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Using capillary electrophoresis to study methylation effect on RNA-peptide interaction

100%

Mucha P., Szyk A., Rekowski P., Agris P. F.

Acta Biochimica Polonica

2003

tom 50

nr 3

Methylation of RNA and proteins is one of a broad spectrum of post-trans- criptional/translational mechanisms of gene expression regulation. Its functional signification is only beginning to be understood. A sensitive capillary electrophoresis mobility shift assay (CEMSA) for qualitative study of the methylation effect on biomolecules interaction is presented. Two RNA-peptide systems were chosen for the study. The first one consists of a 17-nucleotide analogue (+27-+43) of the yeast tRNA Phe anticodon stem and loop domain (ASL Phe) containing three of the five naturally occurring modifications (2'-O-methylcytidine (Cm32), 2'-O-methylguanine (Gm34) and 5-methylcytidine (m5 C40)) (ASL Phe -Cm32,Gm34,m5 C40) and a 15-amino-acid peptide (named tF 2: Ser1 -Ile-Ser-Pro-Trp5 -Gly-Phe-Ser-Gly-Leu10 -Leu- Arg-Trp-Ser-Tyr15 ) selected from a random phage display library (RPL). A pep- tide-concentration-dependent formation of an RNA-peptide complex was clearly observable by CEMSA. In the presence of the peptide the capillary electrophoresis (CE) peak for triply methylated ASL Phe shifted from 18.16 to 20.90 min. Formation of the complex was not observed when an unmethylated version of ASL Phe was used. The second system studied consisted of the (+18)-(+44) fragment of the trans-activation response element of human immunodeficiency virus type 1 (TAR RNA HIV-1) and a 9-amino-acid peptide of the trans-activator of transcription protein (Tat HIV-1) Tat(49–57)-NH2 (named Tat1: Arg49-Lys-Lys-Arg52-Arg-Gln-Arg-Arg- Arg57-NH2). In the presence of Tat(49–57)-NH2 a significant shift of migration time of TAR from 18.66 min to 20.12 min was observed. Methylation of a residue Arg52->Arg(Me)2, crucial for TAR binding, strongly disrupted formation of the complex. Only at a high micromolar peptide concentration a poorly shaped, broad peak of the complex was observed. CE was found to be an efficient and sensitive method for the analysis of methylation effects on interaction of biomolecules.

Modified RNAs as potential drug targets

88%

Guenther R., Forrest B., Newman W., Malkiewicz A., Agris P. F.

Acta Biochimica Polonica

1998

tom 45

nr 1

Bleomycin (BLM) is a natural antibiotic that is effective in treatment of selected cancers. Although the exact therapeutic mechanism of bleomycin is not known, its target is thought to be a nucleic acid. Besides cleaving DNA, in vitro, Fe-bleomycin cleaves the anticodon of yeast tRNAPhe specifically. Using CD and fluorescence spectroscopy we have found that apo-bleomycin binds to synthetic RNA analogs of the anticodon of yeast tRNAPhe with an affinity similar to that previously reported for DNA. In order to understand BLM's selectivity, the role magnesium ions play in RNA recognition should be explained. Many RNA substrates for Fe-BLM, including yeast tRNAPhe, are not cleaved by the drug when the Mg2+ concentration exceeds 1 mM. Competition experiments with anticodon analogs provide insight into the role of magnesium ions in RNA recognition by BLM. These simple modified RNAs may be useful as model systems for investigating BLM/RNA recognition and development of highly selective drugs toward RNA targets.

Thermodynamic contribution of nucleoside modifications to yeast tRNAPhe anticodon stem loop analogs

64%

Agris P. F., Guenther R., Sochacka E., Newmann W., Czerwinska G., Liu G., Ye W., Malkiewicz A.

Acta Biochimica Polonica

1999

tom 46

nr 1

The determination of the structural and functional contributions of natural modified nucleosides to tRNA has been limited by lack of an approach that can systematically incorporate the modified units. We have produced a number of oligonucleotide analogs, of the anticodon of yeast tRNAPhe by, combining standard automated synthesis for the major nucleosides with specialty chemistries for the modified nucleosides. In this study, both naturally occurring and unnatural modified nucleotides were placed in native contexts. Each oligonucleotide was purified and the nucleoside composition determined to validate the chemistry. The RNAs were denatured and analyzed to determine the van't Hoff thermodynamic parameters. Here, we report the individual thermodynamic contributions for Cm, Gm, m1G, m5C, Ψ. In addition m5m6U, m1Ψ, and m3Ψ, were introduced to gain additional understanding of the physicochemical contribution of Ψ and m5C at an atomic level. These oligonucleotides demonstrate that modifications have measurable thermodynamic contributions and that loop modifications have global contributions.

Ograniczanie wyników

3 Acta Biochimica Polonica

3 Agris P. F.

2 Guenther R.

2 Malkiewicz A.

1 Czerwinska G.

1 Forrest B.

1 Liu G.

1 Mucha P.

1 Newman W.

1 Newmann W.

1 Rekowski P.

1 Sochacka E.

1 Szyk A.

1 Ye W.

1 2003

1 1999

1 1998

Wyniki wyszukiwania

Using capillary electrophoresis to study methylation effect on RNA-peptide interaction

Modified RNAs as potential drug targets

Thermodynamic contribution of nucleoside modifications to yeast tRNAPhe anticodon stem loop analogs