Reactions of nucleic acid bases with alpha-acetylenic esters. Synthesis and enzymatic evaluation of chemically modified nucleotides

Analogues of adenine nucleotides, containing an additional chloromethyl-pyrimidone ring fused to the purine base, were obtained by treatment of AMP, ADP and ATP with an alpha-acetylenic ester, methyl 4-chlorobut-2-ynoate. These compounds were tested for their ability to substitute for the natural substrates or cofactors of several enzymes. With the ADP analogue, pyruvate kinase showed a significant increase of the Km value and a moderate decrease of V, while the reverse was observed when hexokinase was tested with modified ATP. Adenylate kinase was active with the ATP analogue but not with the AMP derivative. Myosin accepted the ATP analogue as a substrate, but was irreversibly modified. Among the dehydrogenases tested, only glucose-6-phosphate dehydrogenase was inhibited by the nucleotide analogue. The structure--activity relationship of these nucleotide derivatives, which represent the largest dimensional deviation known from natural nucleotides, is discussed in comparison with some earlier described dimensional probes of enzyme-nucleotide binding sites.     

Comparison of the tertiary structure of yeast tRNA(Asp) and tRNA(Phe) in solution. Chemical modification study of the bases

A comparative study of the solution structures of yeast tRNA(Asp) and tRNA(Phe) was undertaken with chemical reagents as structural probes. The reactivity of N-7 positions in guanine and adenine residues was assayed with dimethylsulphate and diethyl-pyrocarbonate, respectively, and that of the N-3 position in cytosine residues with dimethylsulphate. Experiments involved statistical modifications of end-labelled tRNAs, followed by splitting at modified positions. The resulting end-labelled oligonucleotides were resolved on polyacrylamide sequencing gels and analysed by autoradiography. Three different experimental conditions were used to follow the progressive denaturation of the two tRNAs. Experiments were done in parallel on tRNA(Asp) and tRNA(Phe) to enable comparison between the two solution structures and to correlate the results with the crystalline conformations of both molecules. Structural differences were detected for G4, G45, G71 and A21: G4 and A21 are reactive in tRNA(Asp) and protected in tRNA(Phe), while G45 and G71 are protected in tRNA(Asp) and reactive in tRNA(Phe). For the N-7 atom of A21, the different reactivity is correlated with the variable variable loop structures in the two tRNAs; in the case of G45 the results are explained by a different stacking of A9 between G45 and residue 46. For G4 and G71, the differential reactivities are linked to a different stacking in both tRNAs. This observation is of general significance for helical stems. If the previous results could be fully explained by the crystal structures, unexpected similarities in solution were found for N-3 alkylation of C56 in the T-loop, which according to crystallography should be reactive in tRNA(Asp). The apparent discrepancy is due to conformational differences between crystalline and solution tRNA(Asp) at the level of the D and T-loop contacts, linked to long-distance effects induced by the quasi-self-complementary anticodon GUC, which favour duplex formation within the crystal, contrarily to solution conditions where the tRNA is essentially in its free state.     

Sequence-specific chemical modification of a 365-nucleotide-long DNA fragment with an alkylating oligonucleotide derivative

An aromatic 2-chloroethylamino group was attached to the 5'-terminal phosphate of the oligodeoxyribonucleotide pCCCTCTTTCTT. The oligonucleotide derivative prepared was used for modification of the 365-nucleotide-long DNA fragment. It was found that modification of the fragment proceeds in a sequence-specific way at 3 guanosine residues within the sequence complementary to the oligonucleotide reagent.     

Reaction mechanisms of riboflavin triplet state with nucleic acid bases.

ESR and laser flash photolysis studies have determined a reasonable order of reactivity of nucleotides with triplet riboflavin (3Rb*) for the first time. ESR detection of triplet state reactivity of Rb with nucleoside, polynucleotide and DNA has been obtained simultaneously. In addition, ESR spin elimination measurement of the reactivity of 3Rb* with nucleotides in good accord with laser flash photolysis determination of the corresponding rate constants offers a simple and reliable method to detect the reactivities of nucleic acids and its components with photoexcited flavins. Kinetic, ESR and thermodynamic studies have demonstrated that Rb should be a strong endogenous photosensitizer capable of oxidizing all nucleic acid bases, and preferentially two purine nucleotides with high rate constants.     

Fluorescent probe and permeability to cells of isopoly (S-carboxymethyl-L-cysteine) derivative of nucleic acid bases.

Isopoly(S-carboxymethyl-L-cysteine) derivatives of nucleic acid bases were found to form stable complex with oligo-DNA in vitro. Fluorescent probed isopoly(S-carboxymethyl cysteine) derivatives of nucleic acid bases were prepared as antisense oligomers. The transfection of the oligomer into cells was carried out by HVJ-liposome method. Fluorescence was observed from the cells treated with HVJ-liposome including fluorescent probed oligomers.     

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.     

Preparation of an isomorphous heavy-atom derivative of tobacco mosaic virus by chemical modification with 4-sulpho-phenylisothiocyanate

The reaction of the vulgare and U2 strains of tobacco mosaic virus with 4-sulpho-phenylisothiocyanate has been investigated. The coat protein of the U2 strain has a proline residue at its N-terminus and a lysine residue at position 53. Whereas both residues could be reacted with 4-sulpho-phenylisothiocyanate in the isolated coat protein, only proline-1 was modified during treatment of the intact virus with the same reagent, thereby showing that the loss of reactivity of the ?-amino group of lysine-53 is a consequence of the virus structure. The 4-sulpho-phenylthiocarbamoyl derivative of amino groups shows considerable tautomerism and, as a consequence, it proved possible to prepare a heavy-atom derivative of the intact U2 strain in which methyl mercury nitrate was bound by the modified N-terminal residue of the coat protein.On the other hand, when the intact vulgare strain was treated with 4-sulphophenylisothiocyanate, little or no modification of the ?-amino groups of the two lysine residues (positions 53 and 68) per polypeptide chain was observed. Taking into account previous studies on the reactivity of the amino groups of the coat protein in tobacco mosaic virus vulgare and assuming that all strains and mutants have closely similar three-dimensional structures, these experiments suggest that the N-terminal residue is more exposed (i.e. probably nearer the virus “surface”) than the side-chain of lysine-68, which in turn is more accessible than the side-chain of lysine-53. This interpretation is readily compatible with the results of X-ray diffraction analysis carried out on these chemically modified viruses (Mandelkow &; Holmes, 1974) and lends support to the hope that such methods of preparing heavy-atom derivatives of proteins will be of general use.     

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.     

Theoretical study on the proton chemical shifts of hydrogen bonded nucleic acid bases.

The variation of the proton chemical shifts due to the formation intermolecular hydrogen bonds is computed for a number of complexes which can be formed between the bases of the nucleic acids. The shifts expected for the isolated base pairs, in particular for the G-N1 H, T(or U)-N3H protons and the protons of the amino groups of A, G c, when combined with previous computations on the shifts to be expected upon base stacking, may enable a refined analysis of the high resolution NMR spectra of self complementary polynucleotides or tRNAs. Two examples are presented of a direct computation of proton shits associated with helix-coil transitions, helpful for deducing the helical structure in solution.     

Internal dynamics of tRNA(Phe) studied by depolarized dynamic light scattering

The collective internal dynamics of transfer RNA(Phe) from brewer's yeast in solution was studied by depolarized dynamic light scattering (DDLS). Within the melting region of tRNA the depolarized spectra consist of two Lorentzian, where the narrow (slow) component describes the overall rotation of the macromolecule. The broad component is attributed to the collective reorientation of the bases within the biopolymer. At high temperature only this relaxation process is observed in the spectrum. The viscosity dependence of the collective internal relaxation process is described by the Stokes-Einstein-Debye equation for rotational diffusion. Estimates of the internal orientational pair correlation factor from the integral depolarized intensities of tRNA(Phe) solutions indicates that the observed dynamics correspond to the collective reorientation of approximately 5 bases. A comparison of the results presented with DDLS studies on the aggregation of the mononucleotide guanosine-5'-monophosphate confirms this result. For a further characterization of the relaxation process we studied the effect of hydrostatic pressure (1-1000 bar) on the depolarized spectra of tRNA. While other spectroscopic methods like nmr, fluorescence polarization anisotropy decay, or ESR give information about the very local motion of a single base within the DNA or RNA, this study shows that by DDLS one can characterize collective internal motions of macromolecules.     

Hybridization of antisense oligonucleotides with the 3'part of tRNA(Phe)

The interaction of antisense oligodeoxyribonucleotides with yeast tRNA(Phe) was investigated. 14-15-mers complementary to the 3'-terminal sequence including the ACCA end bind to the tRNA under physiological conditions. At low oligonucleotide concentrations the binding occurs at the unique complementary site. At higher oligonucleotide concentrations, the second oligonucleotide molecule binds to the complex due to non-perfect duplex formation in the T-loop stabilized by stacking between the two bound oligonucleotides. In these complexes the acceptor stem is open and the 5'-terminal sequence of the tRNA is accessible for binding of a complementary oligonucleotide. The results prove that the efficient binding of oligonucleotides to the 3'-terminal sequence of the tRNA occurs through initial binding to the single-stranded sequence ACCA followed by invasion in the acceptor stem and strand displacement.     

The behavior and effect of isopoly (S-carboxymethyl-L-cysteine) derivatives of nucleic acid bases.

Isopoly(S-carboxymethyl-L-cysteine) derivatives of nucleic acid bases were prepared as antisense compounds. These compounds in vitro have been found to form stable complex with oligo-DNA or RNA. This paper deals with effect of antisense compounds in vivo. The target in this paper is the sequence of the PSD-95 protein linked with NMDA receptor. Excess passing of calcium ions through the loss of the signal pathway without PSD-95 proteins caused by antisense compound. The cells detailing with L-cysteine derivatives showed the lowest percentage of 19.1%. The data were compared with that of phosphotioate antisense compound.