Dear Visitor, we can set up here your advertisements upon your wish. Please get in touch with us.
  Login:    Sitemap:    Search:

Design and Analyses of Non-coding RNAs - Investigating the Role of Non-coding RNAs in Gene Regulation

The discovery of RNA interfering pathways in mammalians including humans raises many hopes for the deployment of novel and efficient tools for gene therapy of viral infections and other wide-spread diseases including cancer by specific targeting of key for the disease` development mRNAs or viral RNAs trough mircoRNAs and RNA silencing pathways. Despite the results achieved in the expression of shRNAs in mammalian cells and the RNA silencing effect demonstrated there are many side effects that spell significant problems facing the application of RNA interfering technology in pharmaceutical industry.

A major problem for the efficient retroviral RNAs silencing such as HIV, is the fact the viral RNAs tend to mutate very fast under the selection pressure of constantly expressed interfering RNA. As a result of that after certain viral generation the specific interfering RNA is not anymore effective towards the virus because of the specific mutations induced by the interfering RNA. In the case of constantly expressed interfering RNA against specific mRNAs strong silencing effect of many non-target mRNAs are observed. To avoid both problems interfering RNAs like shRNAs should be temporary expressed only when needed. This can be achieved by applying RNA-sensing allosteric ribozymes with YES logic function depicted in Fig. 1. An allosteric ribozyme with YES logic function, designed as already described, is constantly expressed into the cell using viral vectors. In the absence of effector RNA the YES gate folds into an inactive state (Fig. 1a). In this conformation the ribozyme`s self-cleavage is inhibited. An effector RNA binds to the ribozyme binding site and turns the ribozyme into an activate (ON) state (Fig. 1b). As a result of the ribozyme`s self-cleavage short-hairpin (sh) RNA is formed (Fig. 1c). The shRNA is converted into double-stranded(ds) RNA with sticky ends by the Dicer enzyme (Fig. 1d). The dsRNA is single- stranded by the RISC complex and used as a template for short interfering RNA to degrade target mRNA or for microRNA to translational repression (Fig. 1e). When the effector RNA, that can be viral and disease indicative RNA, is not present anymore the ribozymes form inactive state again (Fig. 1a) and the shRNA production is held. The restricted time of shRNA production does not allow the targeted viral RNA to mutate under the selection pressure of the shRNA and to escape the silencing effect. The conditional expression of shRNA can reduce the side effects of RNA interfering technology to a minimum and makes it more practically feasible approach to tackle a variety of disorders like viral infection diseases including ADIS and cancer including lymphomas.

Figure 1. Producing short-hairpin RNAs by RNA-sensing allosteric ribozymes with YES logic function for engineering siRNA and microRNA functions. (a) In the absence of effector RNA the YES gate is designed to fold into an inactive structure where stem IV is formed instead of stem III. (b) The YES switch binds the effector RNA and folds into an active state in which stem III is formed. (c) As a result the ribozyme undergoes self-cleavage and shRNA is produced. (d) Dicer converts shRNA into dsRNA with sticky ends. (e) In the presence of RISC complex the dsRNA is single-stranded and used for gene repression either by RNA silencing via decay of mRNAs or by microRNA function via translational suppression.

1. Robert Penchovsky - Quality Assurance in Healthcare Service Delivery, Nursing and Personalized Medicine: Technologies and Processes. Engineering Gene Control Circuits with Allosteric Ribozymes in Human Cells as a Medicine of the Future – 2012, Publisher IGI Global: DOI: 10.4018/978-1-120-7.

2. Robert Penchovsky - Computational Design of Allosteric Ribozymes as Molecular Biosensors –2014, Biotechnology Advances, Q1 (Biochemistry, Genetics and Molecular Biology), IF – 11,866

3. Nikolet Pavlova, Dimitrios Kaloudas, Robert Penchovsky - Riboswitch distribution, structure, and function in bacteria – 2019, Gene, 0378-1119, Q1 (Biochemistry, Genetics and Molecular Biology), IF – 2,5