Ryszard Stolarski - Academia.edu (original) (raw)

Papers by Ryszard Stolarski

ABSTRACT Self-association of a series of newly synthesized dinucleotide mRNA cap-analogues has be... more ABSTRACT Self-association of a series of newly synthesized dinucleotide mRNA cap-analogues has been investigated in aqueous buffers by means of H-1 NMR and fluorescence, and compared with that for a regular cap, 7-methylguanosine(5')ppp(5')guanosine (m(7)GpppG). The previously investigated inter-molecular association between the tripeptide, Trp-Leu-Glu, and cap analogues (Biochim. Biophys. Acta 1293, 97 (1996)) has been extended to include m(7)GpppC and m(7)Gpppm(7)G. More detailed insight into the stacking interactions was attempted by means of molecular dynamics simulations.

Molecules

Etheno-derivatives of 2-aminopurine, 2-aminopurine riboside, and 7-deazaadenosine (tubercidine) w... more Etheno-derivatives of 2-aminopurine, 2-aminopurine riboside, and 7-deazaadenosine (tubercidine) were prepared and purified using standard methods. 2-Aminopurine reacted with aqueous chloroacetaldehyde to give two products, both exhibiting substrate activity towards bacterial (E. coli) purine-nucleoside phosphorylase (PNP) in the reverse (synthetic) pathway. The major product of the chemical synthesis, identified as 1,N2-etheno-2-aminopurine, reacted slowly, while the second, minor, but highly fluorescent product, reacted rapidly. NMR analysis allowed identification of the minor product as N2,3-etheno-2-aminopurine, and its ribosylation product as N2,3-etheno-2-aminopurine-N2-β-d-riboside. Ribosylation of 1,N2-etheno-2-aminopurine led to analogous N2-β-d-riboside of this base. Both enzymatically produced ribosides were readily phosphorolysed by bacterial PNP to the respective bases. The reaction of 2-aminopurine-N9-β -d-riboside with chloroacetaldehyde gave one major product, clearly...

Zeitschrift für Naturforschung C

The 3′:5′-cyclic phosphates of 2′,3′-secoadenosine and guanosine, structural analogues of cAMP an... more The 3′:5′-cyclic phosphates of 2′,3′-secoadenosine and guanosine, structural analogues of cAMP and cGMP, were synthesized by cyclization of the 5′-phosphates of 2′,3′-secoadenosine and guanosine, respectively. The 2′,3′-seco-3′:5′-cAMP was converted to the IM P analogue by nitrous acid deamination, and to the 8-bromo analogue by bromination. Chemical phosphorylation of 2′,3′-secoadenosine gave four products, the major one of which, in 50% yield, was 2′,3′-seco-3′:5′-cAMP, identical to that obtained by the cyclization reaction above. The three other products have been tentatively identified. The conformations in solution of the seco nucleosides, their 5′-monophosphates, and their 3′:5′-cyclic phosphates, were determined with the aid of 1H , 13C and 31P NMR spectroscopy, particular attention being devoted to orientations about the C-O bonds and the glycosidic bond, and the results compared with crystallographic data available for the 2′,3′-seco congener of ribofuranosyl benzimidazole....

Collection of Czechoslovak Chemical Communications, 1993

Http Dx Doi Org 10 1081 Ncn 120023120, Aug 31, 2006

Canadian Journal of Chemistry, 1985

Methods in Enzymology, Feb 1, 2007

Synthetic capped RNA transcripts produced by in vitro transcription in the presence of m(7)Gp(3)G... more Synthetic capped RNA transcripts produced by in vitro transcription in the presence of m(7)Gp(3)G have found a wide application in studying such processes as mRNA translation, pre-mRNA splicing, mRNA turnover, and intracellular transport of mRNA and snRNA. However, because of the presence of a 3'-OH on both m(7)Guo and Guo moieties of the cap structure, one-third to one-half of the mRNAs contain a cap incorporated in the reverse orientation. The reverse cap structures bind poorly to eIF4E, the cap binding protein, and reduce overall translational efficiency. We therefore replaced the conventional m(7)Gp(3)G cap by "anti-reverse" cap analogs (ARCAs) in which the 3'-OH of m(7)Guo moiety was substituted by 3'-deoxy or 3'-O-methyl groups, leading to m(7)3'dGp(3)G or m(2)(7,3'-O) Gp(3)G, respectively. The class of ARCAs was extended to analogs possessing an O-methyl group or deoxy group at C2' of m(7)Guo. We have also developed a series of ARCAs containing tetra- and pentaphosphates. mRNAs capped with various ARCAs were translated 1.1- to 2.6-fold more efficiently than their counterparts capped with m(7)Gp(3)G in both in vitro and in vivo systems. In a separate series, a methylene group was introduced between the alpha- and beta-, or beta- and gamma-phosphate moieties, leading to m(2)(7,3'-O)Gpp(CH2)pG and m(2)(7,3'-O)Gp(CH2)ppG. These analogs are resistant to cleavage by the decapping enzymes Dcp1/Dcp2 and DcpS, respectively. mRNA transcripts capped with m(2)(7,3'-O)Gpp(CH2)pG were more stable when introduced into cultured mammalian cells. In this chapter, we describe the synthesis of representative ARCAs and their biophysical and biochemical characterization, with emphasis on practical applications in mRNA translation.

Nucleos Nucleot Nucleic Acids, 2003

A series of new mRNA anti reverse cap analogues (ARCA) was designed to obtain a tool for studying... more A series of new mRNA anti reverse cap analogues (ARCA) was designed to obtain a tool for studying the mechanism of protein translation. Dinucleoside P 1 , P 3-triP P 1 , P 4-tetra-and P 1 , P 5-pentaphosphates, linked by a 5 0-to-5 0 phosphate bridge and composed of modified 7-methylguanosine and guanosine, have been synthesized. The hydroxyl group (2 0 OH or 3 0 OH) in 7-metylguanosine moiety was replaced by-OCH 3 or-H in order to obtain the cap analogues capable to be correctly incorporated into synthetic mRNA transcripts. Tri-, tetra-, and pentaphosphates were prepared by ZnCl 2 catalyzed condensation in DMF of derivatives of the 7-methylguanosine diphosphates with the guanosine mono-, di-and triphosphate P-imidazolides, respectively. The structures of the novel compounds were established by means of 1 H and 31 P NMR spectra.

Org. Biomol. Chem., 2016

We describe acetylpyrene-labeled mRNA cap analogues with untypical fluorescent properties as prob... more We describe acetylpyrene-labeled mRNA cap analogues with untypical fluorescent properties as probes for inhibitor screening of DcpS.

Biochemistry Usa, Jul 1, 1984

European Journal of Biochemistry, Dec 1, 1987

Two 19-base-pair oligodeoxynucleotides, analogues of one of the operators which specifically bind... more Two 19-base-pair oligodeoxynucleotides, analogues of one of the operators which specifically bind the repressor protein in the regulatory part of the transposon Tn10 tetracycline-resistance (tet) determinant, have been studied by 1H-NMR spectroscopy. The analogues contain a mismatch in the central base pair of the double helix (T.T or A.A). The imino protons have been assigned to the base pairs by one-dimensional NOE measurements, and the thermally induced transition from the duplex to the single strand has been followed. The cytidine amino resonances have been assigned by means of two-dimensional NOE spectroscopy in H2O. Two-dimensional phase-sensitive NOE and magnitude-correlated spectra have been recorded in 2H2O; all nonexchangeable protons, with the exception of some of H5', H5" protons, have been assigned. The NMR data made it possible to carry out a qualitative analysis of the structures of both oligodeoxynucleotides. The general structures close to B-DNA, show irregularities in the mismatch areas.

Nucleosides Nucleotides and Nucleic Acids, Aug 31, 2006

A series of new mRNA anti reverse cap analogues (ARCA) was designed to obtain a tool for studying... more A series of new mRNA anti reverse cap analogues (ARCA) was designed to obtain a tool for studying the mechanism of protein translation. Dinucleoside P 1 , P 3-triP P 1 , P 4-tetra-and P 1 , P 5-pentaphosphates, linked by a 5 0-to-5 0 phosphate bridge and composed of modified 7-methylguanosine and guanosine, have been synthesized. The hydroxyl group (2 0 OH or 3 0 OH) in 7-metylguanosine moiety was replaced by-OCH 3 or-H in order to obtain the cap analogues capable to be correctly incorporated into synthetic mRNA transcripts. Tri-, tetra-, and pentaphosphates were prepared by ZnCl 2 catalyzed condensation in DMF of derivatives of the 7-methylguanosine diphosphates with the guanosine mono-, di-and triphosphate P-imidazolides, respectively. The structures of the novel compounds were established by means of 1 H and 31 P NMR spectra.

The Journal of Physical Chemistry B, Jun 22, 2011

Molecular mechanisms underlying the recognition of the mRNA 5&amp... more Molecular mechanisms underlying the recognition of the mRNA 5' terminal structure called "cap" by the eukaryotic initiation factor 4E (eIF4E) are crucial for cap-dependent translation. To gain a deeper insight into how the yeast eIF4E interacts with the cap structure, isothermal titration calorimetry and the van't Hoff analysis based on intrinsic protein fluorescence quenching upon titration with a series of chemical cap analogs were performed, providing a consistent thermodynamic description of the binding process in solution. Equilibrium association constants together with thermodynamic parameters revealed similarities and differences between yeast and mammalian eIF4Es. The yeast eIF4E complex formation was enthalpy-driven and entropy-opposed for each cap analog at 293 K. A nontrivial isothermal enthalpy–entropy compensation was found, described by a compensation temperature, T(c) = 411 ± 18 K. For a low affinity analog, 7-methylguanosine monophosphate, a heat capacity change was detected, ΔC(p)° = +5.2 ± 1.3 kJ·mol(-1)·K(-1). The charge-related interactions involving the 5′-5′ triphosphate bridge of the cap and basic amino acid side chains at the yeast eIF4E cap-binding site were significantly weaker (by ΔΔH°(vH) of about +10 kJ·mol(-1)) than those for the mammalian homologues, suggesting their optimization during the evolution.

Http Dx Doi Org 10 1081 Ncn 200060103, Nov 15, 2011

Chemical synthesis of a series of novel dinucleoside cap analogues, m7GpppN, where N is formycin ... more Chemical synthesis of a series of novel dinucleoside cap analogues, m7GpppN, where N is formycin A, 3'-O-methylguanosine, 9-beta-D-arabinofuranosyladenine, and isoguanosine, has been performed using our new methodology. The key reactions of pyrophosphate bonds formation were achieved in anhydrous dimethylformamide solutions employing the catalytic properties of zinc salts. Structures of the new cap analogues were confirmed by 1H NMR and 31p NMR spectra. The binding affinity of the new cap analogues for murine eIF4E(28-217) were determined spectroscopically showing the highest association constant for the analogue that contains formycin A.

Nucleosides and Nucleotides, 1999

Interactions of mammalian (rat hepatoma) thymidylate synthase with FdUMP and its 4-thio analogue ... more Interactions of mammalian (rat hepatoma) thymidylate synthase with FdUMP and its 4-thio analogue were studied in solution with the use of H and F NMR. The results pointed to formation of ternary covalent complexes of TS with either nucleotide in the presence of N, N-methylenetetrahydrofolate and only noncovalent binary complexes in the absence of the cofactor.

RNA (New York, N.Y.), 2001

The ability to synthesize capped RNA transcripts in vitro using bacteriophage polymerases has bee... more The ability to synthesize capped RNA transcripts in vitro using bacteriophage polymerases has been of considerable value in a variety of applications. However, Pasquinelli et al. [RNA (1995) 1:957-967] found that one-third to one-half of the caps are incorporated in the reverse orientation, that is, with the m7G moiety of m7GpppG linked by a 3'-5' phosphodiester bond to the first nucleotide residue of the RNA chain. Such reverse caps are unlikely to be recognized by eIF4E, based on previous studies, and thus complicate any comparison of the translational efficiencies of in vitro-synthesized mRNAs. We therefore designed two novel cap analogs, P(1)-3'-deoxy-7-methyguanosine-5' P3-guanosine-5' triphosphate and P(1)-3'-O,7-dimethylguanosine-5' P3-guanosine-5' triphosphate, that are, theoretically, incapable of being incorporated in the reverse orientation. The key reactions of pyrophosphate bond formation were achieved in anhydrous dimethylformamide solut...

Acta biochimica Polonica, 2003

Description of the recognition specificity between proteins and nucleic acids at the level of mol... more Description of the recognition specificity between proteins and nucleic acids at the level of molecular interactions is one of the most challenging tasks in biophysics. It is key to understanding the course and control of gene expression and to the application of the thus acquired knowledge in chemotherapy. This review presents experimental results of thermodynamic studies and a discussion of the role of thermodynamics in formation and stability of functional protein-RNA complexes, with a special attention to the interactions involving mRNA 5' cap and cap-binding proteins in the initiation of protein biosynthesis in the eukaryotic cell. A theoretical framework for analysis of the thermodynamic parameters of protein-nucleic acid association is also briefly surveyed. Overshadowed by more spectacular achievements in structural studies, the thermodynamic investigations are of equal importance for full comprehension of biopolymers' activity in a quantitative way. In this regard, ...

European Journal of Biochemistry, 1984

Analyses of 1H and 13C NMR spectra have been utilized to extend studies on the dynamic equilibriu... more Analyses of 1H and 13C NMR spectra have been utilized to extend studies on the dynamic equilibrium syn-anti about the glycosidic bond of purine nucleosides and nucleotides. With the aid of chemically synthesized model analogous in fixed syn and anti conformations, and the introduction of appropriate corrections for the conformation of the exocyclic chain of the sugar moiety, it is possible to evaluate quantitatively the relative populations of the syn and anti conformers from the experimentally observed chemical shifts of H(2') and C(2'). The resulting agreement between the data based on H(2') chemical shifts with those deduced from C(2') chemical shifts extends the validity of this procedure, and furnishes more accurate results than those previously based on uncorrected H(2') chemical shifts alone. The overall findings are briefly compared with those derived from measurements of proton relaxation times and the Overhauser effect, as well as by X-ray diffraction in the solid state. Attention is drawn to the potential utility of the results, including chemical shift data, in studies on interactions of nucleosides and nucleotides with appropriate enzyme systems.

ABSTRACT Self-association of a series of newly synthesized dinucleotide mRNA cap-analogues has be... more ABSTRACT Self-association of a series of newly synthesized dinucleotide mRNA cap-analogues has been investigated in aqueous buffers by means of H-1 NMR and fluorescence, and compared with that for a regular cap, 7-methylguanosine(5')ppp(5')guanosine (m(7)GpppG). The previously investigated inter-molecular association between the tripeptide, Trp-Leu-Glu, and cap analogues (Biochim. Biophys. Acta 1293, 97 (1996)) has been extended to include m(7)GpppC and m(7)Gpppm(7)G. More detailed insight into the stacking interactions was attempted by means of molecular dynamics simulations.

Molecules

Etheno-derivatives of 2-aminopurine, 2-aminopurine riboside, and 7-deazaadenosine (tubercidine) w... more Etheno-derivatives of 2-aminopurine, 2-aminopurine riboside, and 7-deazaadenosine (tubercidine) were prepared and purified using standard methods. 2-Aminopurine reacted with aqueous chloroacetaldehyde to give two products, both exhibiting substrate activity towards bacterial (E. coli) purine-nucleoside phosphorylase (PNP) in the reverse (synthetic) pathway. The major product of the chemical synthesis, identified as 1,N2-etheno-2-aminopurine, reacted slowly, while the second, minor, but highly fluorescent product, reacted rapidly. NMR analysis allowed identification of the minor product as N2,3-etheno-2-aminopurine, and its ribosylation product as N2,3-etheno-2-aminopurine-N2-β-d-riboside. Ribosylation of 1,N2-etheno-2-aminopurine led to analogous N2-β-d-riboside of this base. Both enzymatically produced ribosides were readily phosphorolysed by bacterial PNP to the respective bases. The reaction of 2-aminopurine-N9-β -d-riboside with chloroacetaldehyde gave one major product, clearly...

Zeitschrift für Naturforschung C

The 3′:5′-cyclic phosphates of 2′,3′-secoadenosine and guanosine, structural analogues of cAMP an... more The 3′:5′-cyclic phosphates of 2′,3′-secoadenosine and guanosine, structural analogues of cAMP and cGMP, were synthesized by cyclization of the 5′-phosphates of 2′,3′-secoadenosine and guanosine, respectively. The 2′,3′-seco-3′:5′-cAMP was converted to the IM P analogue by nitrous acid deamination, and to the 8-bromo analogue by bromination. Chemical phosphorylation of 2′,3′-secoadenosine gave four products, the major one of which, in 50% yield, was 2′,3′-seco-3′:5′-cAMP, identical to that obtained by the cyclization reaction above. The three other products have been tentatively identified. The conformations in solution of the seco nucleosides, their 5′-monophosphates, and their 3′:5′-cyclic phosphates, were determined with the aid of 1H , 13C and 31P NMR spectroscopy, particular attention being devoted to orientations about the C-O bonds and the glycosidic bond, and the results compared with crystallographic data available for the 2′,3′-seco congener of ribofuranosyl benzimidazole....

Collection of Czechoslovak Chemical Communications, 1993

Http Dx Doi Org 10 1081 Ncn 120023120, Aug 31, 2006

Canadian Journal of Chemistry, 1985

Methods in Enzymology, Feb 1, 2007

Synthetic capped RNA transcripts produced by in vitro transcription in the presence of m(7)Gp(3)G... more Synthetic capped RNA transcripts produced by in vitro transcription in the presence of m(7)Gp(3)G have found a wide application in studying such processes as mRNA translation, pre-mRNA splicing, mRNA turnover, and intracellular transport of mRNA and snRNA. However, because of the presence of a 3'-OH on both m(7)Guo and Guo moieties of the cap structure, one-third to one-half of the mRNAs contain a cap incorporated in the reverse orientation. The reverse cap structures bind poorly to eIF4E, the cap binding protein, and reduce overall translational efficiency. We therefore replaced the conventional m(7)Gp(3)G cap by "anti-reverse" cap analogs (ARCAs) in which the 3'-OH of m(7)Guo moiety was substituted by 3'-deoxy or 3'-O-methyl groups, leading to m(7)3'dGp(3)G or m(2)(7,3'-O) Gp(3)G, respectively. The class of ARCAs was extended to analogs possessing an O-methyl group or deoxy group at C2' of m(7)Guo. We have also developed a series of ARCAs containing tetra- and pentaphosphates. mRNAs capped with various ARCAs were translated 1.1- to 2.6-fold more efficiently than their counterparts capped with m(7)Gp(3)G in both in vitro and in vivo systems. In a separate series, a methylene group was introduced between the alpha- and beta-, or beta- and gamma-phosphate moieties, leading to m(2)(7,3'-O)Gpp(CH2)pG and m(2)(7,3'-O)Gp(CH2)ppG. These analogs are resistant to cleavage by the decapping enzymes Dcp1/Dcp2 and DcpS, respectively. mRNA transcripts capped with m(2)(7,3'-O)Gpp(CH2)pG were more stable when introduced into cultured mammalian cells. In this chapter, we describe the synthesis of representative ARCAs and their biophysical and biochemical characterization, with emphasis on practical applications in mRNA translation.

Nucleos Nucleot Nucleic Acids, 2003

A series of new mRNA anti reverse cap analogues (ARCA) was designed to obtain a tool for studying... more A series of new mRNA anti reverse cap analogues (ARCA) was designed to obtain a tool for studying the mechanism of protein translation. Dinucleoside P 1 , P 3-triP P 1 , P 4-tetra-and P 1 , P 5-pentaphosphates, linked by a 5 0-to-5 0 phosphate bridge and composed of modified 7-methylguanosine and guanosine, have been synthesized. The hydroxyl group (2 0 OH or 3 0 OH) in 7-metylguanosine moiety was replaced by-OCH 3 or-H in order to obtain the cap analogues capable to be correctly incorporated into synthetic mRNA transcripts. Tri-, tetra-, and pentaphosphates were prepared by ZnCl 2 catalyzed condensation in DMF of derivatives of the 7-methylguanosine diphosphates with the guanosine mono-, di-and triphosphate P-imidazolides, respectively. The structures of the novel compounds were established by means of 1 H and 31 P NMR spectra.

Org. Biomol. Chem., 2016

We describe acetylpyrene-labeled mRNA cap analogues with untypical fluorescent properties as prob... more We describe acetylpyrene-labeled mRNA cap analogues with untypical fluorescent properties as probes for inhibitor screening of DcpS.

Biochemistry Usa, Jul 1, 1984

European Journal of Biochemistry, Dec 1, 1987

Two 19-base-pair oligodeoxynucleotides, analogues of one of the operators which specifically bind... more Two 19-base-pair oligodeoxynucleotides, analogues of one of the operators which specifically bind the repressor protein in the regulatory part of the transposon Tn10 tetracycline-resistance (tet) determinant, have been studied by 1H-NMR spectroscopy. The analogues contain a mismatch in the central base pair of the double helix (T.T or A.A). The imino protons have been assigned to the base pairs by one-dimensional NOE measurements, and the thermally induced transition from the duplex to the single strand has been followed. The cytidine amino resonances have been assigned by means of two-dimensional NOE spectroscopy in H2O. Two-dimensional phase-sensitive NOE and magnitude-correlated spectra have been recorded in 2H2O; all nonexchangeable protons, with the exception of some of H5', H5" protons, have been assigned. The NMR data made it possible to carry out a qualitative analysis of the structures of both oligodeoxynucleotides. The general structures close to B-DNA, show irregularities in the mismatch areas.

Nucleosides Nucleotides and Nucleic Acids, Aug 31, 2006

A series of new mRNA anti reverse cap analogues (ARCA) was designed to obtain a tool for studying... more A series of new mRNA anti reverse cap analogues (ARCA) was designed to obtain a tool for studying the mechanism of protein translation. Dinucleoside P 1 , P 3-triP P 1 , P 4-tetra-and P 1 , P 5-pentaphosphates, linked by a 5 0-to-5 0 phosphate bridge and composed of modified 7-methylguanosine and guanosine, have been synthesized. The hydroxyl group (2 0 OH or 3 0 OH) in 7-metylguanosine moiety was replaced by-OCH 3 or-H in order to obtain the cap analogues capable to be correctly incorporated into synthetic mRNA transcripts. Tri-, tetra-, and pentaphosphates were prepared by ZnCl 2 catalyzed condensation in DMF of derivatives of the 7-methylguanosine diphosphates with the guanosine mono-, di-and triphosphate P-imidazolides, respectively. The structures of the novel compounds were established by means of 1 H and 31 P NMR spectra.

The Journal of Physical Chemistry B, Jun 22, 2011

Molecular mechanisms underlying the recognition of the mRNA 5&amp... more Molecular mechanisms underlying the recognition of the mRNA 5' terminal structure called "cap" by the eukaryotic initiation factor 4E (eIF4E) are crucial for cap-dependent translation. To gain a deeper insight into how the yeast eIF4E interacts with the cap structure, isothermal titration calorimetry and the van't Hoff analysis based on intrinsic protein fluorescence quenching upon titration with a series of chemical cap analogs were performed, providing a consistent thermodynamic description of the binding process in solution. Equilibrium association constants together with thermodynamic parameters revealed similarities and differences between yeast and mammalian eIF4Es. The yeast eIF4E complex formation was enthalpy-driven and entropy-opposed for each cap analog at 293 K. A nontrivial isothermal enthalpy–entropy compensation was found, described by a compensation temperature, T(c) = 411 ± 18 K. For a low affinity analog, 7-methylguanosine monophosphate, a heat capacity change was detected, ΔC(p)° = +5.2 ± 1.3 kJ·mol(-1)·K(-1). The charge-related interactions involving the 5′-5′ triphosphate bridge of the cap and basic amino acid side chains at the yeast eIF4E cap-binding site were significantly weaker (by ΔΔH°(vH) of about +10 kJ·mol(-1)) than those for the mammalian homologues, suggesting their optimization during the evolution.

Http Dx Doi Org 10 1081 Ncn 200060103, Nov 15, 2011

Chemical synthesis of a series of novel dinucleoside cap analogues, m7GpppN, where N is formycin ... more Chemical synthesis of a series of novel dinucleoside cap analogues, m7GpppN, where N is formycin A, 3'-O-methylguanosine, 9-beta-D-arabinofuranosyladenine, and isoguanosine, has been performed using our new methodology. The key reactions of pyrophosphate bonds formation were achieved in anhydrous dimethylformamide solutions employing the catalytic properties of zinc salts. Structures of the new cap analogues were confirmed by 1H NMR and 31p NMR spectra. The binding affinity of the new cap analogues for murine eIF4E(28-217) were determined spectroscopically showing the highest association constant for the analogue that contains formycin A.

Nucleosides and Nucleotides, 1999

Interactions of mammalian (rat hepatoma) thymidylate synthase with FdUMP and its 4-thio analogue ... more Interactions of mammalian (rat hepatoma) thymidylate synthase with FdUMP and its 4-thio analogue were studied in solution with the use of H and F NMR. The results pointed to formation of ternary covalent complexes of TS with either nucleotide in the presence of N, N-methylenetetrahydrofolate and only noncovalent binary complexes in the absence of the cofactor.

RNA (New York, N.Y.), 2001

The ability to synthesize capped RNA transcripts in vitro using bacteriophage polymerases has bee... more The ability to synthesize capped RNA transcripts in vitro using bacteriophage polymerases has been of considerable value in a variety of applications. However, Pasquinelli et al. [RNA (1995) 1:957-967] found that one-third to one-half of the caps are incorporated in the reverse orientation, that is, with the m7G moiety of m7GpppG linked by a 3'-5' phosphodiester bond to the first nucleotide residue of the RNA chain. Such reverse caps are unlikely to be recognized by eIF4E, based on previous studies, and thus complicate any comparison of the translational efficiencies of in vitro-synthesized mRNAs. We therefore designed two novel cap analogs, P(1)-3'-deoxy-7-methyguanosine-5' P3-guanosine-5' triphosphate and P(1)-3'-O,7-dimethylguanosine-5' P3-guanosine-5' triphosphate, that are, theoretically, incapable of being incorporated in the reverse orientation. The key reactions of pyrophosphate bond formation were achieved in anhydrous dimethylformamide solut...

Acta biochimica Polonica, 2003

Description of the recognition specificity between proteins and nucleic acids at the level of mol... more Description of the recognition specificity between proteins and nucleic acids at the level of molecular interactions is one of the most challenging tasks in biophysics. It is key to understanding the course and control of gene expression and to the application of the thus acquired knowledge in chemotherapy. This review presents experimental results of thermodynamic studies and a discussion of the role of thermodynamics in formation and stability of functional protein-RNA complexes, with a special attention to the interactions involving mRNA 5' cap and cap-binding proteins in the initiation of protein biosynthesis in the eukaryotic cell. A theoretical framework for analysis of the thermodynamic parameters of protein-nucleic acid association is also briefly surveyed. Overshadowed by more spectacular achievements in structural studies, the thermodynamic investigations are of equal importance for full comprehension of biopolymers' activity in a quantitative way. In this regard, ...

European Journal of Biochemistry, 1984

Analyses of 1H and 13C NMR spectra have been utilized to extend studies on the dynamic equilibriu... more Analyses of 1H and 13C NMR spectra have been utilized to extend studies on the dynamic equilibrium syn-anti about the glycosidic bond of purine nucleosides and nucleotides. With the aid of chemically synthesized model analogous in fixed syn and anti conformations, and the introduction of appropriate corrections for the conformation of the exocyclic chain of the sugar moiety, it is possible to evaluate quantitatively the relative populations of the syn and anti conformers from the experimentally observed chemical shifts of H(2') and C(2'). The resulting agreement between the data based on H(2') chemical shifts with those deduced from C(2') chemical shifts extends the validity of this procedure, and furnishes more accurate results than those previously based on uncorrected H(2') chemical shifts alone. The overall findings are briefly compared with those derived from measurements of proton relaxation times and the Overhauser effect, as well as by X-ray diffraction in the solid state. Attention is drawn to the potential utility of the results, including chemical shift data, in studies on interactions of nucleosides and nucleotides with appropriate enzyme systems.