A Platform for Identifying GlycoRNA and Identifying Biases in RNA Pulldown Experiments
Lead Research Organisation:
University of Cambridge
Department Name: Chemistry
Abstract
Two of the biopolymers that are essential for the existence of life RNA and carbohydrates. RNA, a key player in the "central dogma" of biology, is transcribed from DNA and translated into proteins. However, RNA plays many more roles in biology, for instance as a messenger molecule or as a catalyst for cellular processes. Carbohydrates (sometimes known as "glycans") are traditionally thought of as a source of energy - although they also play many other important roles in regulation and protein folding. Until recently, these two crucial biopolymers were not thought to have shared an interface. New work has challenged this view, pointing toward RNA polymers that are functionalised with glycans. The implications of this new hybrid biopolymer (termed "glycoRNA") are currently unknown, but it could potentially play roles in understanding the fundamental biology of diseased states or in the development of technologies such as vaccines. Here, we propose a new method for studying glycoRNA, by using a strategy of targeting RNA with small molecules. The approach holds numerous advantages - it allows targeting a much larger fraction of the genome than protein-based approaches and holds the promise for being much more selective than DNA-based approaches.
We recently discovered that a commonly-used technique in the field of studying RNA modifications, and indeed in many studies in the general area of chemical biology, may present some previously unknown biases. A molecule called DBCO is often used to enrich target RNA of interest, but we have found that DBCO may preferentially bind to some large RNA transcripts. We are worried this may be affecting the results of researchers around the world and we intend to carefully quantify this interaction and find ways to avoid the bias of future studies.
We also intend to find out which different types of sugars can be found in glycoRNA. Sugars take on a diverse array of structures, from the well-known glucose to other, but related, ones such as galactose and mannose. We believe one of these sugars may be present in particularly high levels in glycoRNA and we plan to determine whether this is the case.
Finally, once we have developed a robust method for identifying the different forms of glycoRNA, we plan to study how prevalent they are in different kinds of environments. These environments could be things such as different cell-types, or even in healthy versus unhealthy cells. If glycoRNA levels are found, for instance, to be present at different levels in unhealthy cells, they could potentially be used a diagnostic tool to identify disease. This approach will facilitate both basic and translational research at the intersection of chemistry, biology and pathology.
We recently discovered that a commonly-used technique in the field of studying RNA modifications, and indeed in many studies in the general area of chemical biology, may present some previously unknown biases. A molecule called DBCO is often used to enrich target RNA of interest, but we have found that DBCO may preferentially bind to some large RNA transcripts. We are worried this may be affecting the results of researchers around the world and we intend to carefully quantify this interaction and find ways to avoid the bias of future studies.
We also intend to find out which different types of sugars can be found in glycoRNA. Sugars take on a diverse array of structures, from the well-known glucose to other, but related, ones such as galactose and mannose. We believe one of these sugars may be present in particularly high levels in glycoRNA and we plan to determine whether this is the case.
Finally, once we have developed a robust method for identifying the different forms of glycoRNA, we plan to study how prevalent they are in different kinds of environments. These environments could be things such as different cell-types, or even in healthy versus unhealthy cells. If glycoRNA levels are found, for instance, to be present at different levels in unhealthy cells, they could potentially be used a diagnostic tool to identify disease. This approach will facilitate both basic and translational research at the intersection of chemistry, biology and pathology.
Technical Summary
The recent discovery of RNA transcripts modified with glycans (glycoRNA) has opened up several research questions. What are the cellular roles of glycoRNA and are they involved in cell signalling? What is the nature of the glycosidic linkage between an RNA nucleobase and carbohydrate? Do glycoRNAs play roles in diseased states that could lead to the development of novel therapeutics?
In the course of attempting to answer these questions, we sought to develop a simple protocol for identifying glycoRNA transcripts using RT-qPCR. The development led to the finding that the dibenzocyclooctyne functional group, commonly used for copper-free click chemistry, appears to cause a bias toward identification of long RNA transcripts and their preferential binding to streptavidin-coated magnetic beads, which are used to enrich azide-tagged glycoRNAs.
This proposal seeks to uncover universal conditions for RNA pulldown experiments of this nature, which at present may suffer from unknown biases. Careful sequencing experiments with appropriate controls will be performed, investigating effects such as copper-catalysed versus copper-free click chemistry. The results obtained from these studies will be applied to the development of a fast, inexpensive RT-qPCR assay for identifying glycoRNA transcripts. This protocol will enable researchers with interests in specific areas of RNA biology to quickly evaluate whether a transcript is glycosylated. Enzymatic treatment of glycoRNA with glycosidases is expected to provide a detailed picture on both the nature of the glycosidic linkage and single-base resolution of modifications.
This proposal also aims to reveal additional glycoRNA transcripts through the use of other carbohydrate probes. Preliminary results indicate that probes for glucose and galactose also lead to the observation of glycRNA, and identification of these transcripts will provide a more complete picture on the developing field of carbohydrate-modified RNA.
In the course of attempting to answer these questions, we sought to develop a simple protocol for identifying glycoRNA transcripts using RT-qPCR. The development led to the finding that the dibenzocyclooctyne functional group, commonly used for copper-free click chemistry, appears to cause a bias toward identification of long RNA transcripts and their preferential binding to streptavidin-coated magnetic beads, which are used to enrich azide-tagged glycoRNAs.
This proposal seeks to uncover universal conditions for RNA pulldown experiments of this nature, which at present may suffer from unknown biases. Careful sequencing experiments with appropriate controls will be performed, investigating effects such as copper-catalysed versus copper-free click chemistry. The results obtained from these studies will be applied to the development of a fast, inexpensive RT-qPCR assay for identifying glycoRNA transcripts. This protocol will enable researchers with interests in specific areas of RNA biology to quickly evaluate whether a transcript is glycosylated. Enzymatic treatment of glycoRNA with glycosidases is expected to provide a detailed picture on both the nature of the glycosidic linkage and single-base resolution of modifications.
This proposal also aims to reveal additional glycoRNA transcripts through the use of other carbohydrate probes. Preliminary results indicate that probes for glucose and galactose also lead to the observation of glycRNA, and identification of these transcripts will provide a more complete picture on the developing field of carbohydrate-modified RNA.
