A new tool in RNA research: using an expanded genetic repertoire for site-specific incorporation of functional groups

Many important processes in mammalian cells involve RNA. Of particular interest are those in which RNA molecules themselves act to catalyse events that affect a second RNA molecule. RNA molecules are often able to adopt a number of structures, and they can fluctuate between these either spontaneously (thermaly-driven) or as a result of the actions of enzymes. An obvious example of such a system is the ribosome, in which ribosomal RNAs and tRNAs drive chemical and conformational changes involved in decoding a mRNA molecule and synthesizing a protein.A more intriguing and far lass well understood example is RNA splicing, in which large stretches of RNA are displaced from newly-transcribed RNA to form mRNA. The splicing machinery is RNA-based, and the RNA substrates are very long, sites are hard to recognise, and the use of these sites is often subject to complex tissue-specific regulation that may involve the formation of structures with the RNA. A good way of monitoring whether RNA undergoes changes in its structures or conformations is to place fluorescent labels at two sites in the RNA. These labels are chosen such that, when they come into close proximity, they transfer the energy of fluorescence excitation from one to the other; this can be measured. This is a particularly good method for following the events on a single molecule, which is an essential approach for studying splicing.

The main drawback at present is that it is very difficult to introduce two labels at specific sites far inside a long RNA molecule. We propose to overcome this by a radical new strategy, in which we take advantage of two new bases (representated as Z and P) that can base-pair to each other and are known to work well in DNA synthesis reactions such as PCR. We will create templates for transcription of RNA by PCR in which we place a Z and a P base at specific sites in the template strand. We will make RNA versions of P and Z, incorporating chemical groups that will allow us to add fluorescent labels to the bases (different ones for Z and P). The RNA will be modified at P and Z with the labels, and we will use the doubly-labelled RNA as a substrate in splicing reactions for single molecule studies. This will have a major impact in RNA research, and we will try to ensure both that the modified bases become commercially available and that the ability to follow RNA fluorescence energy transfer easily is recognised as opening up new opportunities to search for drugs that affect RNA-basd reactions.

A major obstacle in research into the mechanisms of reactions involving RNA molecules longer than about 150 nucleotides is the difficulty of introducing modifications into specific sites more than the length of an RNA oligonucleotide (up to about 70 nts) from th etermini of the RNA. Current methods based on ligation give very low yields. Such modifications are needs for conjugation of fluorophores, cross-linking reagents, Fe-BABE, etc. The problem is especially acute when two different fluorophores have to be incorporated for FRET studies. Single molecule FRET is essential to investigate mammalian pre-mRNA splicing. Our aim is to demonstrate the feasibility of using an expanded genetic repertoire for introducing one or two modified nucleotides into transcripts that can then be conjugated to fluorophores.

Our objectives are:
(i) to synthesize orthogonal Z and P ribonucleoside triphosphates that carry novel functions (alkyne and amine) for conjugation;
(ii) to prepare oligodeoxynucleotide primers containing Z and P bases, to use these in overlapping PCR to place Z and P bases at specific sites in a transcription template, and to prepare RNA transcripts with the modified Z and P bases at the correct sites;
(iii) to label the transcripts at these two sites with two specific fluorophores for single molecule FRET and to determine with short model transcripts that the fluorophores perform as expected in single molecule FRET;
(iv) to demonstrate that long transcripts containing two fluorophores introduced by these methods can be used for single molecule FRET studies of splicing complexes assembled in nuclear extracts.

Significant further objectives after the funding period are to enable commercial manufacture and sale of these modified ribonucleoside triphosphates and to demonstrate the feasibility of using appropriately modified transcripts for high-throughput screening for inhibitors of RNA reactions.

1. Types of impact activity

- Our plans have revealed a major gap in the market for the development of methodology that would enable site-specific modification of RNA molecules. We consider our proposed methods as the beginning of a platform technology in which functional groups can be post-synthetically further modified to incorporate a plethora of desirable modules ranging from fluorophores, cross-linking agents, affinity probes and radio-labels. No other method of RNA labelling to date can potentially achieve site-specific modification of RNA molecules in multiple sites on a preparative scale. This is a serious hindrance to the study of both fundamental RNA processes but also to the development of therapeutics targeting key biological pathways at the RNA level.

- Our use of an expanded genetic repertoire to achieve site-specific incorporation of functional groups has so many possible applications in the RNA world that we have engaged in discussions with a commercial manufacturer (BaseClick GmBH) about production and marketing once we have achieved proof of concept. These agreements will be validated by the Business Development offices in Strathclyde and Leicester to identify any potential IP.

- The possibility of large-scale production of site-specifically modified RNA would be very useful for high throughput screening assays based on intramolecular FRET to detect molecules that interfere with RNA-protein interactions, complex assembly, splicing, etc. The possible applications of these and other aspects of our work will be monitored by meeting at intervals of three months in collaboration with Strathclyde's Research & Knowledge Exchange Services (RKES) office, who will be able to advise on commercial potential and strategies for exploitation.

- There is also the possibility of identifying important targets in cancer and seeking funds to start up a company to demonstrate the feasibility of such screening. Both institutions have a successful track record in this area (e.g. MGB Biopharma, Strathclyde; Centre for Translational Therapeutics, Leicester).

Communications and engagement

- Peer reviewed papers will be deposited with open access archives of research in Strathclyde (SPIDER) and Leicester (Leicester Research Archive).

- Following the generation, publication and protection of research outputs, we will need to make direct contact with commercial interests to further the aims described above. This will be done in part by speaking at drug discovery conferences and also by making direct contact with companies.

- I.C.E. will produce a video describing splicing and another one describing single molecule approaches as components of a series of 5-10 minute videos, aimed at secondary schools.

- The research will be described for a lay audience in LE1, a magazine produced by the University of Leicester, which is printed and distributed locally, and accessible online.

- G.A.B. will coordinate with OPEN-West - the outreach network of western Scotland - and engage with their various outreach programmes such as "Engaging with a Scientist" evenings and contributing to "The Gist" magazine.

2. Impact activity deliverables and timetables

- Discussions with the potential partner company involved in production and marketing will be completed by the end of year 1, with a view to initiating the process by the end of the next year.

- For application to high throughput screening, direct meetings with relevant pharmaceutical companies will be held in year 2.

- Discussions about setting up a company to undertake high-throughput screening or manufacture and supply of the modified NTPs will begin towards the end of year 1 also. In order to monitor the commercial progress of this work, monthly meetings will be held with the RKES office at Strathclyde after the funding period had ended.