readily taken up by cells and react with cellular DNA, RNA and some proteins which may play a role in delivery of the compounds into cells.     

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.     

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.     

Interaction of alkylating oligonucleotide derivatives with mammalian cells. A study of the uptake mechanism of derivative cells

Interaction of alkylating deoxyribooligonucleotide derivatives, bearing 4-[(N-2-chloroethyl-N-methyl)amino]benzylamine residues at their 5'-terminal phosphates, with mouse fibroblasts L929 and with ascite carcinoma cells Krebs 2 has been investigated. It was found, that the derivatives are taken up by the cells according to the endocytosis mechanism. At high concentration of the oligonucleotide derivatives in the cultivation medium (greater than 10 microM), the fluid phase endocytosis is the major pathway of uptake; binding of the derivatives by the cells is partially reversible and their intracellular mean concentration approaches 1/20 of their extracellular concentration. At low concentration of the oligonucleotide derivatives, the predominant mechanism is the more efficient adsorption endocytosis; at concentration of the derivatives less than 0.5 microM, their mean intracellular concentration exceeds that in the culture medium. Stability of the oligonucleotide derivatives in cells depends on their nucleotide composition. Their nucleolytic degradation rate is low enough to allow them to react with cellular biopolymers.     

Sequence-specific modification of nucleic acids by oligonucleotide derivative containing alkylating groups in the C-5-position of deoxyuridine]

A new type of alkylating derivatives of oligonucleotides with 4(N-methyl-N-2-chloroethylamino)benzyl (RCl) group at C-5 of deoxyuridine with a high extent of the target modification was prepared. The synthesized reagents d(ULNHRClCCACTT), where L = CH2 (Ia), CH2OCH2CH2 (Ib) and CH2NHCOCH2CH2 (Ic), proved to effectively (80-90%) modify the oligonucleotide d(TAAGTGGAGTTTGGC). The reagents (Ia) and (Ib) alkylate G6, G7 and G9 positions, while the reagent (Ic) modifies predominantly G9.     

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.     

Highly selective labeling of the E. coli RNA-polymerase promoter complex with reactive derivatives of oligonucleotide primers of various specificity

A technique of highly selective affinity labelling, which includes covalent modification of the enzyme-T7A2 promoter complex with reactive oligonucleotide derivatives and subsequent elongation of the attached oligonucleotide residue with a radioactive substrate was used to study the product-binding site of E. coli RNA polymerase. Different oligonucleotides complementary to the T7A2 promoter (with lengths ranging from 2 to 8 residues) containing 5'-terminal phosphorylating, alkylating or aldehyde groups were used for the labelling. The procedure resulted in labelling DNA and beta-, beta'- or sigma-subunits of the enzyme, which are therefore believed to contact with growing RNA in the course of initiation. Consideration of the labelling patterns as a functions of the oligonucleotide's length as well as of the structure and chemical specificity of the reactive groups led to a tentative topographic scheme of the RNA polymerase product-binding region.     

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).     

Sequence specificity modification of double-stranded DNA with an alkylating derivative of oligodeoxyribonucleotide pT(CT)6

It is shown that in slightly acidic solution (pH approximately 5.3) reagent CIRCH2NHpT(CT)6 (RCl = -C6H4-N(CH3)CH2CH2Cl) modifies a double-stranded DNA fragment (120 b. p.) containing A(GA)6.T(CT)6 sequence at a single nucleotide residue, viz. G29 located near to this sequence in the DNA chain. The location of this modification point suggests formation of a triple-stranded reactive complex with parallel orientation of the pyrimidine oligonucleotide moiety of the reagent and pyrine sequence of the target DNA. Analysing the modification extent dependence of the reagent concentration the association constant Kx between the reagent and DNA was calculated (Kx = (0.95 +/- 0.03).10(5) M-1, 25 degrees C, pH = 5.3, [NaCl] = 0.1 M). The modification by the reagent ClRCH2NHpT(m5CT)6 has the same quantitative characteristics as in the case of ClRCH2NHpT(CT)6.     

Inhibition of translation of immunoglobulin mRNA in vitro using an alkylating oligonucleotide derivative

Effect of complementary oligonucleotides and their reactive derivatives on translation of mouse immunoglobulin G kappa light chain was investigated. It was found that oligonucleotide pTGCTCTGGTTT and shorter oligonucleotides complementary to the coding sequence of the mRNA (nucleotides 205-215) do not arrest translation of the mRNA in the rabbit reticulocyte cell-free translation system. Preincubation of the mRNA with the alkylating 4-(N-2-chloroethyl-N-methylamino)benzyl-5'-phosphamide derivative of the oligonucleotide completely suppresses the synthesis of the protein thus demonstrating higher efficiency of the reactive oligonucleotide derivatives as inhibitors of the mRNA function.     

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.     

Specific modification of nucleic acids inside the cell with alkylating derivatives of oligonucleotides ethylated at internucleotide phosphates

The alkylating derivatives of (C6H5NH)2P[dTp(Et)]4U and [dTp(Et)]9 U with completely esterified internucleotide phosphates bearing reactive 2,3 -O-4(N-2-chloroethyl N-methylamino)-benazylidene moiety attached to 3 -end cis-diol group were prepared. These alkylating derivatives of non-ionisable oligonucleotide analogs were demonstrated to penetrate efficiently into Krebs ascites tumor cells and to alkylate nucleic acids inside the cells with a strong preference towards complementary poly(A)-fragments of mRNA.     

Selective alkylation of G24 residue in tRNAPhe

Alkylation of E. coli tRNAPhe with 4-(N-2-chloroethyl-N-methylamino) benzyl-5'-phosphamide of oligonucleotide d(pAACCA) was studied. G24 residue located near the sequence C17GGDA21 partially complementary to the oligonucleotide moiety of the reagent was shown to be alkylated. Oligonucleotide d(pAACCA) inhibited the alkylation. Association constant of oligonucleotide derivative with tRNAPhe (10(3) M-1) was evaluated from the dependence of the extent of tRNA modification on the concentration of the reagent. The reported method for selective alkylation of tRNA may be used for preparing photoaffinity derivatives of tRNA bearing an arylazidogroups in desired position.     

Alkylation and cleavage of a DNA fragment with trioligonucleotide reagent

A possibility of site-directed chemical modification of a ssDNA fragment with "trioligonucleotide reagent" (TOR), consisting of a central oligonucleotide derivative carrying N-(2-chloroethyl)-N-(p-formylphenyl)-N-propyl-N-3-ydeneamino groups at both 5'- and 3'-thiophosphate ends and two border derivatives with 4-carbohydrazidephenyl groups at their 3'- and 5'-phosphate ends, respectively, is shown. Products of site-directed fragment cleavage, more abundant than the alkylation products, were found at 50 degrees C. The overall level of DNA modification by TOR reached 30% at a small excess of the oligonucleotide derivatives.     

Oligonucleotide-binding proteins of Drosophila: tissue specificity]

A reaction of native Drosophila proteins with an alkylating oligonucleotide derivative bearing 4-[(N-2-chlorethyl-N-methyl)amino]benzylamine at the 5' terminal phosphate has been investigated. It was found, that the reagent alkylates a few proteins (90, 50, 44, 39, 32 kDa). The modification was organ specific. The labeled 39 kDa protein is present in the ovaries only, while the modified 32 kDa protein is found only in the bulbus.     

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.     

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.