Controlling gene expression in living cells through small molecule-RNA interactions

Science

Volumn 282, Issue 5387, 1998, Pages 296-298

  Werstuck, Geoffrey ^a,b   Green, Michael R ^a  

^a UNIVERSITY OF MASSACHUSETTS MEDICAL SCHOOL   (United States)

^b MBI Fermentas Inc   (Canada)

Author keywords

[No Author keywords available]

Indexed keywords

KANAMYCIN A; RNA; TOBRAMYCIN;

ARTICLE; GENE EXPRESSION; GENE EXPRESSION REGULATION; LIGAND BINDING; MOLECULAR INTERACTION; NONHUMAN; PRIORITY JOURNAL; RNA BINDING;

ANIMALS; ANTI-BACTERIAL AGENTS; BASE SEQUENCE; BENZIMIDAZOLES; BISBENZIMIDE; CHO CELLS; CRICETINAE; DRUG RESISTANCE, MICROBIAL; ESCHERICHIA COLI; GENE EXPRESSION REGULATION; KANAMYCIN; LIGANDS; MOLECULAR SEQUENCE DATA; PROTEIN BIOSYNTHESIS; RNA; RNA, MESSENGER; TOBRAMYCIN; TRANSFECTION;

MAMMALIA;

EID: 0032500736     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.282.5387.296     Document Type: Article

References (28)

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10. 2), loaded onto a preselection column (0.25 ml of glycine-Sepharose), eluted with two column volumes of selection buffer, and immediately loaded onto a 0.5-ml aminoglycoside-Sepharose column. Columns were washed with 10 column volumes of selection buffer (rounds 1 to 5), 10 column volumes of buffer with 5 mM competitor aminoglycoside (rounds 6 to 9), or 10 column volumes of buffer with 10 mM competitor (rounds 10 to 14). The competitor aminoglycoside for tobramycin aptamer selection was kanamycin A and vice versa. Bound RNA was eluted with the cognate aminoglycoside (5 mM) and amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) using flanking primers. The PCR products were transcribed into RNA with T7 RNA polymerase and purified by polyacrylamide gel electrophoresis. Pools were subcloned into the plasmid pBluescript (Stratagene) and sequenced after rounds 10, 12, and 14.
11. One or three copies of the kanamycin A (kan) or the tobramycin (tob) aptamer were cloned into the Nde I site (one copy) or Nde I and Bsa I sites (three copies) of T7 RNA polymerase-driven expression vector pRSETA (Invitrogen) and transformed into a bacterial strain containing an isopropyl β-D-thiogalactopyranoside (IPTG)-inducible T7 RNA polymerase. Bacterial strains were grown in liquid culture overnight, induced with 0.1 mM IPTG for 1 hour, and then diluted into medium containing antibiotic and 0.1 mM IPTG.
12. The partial resistance of bl-kan1 to tobramycin is consistent with the fact that the kan aptamer binds both drugs, whereas the tob aptamer is more selective and binds only tobramycin.
13. None of the strains exhibited increased resistance to the unrelated antibiotics tetracycline and gentamicin.
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23. 2. In selection rounds 1 to 6, columns were washed with 20 column volumes of selection buffer and eluted with 2 column volumes of 10 mM H33258. In selection rounds 7 to 10, columns were washed with 20 column volumes of buffer and 20 column volumes of 10 mM benzimidazolepropionic acid (in selection buffer) before elution.
24. H10 and H19 bound H33258 and the closely related H33342 comparably.
25. CHO cells (80% confluent) were pretreated with 0, 5, or 10 mM H33342 and then cotransfected (Lipofectamine, Gibco-BRL) with 1 μg of pSVβgal or pH2βgal and 1 μg of the luciferase reporter gene pGL3 (Promega). Two hours after transfection H33342 was reapplied to the cells. Twenty-four hours after transfection cells were harvested and cell extracts were prepared. Cell extracts were normalized for total protein (Bradford assay). β-Galactosidase and luciferase activities in the extracts were determined relative to standard curves generated with the purified β-galactosidase and luciferase enzymes (Promega).
26. In these experiments, H33342 was used instead of H33258 because it is about 10 times more cell-permeable (14).
27. The parental expression vector SVβGal was also not inhibited by 5 or 10 μM H33342.
28. We are grateful to R. Singh for advice and for providing the randomized RNA pool and to M. Zapp for helpful discussions. This work was supported by an NIH grant to M.R.G. M.R.G. is an investigator of the Howard Hughes Medical Institute.