Preparation of ribonuclease S domain-swapped dimers conjugated with DNA and PNA: modulating the activity of ribonucleases.
Obtaining highly specific and active ribonuclease activities is an important goal with numerous medical and biochemical applications. As a step toward more active and specific ribonucleases, we describe the preparation and the enzymatic and structural properties of RNase S monomers and dimers conjugated to DNA and PNA molecules. Poly(dT)n (2'-oligodeoxyribonucleotides, n = 8, 15) and t8 peptide nucleic acid (PNA) chains have been conjugated to the S-peptide of ribonuclease S. Monomers and dimers of the conjugated enzyme have been obtained and characterized by 1H NMR spectroscopy, showing that DNA or PNA conjugation does not alter the native structure of ribonuclease S. The oligonucleotide-conjugated RNase S monomer and dimer show significant activity against single-stranded RNA and very low/negligible hydrolysis of double-stranded poly(A).poly(U). In contrast, the t8-conjugated RNase S monomer and dimer show substantial activity against both ssRNA and dsRNA. These results highlight the importance of positive charges near but not in the active site in enhancing activity against dsRNA and reveal the promise of PNA-RNase conjugates for modulating RNase activity.
Pulido
Daniel
D
Department de Química Orgànica, Institut de Biomedicina de la Universitat de Barcelona, Barcelona, Spain.
López-Alonso
Jorge Pedro
JP
Marchán
Vicente
V
González
Carlos
C
Grandas
Anna
A
Laurents
Douglas V
DV
D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D019281:Dimerization; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D020135:Peptide Nucleic Acids / Q000737:chemistry*; D012313:RNA / Q000235:genetics / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry* / Q000378:metabolism*
D020135:Peptide Nucleic Acids; D012313:RNA; D004247:DNA; D012260:Ribonucleases; C022542:ribonuclease S