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Tet Off?

03/21/2014
Kristie Nybo, PhD

Could a new method enabling dose-dependent inhibition of gene expression by simply inserting DNA sequences on either side of a gene replace the Tet on/off system? Find out...


For the first time, researchers have employed aptazyme riboswitches, small ligand-dependent self-cleaving segments of RNA, to conditionally knockdown viral gene expression (1). The system is simple, efficient, and potentially more flexible than other available approaches, such as the Tet on/off system, for altering gene expression in cells.

Schematic diagram of viral gene expression shut down using an Off-switch aptazyme (1).

Dirk Nettelbeck from the German Cancer Research Center (Heidelberg) was searching for a way to improve the safety of oncolytic viruses, which selectively infect and lyse tumor cells, when he attended a talk on riboswitches given by Patrick Ketzer, then a master’s student at Universit├Ąt Konstanz.

“I was immediately excited because I thought that would be a very interesting tool for our purposes. At that time, we had found that promoter control really didn’t work in our viruses,” Nettelbeck explained. He recruited Ketzer to his lab, and together they designed a riboswitch strategy for controlling viral gene expression to improve therapeutic control of oncolytic viruses.

Nettelbeck’s method uses aptazymes, a type of riboswitch where an aptamer domain binds a ligand, resulting in self-cleavage of a ribozyme domain. The researchers inserted sequences encoding aptazymes on either side of a viral gene. In the absence of a ligand, the short sequence did not interfere with transcription of the gene, but when ligand was added, the aptazyme cleaved itself, cutting the gene from the viral genome and eliminating expression.

“If you think about the potential of the system, the first thing to consider is a replacement for the Tet system,” Nettelbeck said. “This [method] is much less complex. In the Tet system, you need the promoter; you need to express a recombinant transcription factor that responds to the ligand and needs to bind to the promoter, whereas with the riboswitch, you just need to insert 100 base pairs into the mRNA.”

Nettlebeck’s team showed that the new approach effectively knocked down gene expression and viral replication in adenovirus, a dsDNA virus with nuclear replication, and also in measles virus, which carries a single-stranded RNA genome and replicates in the cytoplasm. And since the aptamer domains can be selected to bind to any type of ligand, this approach offers far more flexibility than the Tet system, which relies only on tetracycline antibiotics.

Nettelbeck was quick to note though that this study presents only a proof of principle. RNA switches have rarely been used in eukaryotic systems, so improving regulation and broadening the range of ligand recognition are important next steps. “There are many advantages in the system, but realizing them will all depend on the opportunity and the technology to really find more versatile switches.”

Reference

(1) Ketzer P, Kaufmann JK, Engelhardt S, Bossow S, von Kalle C, Hartig JS, Ungerechts G, Nettelbeck DM. Artificial riboswitches for gene expression and replication control of DNA and RNA viruses. Proc Natl Acad Sci U S A. 2014 Feb 4;111(5):E554-62.