A new genetically-encoded aptamer platform for multi-colour RNA imaging

Project: Research

Description

"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 (thermally-driven) or as a result of the actions of enzymes. A less 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 specifically 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 genetically encoding RNA structures to bind to fluorescent tags. Having available a two-colour system to label RNA will provide a powerful new tool for RNA research as it will allow various RNA processing events to be directly compared rather than relying on fluorescence emission of a single fluorophore. Our inter-disciplinary approach is to exploit an artificial evolution technique known as SELEX to identify RNA structures (aptamers) that bind fluorophores that exhibit red emission. We will then incorporate these aptamers into long RNA molecules and investigate their potential as reporters of RNA biology. This will have a major impact in RNA research, and we will ensure both that the aptamers become commercially available and that the ability to follow RNA fluorescence is recognised as opening up new opportunities to search for drugs that affect RNA-based reactions."
StatusFinished
Effective start/end date1/11/1631/10/17

Funding

  • BBSRC (Biotech & Biological Sciences Research Council): £151,049.00

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RNA
Color
Imaging techniques
Molecules
Labels
Fluorophores
Fluorescence
Machinery
Conformations