Trans-acting antigenomic HDV ribozyme for production of in vitro transcripts with homogenous 3' ends.
During in vitro run-off transcription with T7 RNA polymerase, transcripts with heterogenous 3' ends are commonly synthesized. Here, we describe an efficient procedure for correct processing of transcript 3' ends with the use of antigenomic HDV ribozyme. The procedure involves the extension of nascent transcripts with seven nucleotides complementary to the ribozyme's recognition site and, subsequently, the removal of those nucleotides with the HDV ribozyme acting in trans. Sufficient reaction rates and final cleavage extents of approx. 90% can be obtained with just twofold excess of the ribozyme. The highest concentration of RNA substrate suggested for practical applications turns out to be 3 μM. The procedure is an alternative to the use of ribozymes as cis-cleaving autocatalytic cassettes attached to transcript 3' ends.
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.
Milena
M
Szafraniec
Leszek
L
Blaszczyk
Jan
J
Wrzesinski
Jerzy
J
Ciesiolka
D001483:Base Sequence; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D005821:Genetic Techniques; D003698:Hepatitis Delta Virus / Q000201:enzymology*; D021141:Nucleic Acid Amplification Techniques; D059367:RNA Cleavage; D016337:RNA, Catalytic / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D048348:Reverse Transcription
D018076:DNA, Complementary; D016337:RNA, Catalytic; D012333:RNA, Messenger