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

Project: Research

Description

RNA is the least well understood cellular macromolecule. Its activities range from passive coding to dynamic catalysis.
Most of the RNA in a cell is involved in dynamic events, such as translation, in which the structures of the RNA molecules
and their conformational fluctuations play critical roles. A far more complex example is that of pre-mRNA splicing, in which
the snRNAs, components of the active spliceosome, are intimately involved in catalysis and undergo binding, remodelling
and dissociation during each reaction. The pre-mRNA is also likely to undergo important changes in structure and
conformation, and, in mammals, in which introns can be as long as hundreds of thousands of nucleotides and many
splicing patterns are subject to tissue-specific regulation, extensive protein-mediated structures may form. Since the keys
to the contribution of RNA in complex reactions are likely to be its conformational heterogeneity and thermal fluctuations,
understanding the mechanisms of such complex and critical processes will require methods for following structural changes
in a heterogeneous population. Single molecule FRET methods have provided valuable insights into the behaviour of short
molecules of pure RNA. However, equivalent work on the much longer pre-mRNA molecules as they are spliced in nuclear
extracts has been very much restricted by the practical near-impossibility of incorporating two suitable fluorophores at
specific sites. The same problem restricts real time analysis of RNA structures in translation or other RNA processing
events.
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 (or indeed other ligands, such as cross-linking
functions). This would be the first general method that would avoid multiple time-consuming and inefficient ligation steps for
placing two modified nucleotides at specific sites deep inside long transcripts.
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 highthroughput
screening for inhibitors of RNA reactions.

Layman's 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 (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.

Key findings

Demonstrated that non natural base pairs Z-P misincorporate into DNA (PCR) and RNA (transcription). This is in contrast to established literature. Work is underway to determine the source of misincorporation.

In parallel a different base-pairing system was investigated. Work is currently underway to determine the ability of this base-pairing system as a regime to site-specifically label RNA at two different positions.
StatusFinished
Effective start/end date5/11/124/11/13

Funding

  • BBSRC (Biotech & Biological Sciences Research Council): £120,129.07

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Functional groups
RNA
Labels
Fluorophores
Molecules
RNA Precursors
Transcription
Catalysis
Nucleotides
Fluorescence
Tissue