Optimization of the hybridization-based method for purification of thermostable tRNAs in the presence of tetraalkylammonium salts.
We found that both tetramethylammonium chloride (TMA-Cl) and tetra-ethylammonium chloride (TEA-Cl), which are used as monovalent cations for northern hybridization, drastically destabilized the tertiary structures of tRNAs and enhanced the formation of tRNA*oligoDNA hybrids. These effects are of great advantage for the hybridization-based method for purification of specific tRNAs from unfractionated tRNA mixtures through the use of an immobilized oligoDNA complementary to the target tRNA. Replacement of NaCl by TMA-Cl or TEA-Cl in the hybridization buffer greatly improved the recovery of a specific tRNA, even from unfractionated tRNAs derived from a thermophile. Since TEA-Cl destabilized tRNAs more strongly than TMA-Cl, it was necessary to lower the hybridization temperature at the sacrifice of the purity of the recovered tRNA when using TEA-Cl. Therefore, we propose two alternative protocols, depending on the desired properties of the tRNA to be purified. When the total recovery of the tRNA is important, hybridization should be carried out in the presence of TEA-Cl. However, if the purity of the recovered tRNA is important, TMA-Cl should be used for the hybridization. In principle, this procedure for tRNA purification should be applicable to any small-size RNA whose gene sequence is already known.
Yokogawa
Takashi
T
Department of Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan. don@gifu-u.ac.jp
Kitamura
Yusuke
Y
Nakamura
Daigo
D
Ohno
Satoshi
S
Nishikawa
Kazuya
K
D002021:Buffers; D002414:Cations, Monovalent / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D000644:Quaternary Ammonium Compounds / Q000737:chemistry*; D020871:RNA Stability; D012343:RNA, Transfer / Q000737:chemistry / Q000302:isolation & purification*; D012358:RNA, Transfer, Met / Q000737:chemistry; D012360:RNA, Transfer, Phe / Q000737:chemistry; D013696:Temperature; D019789:Tetraethylammonium / Q000737:chemistry*
D002021:Buffers; D002414:Cations, Monovalent; D009838:Oligodeoxyribonucleotides; D000644:Quaternary Ammonium Compounds; D012358:RNA, Transfer, Met; D012360:RNA, Transfer, Phe; D019789:Tetraethylammonium; D012343:RNA, Transfer; C027917:tetramethylammonium