Catherine Goh – University of Copenhagen

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RNAtrain > Fellows > Catherine Goh

Catherine Goh (ER1)


Nationality
:
Singaporean
 

Main Host Institution: Intana Bioscience GmbH/siTOOLs Biotech GmbH

Academic Background: BSc, MSc (Pharmacology), PhD


Project title: Development of the siPOOL technology towards ncRNAs involved in human disease

Project background: siRNA or silencing RNA, discovered in the 1990s by Mello and Fire, has been used extensively by researchers to silence gene expression in various cellular and animal models. siRNAs are short (21 nucleotides) double-stranded RNA that associate with complementary RNA sequences in the cell and target them towards a protein complex where they undergo degradation. When RNAs are degraded, they are no longer able to perform their function or be translated into proteins. This process is called RNA interference and is used as a tool to examine the function of certain RNAs and their corresponding genes.

Being easy to apply to cells without any need for genome editing, siRNAs provide a simple, fast and transient way to regulate gene expression. However, a major problem associated with their use is the numerous off-target effects. These stem from the ability of siRNAs to degrade RNAs based on a “seed region” or recognition site requiring as little as 6 bases of sequence complementarity between siRNA and target sequence (Birmingham et al., 2006), which may be found on many RNAs. Furthermore, siRNAs are indistinguishable from endogenous microRNAs which do not need perfectly matched seed regions to carry out RNA degradation or translational inhibition.

siPOOLs were therefore developed by siTOOLs Biotech as an advanced RNA interference tool. siPOOLs consist of 30 selected siRNAs which share a common gene target but contain different seed regions. As a result, the desired target gene is efficiently silenced whilst off-target effects are diluted due to the very low concentration of the individual siRNAs.

Our work focusses on improving siPOOL production to reduce cost of synthesis and thus enable the generation of large-scale siPOOL libraries for functional screening and validation. We also aim to further enhance the silencing potency of siPOOLs targeting non-coding RNA i.e. RNAs that are not translated into proteins. ncRNAs comprise of small ncRNAs (microRNAs, small nucleolar RNAs etc.) and long ncRNAs (lncRNA). The latter of which is a new, rapidly evolving field of science that may reveal exciting novel functions of RNA. These lncRNAs can prove challenging to “silence” because they can fold into structures or bind to molecules that prevent siRNA access. Some lncRNAs are also enriched in the nucleus, where they may be protected from the mostly cytoplasmic RNAi machinery. In spite of these hurdles, highly efficient siPOOLs have been designed for both cytoplasmic and nuclear lncRNAs and efforts will be undertaken during this project to further improve their efficacy.

Project Aims:  Improve siPOOL production towards higher throughput and reduced cost, allowing large scale siPOOL library synthesis; Optimize siPOOL design  for efficient silencing of lncRNAs.

Expected outcome: Enable robust and efficient RNAi based gene knockdown with minimal off-target effects to delineate the functions of non-coding RNA.

Reference: A. Birmingham, E.M. Anderson, 3'-UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nat. Met. 3(3) 199-204 (2006).