Rapid preparation of RNA samples for NMR spectroscopy and X-ray crystallography.

Knowledge of the three-dimensional structures of RNA and its complexes is important for understanding the molecular mechanism of RNA recognition by proteins or ligands. Enzymatic synthesis using T7 bacteriophage RNA polymerase is used to prepare samples for NMR spectroscopy and X-ray crystallography. However, this run-off transcription method results in heterogeneity at the RNA 3-terminus. For structural studies, RNA purification requires a single nucleotide resolution. Usually PAGE purification is used, but it is tedious, time-consuming and cost ineffective. To overcome these problems in high-throughput RNA synthesis, we devised a method of RNA preparation that uses trans-acting DNAzyme and sequence-specific affinity column chromatography. A tag sequence is added at the 3' end of RNA, and the tagged RNA is picked out using an affinity column that contains the complementary DNA sequence. The 3' end tag is then removed by sequence-specific cleavage using trans-acting DNAzyme, the arm lengths of which are optimized for turnover number. This purification method is simpler and faster than the conventional method.

[1]  M. Mörl,et al.  A universal method to produce in vitro transcripts with homogeneous 3' ends. , 2002, Nucleic acids research.

[2]  M. Lai,et al.  Template Requirement and Initiation Site Selection by Hepatitis C Virus Polymerase on a Minimal Viral RNA Template* , 2000, The Journal of Biological Chemistry.

[3]  M. Kainosho,et al.  Structural features of an influenza virus promoter and their implications for viral RNA synthesis , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[4]  G. F. Joyce,et al.  Mechanism and utility of an RNA-cleaving DNA enzyme. , 1998, Biochemistry.

[5]  S R Rasmussen,et al.  Covalent immobilization of DNA onto polystyrene microwells: the molecules are only bound at the 5' end. , 1991, Analytical biochemistry.

[6]  E. Southern,et al.  A simple and cost-effective method for producing small interfering RNAs with high efficacy. , 2003, Nucleic acids research.

[7]  O. Uhlenbeck,et al.  Synthesis of small RNAs using T7 RNA polymerase. , 1989, Methods in enzymology.

[8]  O. Uhlenbeck,et al.  Sequence-dependent structural variations of hammerhead RNA enzymes. , 1990, Nucleic acids research.

[9]  G. F. Joyce,et al.  A general purpose RNA-cleaving DNA enzyme. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[10]  L. Gutshall,et al.  hnRNP C and polypyrimidine tract-binding protein specifically interact with the pyrimidine-rich region within the 3'NTR of the HCV RNA genome. , 1999, Nucleic acids research.

[11]  C. Rice,et al.  Secondary structure determination of the conserved 98-base sequence at the 3' terminus of hepatitis C virus genome RNA , 1997, Journal of virology.

[12]  K. Taira,et al.  Effects of helical structures formed by the binding arms of DNAzymes and their substrates on catalytic activity. , 1998, Nucleic acids research.

[13]  A. Bax,et al.  Resolution enhancement and spectral editing of uniformly 13C-enriched proteins by homonuclear broadband 13C decoupling , 1992 .

[14]  J. Puglisi,et al.  Synthesis and purification of large amounts of RNA oligonucleotides. , 1991, BioTechniques.

[15]  J. Puglisi,et al.  Preparation of isotopically labeled ribonucleotides for multidimensional NMR spectroscopy of RNA. , 1992, Nucleic acids research.

[16]  Wen‐Chien Lee,et al.  Affinity chromatography of DNA on nonporous copolymerized particles of styrene and glycidyl methacrylate with immobilized polynucleotide. , 2003, Analytical biochemistry.

[17]  O. Uhlenbeck,et al.  Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. , 1987, Nucleic acids research.