Over the past 10 years it has become increasingly apparent that there is exceptional complexity in the RNA population in cells, including microRNAs, Piwi-interacting RNAs, termini-associated RNAs and other noncoding RNAs. In many cases, alterations in these RNAs, or proteins that bind to these RNAs, have been linked to a wide range of medical disorders. A major challenge of molecular biology is to determine the function of these fascinating and novel RNA species. Furthermore, experiments that monitor the localization and trafficking of these RNAs can provide novel insight into RNA processing and other RNA regulatory events.
GFP and other genetically encodable fluorescent protein technologies have revolutionized biomedical research and biotechnology. As a result of GFP, studies that address the trafficking and processing of proteins in relation to specific intracellular organelles and sites within the cell has become commonplace. Because GFP and GFP-tagged proteins are genetically encodable, they can be expressed from transfected DNA, making the preparation of cells that express fluorescently labeled protein accessible to virtually any biomedical research laboratory. Based on the impact of GFP-based protein localization, we expect that similar enabling technologies will provide considerable insights into the function of the numerous newly discovered classes of RNA.
While GFP has considerably advanced protein-based applications and studies of protein biology, comparably simple and robust tools for genetically encoding fluorescently tagged RNA for imaging RNAs in living cells are lacking. If there were an RNA visualization technology analogous to GFP, it would be a major enabling technology that researchers would rapidly embrace. Furthermore, this type of technology would lend itself to the development of cell-based and in vivo high-throughput assays to discover novel therapeutics that affect RNA localization and processing.
A number of non-genetically encodable RNA imaging techniques has been developed. The most commonly used technique to study mRNA localization is in situ hybridization. This is a well-established technique, but it does not allow RNA to be monitored continuously during experimental treatments in living cells. Another approach is to use molecular beacons, which are oligonucleotides that are dual labeled with a fluorophore and a quencher. The beacon adopts a stem-loop structure that is nonfluorescent due to the proximity of the fluorophore and quencher at the base of the stem. When a target mRNA that exhibits complementarity to the loop hybridizes to the beacon, the stem is disrupted, resulting in separation of the fluorophore and quencher and subsequent fluorescence. However, transfected beacons exhibit nonspecific nuclear sequestration and each mRNA requires a custom-designed beacon for visualization.
Because of the inherent difficulties of these synthetic approaches, numerous groups have attempted to develop genetically encoded reporters of RNA localization in cells. All of these approaches involve the recruitment of enhanced GFP (EGFP) aggregates to the RNA of interest. The most widely used of these techniques is the EGFP-MS2 system. This approach uses two components: MS2, a viral protein, fused to EGFP; and MS2-binding elements, which are RNA sequences, inserted into the 3’ UTR of RNAs of interest. Both EGFP-MS2 and MS2-element-containing RNAs, or “fusion RNAs,” are expressed in cells from transfected DNA. EGFP-MS2 binds to the MS2 element-tagged RNA in cells, and fluorescence signals in these cells should represent RNA-EGFP complexes. Because unbound EGFP-MS2 molecules diffuse throughout the cytosol there would be, in principle, a high fluorescence background. To alleviate this problem, a nuclear localization signal (NLS) is incorporated into the EGFP-MS2 fusion protein so that most of the EGFP-MS2 moves into the nucleus. Unfortunately, the consequence of this is that EGFP-MS2-RNA complexes are subjected to two trafficking signals: one encoded within the RNA and another being the NLS within EGFP-MS2. The presence of two trafficking signals confounds interpretation of the intracellular movements of the tagged mRNA. This raises concerns about the physiological relevance of the behavior of EGFP-NLS-tagged RNAs, especially since most RNAs are modified to bind 24-48 copies of MS2-EGFP-NLS, and therefore have 24-48 NLS targeting elements. An additional drawback is that the NLS causes the EGFP-MS2 to accumulate in the nucleus, resulting in intense nuclear fluorescence signals and thereby preventing the analysis of nuclear-localized RNAs. Since much RNA biology occurs in the nucleus, such as nonsense mediated decay, RNA editing, nuclear export of RNA, splicing, pioneer RNA translation, and microRNA processing, nuclear accumulation of EGFP-MS2 impedes the analysis of these events. Moreover, the large payload of having 24-48 EGFP fusion proteins bound to the RNA makes this technique problematic for imaging small and medium-sized noncoding RNAs, which are miniscule in size compared to these EGFP aggregates. Thus, even though EGFP-MS2 has utility, it is not adequate for the needs of the research community.
In summary, the major limitations of currently available methods for genetically encodable fluorescent RNA reporters are:
1. Introduction of artificial trafficking elements, which might perturb natural RNA trafficking.
2. Inability to visualize nuclear RNAs.
3. Large payloads that preclude the tagging of small and medium sized noncoding RNAs.
4. Complicated two- or three-vector systems.
In order to understand how biologically important RNAs function in cells, technology to image the localization and intracellular movements of these RNAs in living cells under a variety of experimental stimuli are needed. Current techniques suffer from major limitations that make them difficult to use, and produce results of dubious validity. While at Cornell University, LucernaTM scientists have developed a novel genetically encodable system to fluorescently tag RNAs in cells. This system utilizes an RNA sequence element, termed SpinachTM, which is appended to an RNA of interest, and which “switches on” the fluorescence of an otherwise non-florescent dye. This dye is based on the structure of the fluorophore in GFP, making SpinachTM an RNA mimic of GFP. With the assistance of an NIH phase I SBIR grant, LucernaTM was successful in developing brighter and more sensitive SpinachTM variants that enabled live-cell imaging of non-coding RNAs in the nucleus. Additionally, LucernaTM has generated several other fluorescent RNA colors as part of its ongoing effort to made available a palette of fluorescent RNA tags that are suitable for multicolor imaging. These experiments demonstrated the feasibility of using fluorescent RNAs as imaging tools that would be easily adopted by the scientific community for the study of non-coding RNA and mRNA dynamics in cellular environment.
LucernaTM plans to use the proprietary aptamer technology licensed from Cornell University to develop ready-to-use RNA imaging tools that address multiple unmet needs in the life science research reagent market. We are currently developing the following commercially ready products:
Plasmids containing sequence cassettes containing different numbers of SpinachTM and CornTM tandem repeats that are suitable for imaging cellular RNAs of varying abundance
A set of plasmids expressing pre-labeled RNA molecules tagged with SpinachTM or other fluorescent RNA tags, which will label key RNA-based cellular structures
A novel switchable fluorescent labeling system comprising a set of DFHBI-like fluorophores that allow for dynamic and reversible multicolor RNA labeling with just one single tagged RNA
We believe our products overcome the technological limitations that currently exist with imaging RNA in living cells. Furthermore, there are no genetically encoded fluorescent reporters for RNA imaging that is commercially available. We plan to establish a strategic partnership with an established life science reagent company for validation and for co-marketing. We believe partnership with an established life science reagent company will help overcome the barrier to entry into the research reagent market and accelerate the distribution of these novel RNA imaging tools to the general research community.