Nucleic Acids Research Advance Access originally published online on October 25, 2008
Nucleic Acids Research 2008 36(21):6752-6766; doi:10.1093/nar/gkn637
Nucleic Acids Research, 2008, Vol. 36, No. 21 6752-6766
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
RNA |
Hairpin ribozyme-antisense RNA constructs can act as molecular lassos
1SomaGenics, Inc., 2161 Delaware Avenue, Santa Cruz, CA 95060 and 2Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305, USA
*To whom correspondence should be addressed. Tel: +1 831 426 7700 (ext. 12); Fax: +1 831 420 0685; Email: bjohnston{at}somagenics.com
Correspondence may also be addressed to Sergei A. Kazakov. Tel: +1 831 426 7700 (ext. 11); Fax: +1 831 420 0685; Email: skazakov{at}somagenics.com
Received July 15, 2008. Revised September 12, 2008. Accepted September 15, 2008.
We have developed a novel class of antisense agents, RNA Lassos, which are capable of binding to and circularizing around complementary target RNAs. The RNA Lasso consists of a fixed sequence derived from the hairpin ribozyme and an antisense segment whose size and sequence can be varied to base pair with accessible sites in the target RNA. The ribozyme catalyzes self-processing of the 5'- and 3'-ends of a transcribed Lasso precursor and ligates the processed ends to produce a circular RNA. The circular and linear forms of the self-processed Lasso coexist in an equilibrium that is dependent on both the Lasso sequence and the solution conditions. Lassos form strong, noncovalent complexes with linear target RNAs and form true topological linkages with circular targets. Lasso complexes with linear RNA targets were detected by denaturing gel electrophoresis and were found to be more stable than ordinary RNA duplexes. We show that expression of a fusion mRNA consisting of a sequence from the murine tumor necrosis factor-
(TNF-) gene linked to luciferase reporter can be specifically and efficiently blocked by an anti-TNF Lasso. We also show in cell culture experiments that Lassos directed against Fas pre-mRNA were able to induce a change in alternative splicing patterns.
Present addresses: Svetlana V. Balatskaya, Codexis, Inc., 515 Galveston Drive, Redwood City, CA 94063200 Penobscot Dr., Redwood City, CA 94063, USA
Tai-Chih Kuo, Department of Biochemistry, College of Medicine, Taipei Medical University, 250 Wu-Hsing Street, Taipei, Taiwan 110-31
Alexander V. Vlassov, Ambion, an Applied Biosystems Business, 2130 Woodward St, Austin, TX 78744, USA
Roger L. Kaspar, TransDerm, 2161 Delaware Avenue, Santa Cruz, CA 95060, USA
Kevin O. Kisich, National Jewish Medical and Research Center, Department of Pediatrics, 1400 Jackson Street, Denver, CO 80206, USA