Synthesis, deprotection, analysis and purification of RNA and ribozymes.
Improvements in the synthesis, deprotection and purification of oligoribonucleotides are described. These advances allow for reduced synthesis and deprotection times, while improving product yield. Coupling times are reduced by half using 5-ethylthio-1H-tetrazole (S-ethyltetrazole) as the activator. Base and 2'-O-t-butyldimethylsilyl deprotection with methylamine (MA) and anhydrous triethylamine/hydrogen fluoride in N-methylpyrrolidinone (TEA.HF/NMP), respectively, requires a fraction of the time necessitated by current standard methods. In addition, the ease of oligoribonucleotide purification and analysis have been significantly enhanced using anion exchange chromatography. These new methods improve the yield and quality of the oligoribonucleotides synthesized. Hammerhead ribozymes synthesized utilizing the described methods exhibited no diminution in catalytic activity.
Wincott
F
F
Department of Chemistry and Biochemistry, Ribozyme Pharmaceuticals Inc., Boulder, CO 80301, USA.
DiRenzo
A
A
Shaffer
C
C
Grimm
S
S
Tracz
D
D
Workman
C
C
Sweedler
D
D
Gonzalez
C
C
Scaringe
S
S
Usman
N
N
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D005021:Ethylamines; D006858:Hydrofluoric Acid; D007202:Indicators and Reagents; D008722:Methods; D008744:Methylamines; D008969:Molecular Sequence Data; D011760:Pyrrolidinones; D012313:RNA / Q000032:analysis / Q000138:chemical synthesis* / Q000302:isolation & purification; D016337:RNA, Catalytic / Q000032:analysis / Q000138:chemical synthesis* / Q000302:isolation & purification; D013777:Tetrazoles
D005021:Ethylamines; D007202:Indicators and Reagents; D008744:Methylamines; D011760:Pyrrolidinones; D016337:RNA, Catalytic; D013777:Tetrazoles; D012313:RNA; C027451:methylamine; D006858:Hydrofluoric Acid; C016162:triethylamine