Abstract
Homogeneous preparations of milligram quantities of RNA are a prerequisite for their characterization by biophysical methods such as crystallography or NMR spectroscopy. Methods for obtaining milligram quantities of pure synthetic RNA are described in this paper. These methods employ anion exchange HPLC for purifying full-length sequence from failure sequences and incompletely deprotected material. RNA molecules with little or extensive amounts of secondary structure could be purified. In cases where the RNA molecule was tightly folded, the cation in the eluent buffer influenced both the distinction of the peaks during chromatography and the final folded conformation. Finally, two RNA sequences were chemically synthesized, deprotected, purified, and crystallized using this methodology.
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- Chou S. H., Flynn P., Reid B. Solid-phase synthesis and high-resolution NMR studies of two synthetic double-helical RNA dodecamers: r(CGCGAAUUCGCG) and r(CGCGUAUACGCG). Biochemistry. 1989 Mar 21;28(6):2422–2435. doi: 10.1021/bi00432a013. [DOI] [PubMed] [Google Scholar]
- Khare D., Orban J. Synthesis of backbone deuterium labelled [r(CGCGAAUUCGCG)]2 and HPLC purification of synthetic RNA. Nucleic Acids Res. 1992 Oct 11;20(19):5131–5136. doi: 10.1093/nar/20.19.5131. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Puglisi J. D., Tinoco I., Jr Absorbance melting curves of RNA. Methods Enzymol. 1989;180:304–325. doi: 10.1016/0076-6879(89)80108-9. [DOI] [PubMed] [Google Scholar]
- Sassanfar M., Szostak J. W. An RNA motif that binds ATP. Nature. 1993 Aug 5;364(6437):550–553. doi: 10.1038/364550a0. [DOI] [PubMed] [Google Scholar]
- Scaringe S. A., Francklyn C., Usman N. Chemical synthesis of biologically active oligoribonucleotides using beta-cyanoethyl protected ribonucleoside phosphoramidites. Nucleic Acids Res. 1990 Sep 25;18(18):5433–5441. doi: 10.1093/nar/18.18.5433. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Uhlenbeck O. C. A small catalytic oligoribonucleotide. Nature. 1987 Aug 13;328(6131):596–600. doi: 10.1038/328596a0. [DOI] [PubMed] [Google Scholar]
- Usman N., Cedergren R. Exploiting the chemical synthesis of RNA. Trends Biochem Sci. 1992 Sep;17(9):334–339. doi: 10.1016/0968-0004(92)90306-t. [DOI] [PubMed] [Google Scholar]
- van Belkum A., Abrahams J. P., Pleij C. W., Bosch L. Five pseudoknots are present at the 204 nucleotides long 3' noncoding region of tobacco mosaic virus RNA. Nucleic Acids Res. 1985 Nov 11;13(21):7673–7686. doi: 10.1093/nar/13.21.7673. [DOI] [PMC free article] [PubMed] [Google Scholar]