Using a Specific RNAâProtein Interaction To Quench the Fluorescent

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Using a specific RNA-protein interaction to quench the fluorescent RNA Spinach Laura Roszyk, Sebastian Kollenda, and Sven Hennig ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00332 • Publication Date (Web): 23 Oct 2017 Downloaded from http://pubs.acs.org on October 24, 2017

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ACS Chemical Biology

Using a specific RNA-protein interaction to quench the fluorescent RNA Spinach

Laura Roszyk1, Sebastian Kollenda2 and Sven Hennig1,3

1

Chemical Genomics Centre of the Max-Planck Society, Otto-Hahn-Str. 11, 44227

Dortmund, Germany 2

Institute for Inorganic Chemistry, University of Duisburg Essen, Universitätsstrasse

7, 45141 Essen, Germany 3

Division of Organic Chemistry, Vrije Universiteit Amsterdam, De Boelelaan 1108,

1081 HZ Amsterdam, Netherlands ACS Paragon Plus Environment


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Abstract RNAs are involved in interaction networks with other biomolecules and are crucial for proper cell function. Yet, their biochemical analysis remains challenging. For Förster Resonance Energy Transfer (FRET), a common tool to study such interaction networks, two interacting molecules have to be fluorescently labelled. ‘Spinach’ is a genetically encodable RNA aptamer, which starts to fluoresce upon binding of an organic molecule. Therefore, it is a biological fluorophore tag for RNAs. However, spinach has never been used in a FRET assembly before. Here, we describe how spinach is quenched when close to acceptors. We used RNA-DNA hybridization to bring quenchers or red organic dyes in close proximity to spinach. Furthermore, we investigate RNA-protein interactions quantitatively on the example of Pseudomonas aeruginosa phage coat protein 7 (PP7) and its interacting pp7-RNA. We utilize spinach quenching as a fully genetically encodable system even under lysate conditions. Therefore, this work represents a direct method to analyse RNA-protein interactions by quenching the spinach aptamer.

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Introduction RNAs fulfil crucial functions in cellular processes such as gene regulation, nuclear organization or RNA metabolism. They play regulatory roles in diverse signalling pathways.1,2 Also, they bring in essential proteins and prepare DNA for transcription.3 Moreover, RNA can function as decoy and drag proteins away from their original localization or bring proteins together in large complexes as scaffolds.1,4,5 Dysregulation or malfunction of their interaction networks with other biomolecules is involved in the onset and progression of neurodegenerative and cardiovascular diseases as well as cancer.6–8 For the detailed understanding of those processes and their connected diseases, homogenous methods for the investigations of RNA interaction networks are essential, but remain challenging. Genetically encodable tags such as fluorescent

proteins

(e.g.

the

green

fluorescent

protein,

GFP)

have

revolutionized the field of protein biochemistry.9–11 As every fluorophore, those biological ones can undergo Förster Resonance Energy Transfer (FRET) once in close proximity to a suitable, second, fluorescent partner due to dipoledipole interactions. For optimal FRET efficiencies, donor and acceptor must be oriented in parallel, whereas FRET cannot occur if both fluorophores are perpendicular to each other. The readout is either an acceptor intensity increase or donor quenching. FRET is an essential tool for the investigation of protein-protein interaction networks.12,13 For the analysis of RNA-protein interaction networks a direct labelling of the RNA, encodable on genomic level, remains to be challenging. To label RNAs there are either indirect methods via fluorescent hybridization probes available, or direct linkage of an organic dye to the RNA during chemical synthesis.14 Disadvantages of both procedures are for instance difficulty in chemical synthesis as well as non-physiological ACS Paragon Plus Environment


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concentrations. Genetically encodable fluorescent labels for RNAs are only available via auxiliary RNA binding proteins that carry a GFP fusion15 or via RNA aptamer sequences that bind small molecules as chromophores.16–18 One of these aptamer-chromophore pairs is the spinach RNA which can bind the small

molecule

(Z)-5-(3,5-difluoro-4-hydroxybenzylidene)-2,3-dimethyl-3,5-

dihydro-4H-imidazol-4-one (DFHBI). DFHBI is a cell-permeable, non-cytotoxic chromophore, which fluorescence increases excessively within the appropriate environment provided by the spinach RNA.19,20 This Spinach-DFHBI complex (hereinafter ‘spinach’) as a fluorescent, genetically encodable RNA label has been used in diverse in cellulo applications like tracking mRNA or sensing small cellular metabolites.21,22 Up to now, spinach has not been shown to undergo quenching, which is an essential step towards its utilization in biochemical studies of RNA networks. Here, we show how binding to various fluorescently labelled biomolecules such as nucleic acids and proteins allows efficient quenching of spinach fluorescence. Additionally, we utilize a biological fluorescent protein label as an acceptor, allowing detailed and fully genetically encodable analysis of RNA-protein interactions. Results and discussion First, we were interested if spinach fluorescence could be utilized for Förster Resonance Energy Transfer (FRET). Therefore, three conditions have to be fulfilled: as FRET occurs via dipole-dipole interactions the orientation of the fluorophores should not be perpendicular, the emission spectrum of the donor has to overlap with the absorption spectrum of the acceptor molecule and the distance of both molecules must be within the range of 1-10 nm.23 Accordingly, we used the very predictive and well characterized formation of an RNA-DNA


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hybrid to place a red fluorescent acceptor dye in close proximity to the spinach chromophore DFHBI. Therefore, we modulated the P1 stem region of spinach which is known to be robust against changes as fluorescence is not altered upon variation of this region.24,25 The 3’-end of spinach was shortened by 15 nt (‘spinach∆15’) which leads to a single stranded, non-hybridized and accessible 5’-end (Fig. 1a, Fig. S1a). Additionally, we designed ssDNA oligonucleotides complementary to this region, which were labelled with various organic acceptor dyes on either their 5’- or their 3’-end. As the precise threedimensional structure of the bound state is unknown, we added linker regions between nucleic acid probe and fluorophore to allow for flexibility and thereby enrich the possibility of the correct orientation (Tab. S1). Binding of each DNA oligonucleotide to spinach∆15 was verified by RNA Electrophoretic Mobility Shift Assay (REMSA). Under non-denaturing conditions, each sample showed a specific signal for spinach∆15 fluorescence. Also, all acceptor-modified DNAs (Fig. 1b, Fig. S1c-e) showed a fluorescent signal for the excess of free probe in the gel. Independently of the labelling position, all complementary oligonucleotides showed binding to the RNA complex as their fluorescent band partially shifted and thereby matched with the spinach channel. No spinach∆15 association

could

be

shown

with

any

of

the

non-complementary

oligonucleotide probes as their fluorescent bands appeared as free probes only (Fig. 1b). In principle, FRET allows acceptor fluorescence increase. Under homogenous assay conditions, we did not observe any significant acceptor increase. This might be due to the solvent exposed DNA labels and accordingly other relaxations of the energy than fluorescence. Also, bleed through of the TMR dye in combination with its high extinction coefficient could hide an acceptor increase (Fig. S2 and Tab. S2). However, radiationless ACS Paragon Plus Environment


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energy transfer is also quantified via donor fluorescence decrease. Therefore, quantitative changes in spinach fluorescence upon binding of all labelled DNA probes were monitored (Fig. 1c). The FRET efficiency can be calculated by the ratio of donor fluorescence (spinach) in presence (DA) and absence (D) of the acceptor (DNA probes). All of the labelled DNA probes fulfil the spectroscopic criteria for FRET (Fig. S1). For 3’ labelled probes, quenching efficiencies were in the range of 30-50%, whereas 5’ labelled probes reached up to 85%. This is in the range of known fluorophore-quencher pairs.26 To analyse a pure quencher molecule under our assay conditions, we used the IowaBlack F (IBF) quencher and observed similar quenching efficiencies as the previously tested red dyes (Fig. 1c). The significant difference in quenching efficiencies between 3’- and 5’- labelled probes is due to a 4 nm variation in distances from the two labelling positions to the spinach chromophore (Fig. S1a). This strong distance dependency is common for resonance energy transfer and is therefore often used to analyse complex formations. In our system, as expected, the closer the fluorophores, the more efficient spinach is quenched. To determine the spinach∆15-RNA:DNA-probe interaction more closely, we titrated each DNA oligonucleotide to spinach∆15 and monitored its change in donor fluorescence. As expected, titration of all DNA probes, which are identical in their sequence and differ only in the type of fluorescent label, showed similar affinities with a KD of about 70 nM (Fig. 1d, Fig. S1, Tab. S2). To show reversibility of the system, we designed an unlabelled DNA oligonucleotide, complementary to the fluorescent DNA probe, to compete with spinach∆15 for binding. Fluorescence of spinach∆15 was almost completely restored (~90%) in a concentration dependent manner indicating that the RNA aptamer is not disturbed by any effect throughout the whole procedure (Fig. 1e). Our ACS Paragon Plus Environment

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experiments

showed

efficient

quenching

of

spinach∆15

fluorescence

presumably via FRET upon binding of DNA probes with various fluorescent acceptor dyes. All red chromophores quenched spinach with similar quenching efficiencies as the IBF quencher. Next, we asked the question, whether it is possible to quench spinach fluorescence via protein-RNA interactions. Therefore, we utilized the well characterized Pseudomonas aeruginosa bacteriophage coat protein 7 (PP7) and its interacting pp7-RNA wild-type hairpin. As described before, deletion of the residues 67–75 of the central FG loop (PP7∆FG) leaves it without any further cysteines and also keeps it from forming higher oligomeric structures.27– 29

PP7∆FG is a 13.6 kDa protein that forms an intertwined homodimer with a

large β-sheet surface that serves as a platform to interact tightly with a short 25 nt pp7-RNA hairpin (Fig. 2a). We designed three cysteine mutants, A22C, A62C and N93C for chemical labelling via thioether formation with the red acceptor fluorophore Tide Fluor 3 (TF3).30 Efficient FRET was ensured either by flexible label orientation in non-rigid regions of the protein itself and additionally by the thioether linker between the cysteine side chain and the TF3 dye. As one pp7-RNA is bound by a PP7∆FG homodimer, each RNA-protein complex carries two red fluorescent labels, one in each protomer. To label the pp7-RNA, we integrated its sequence at the tip of spinach stem P3 (‘spinachpp7’), which was also used in the past to generate spinach based biosensors and therefore made it our preferred entry point for construct design.31–33 Spinach-pp7 is able to bind DFHBI with comparable affinities and fluoresces similar than the unmodified spinach aptamer (Fig. S3, Table S3). Afterwards, we checked if the new spinach-pp7 RNA construct is able to bind all modified proteins. With increasing protein concentrations, spinach-pp7 RNA showed ACS Paragon Plus Environment


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decreased electrophoretic mobility, indicating binding for all labelled protein variants (Fig. 2b, Fig. S3g, h). To calculate distances within the RNA-protein complex we generated a structural model by arranging the crystal structures of PP7-pp7 (2qux) and spinach (4ts2). The distances varied from 4.5–7.0 nm, all within the range for radiationless energy transfer (Fig. 2a, Fig. S3, Tab. S4). To verify if the calculated distances correlate with FRET efficiencies, we analysed spinach-pp7 fluorescence upon addition of TF3-labelled PP7-proteins. Again, no acceptor increase was observed (Fig. S4), whereas all protein variants quenched spinach-pp7 significantly with N93C showing the highest quenching efficiency (QE = 65%) when compared to A62C (QE = 46%) and A22C (QE = 52%) (Fig. 2c). This distance dependency confirmed our structural model, which indicates the shortest distance for the N93C mutant (Fig. 2a, Fig. S3, Tab. S4). This effect is specific, as unlabelled PP7-N93C as well as PP7∆FG do not change spinach fluorescence. Subsequent titration of PP7-N93C-TF3 showed a specific concentration depended fluorescence decrease and the KD could be determined to be about 1.8 µM (Fig. 2d). Unlabelled PP7∆FG competed with binding of TF3-labelled PP7-N93C to spinach-pp7 and restored fluorescence intensity up to 80% (Fig. 2e). Overall, we proved that red organic dyes can be used to quench spinach fluorescence in various RNA-biomolecule interactions such as nucleic acid hybridization as well as RNA-protein interactions. Hereafter, we were interested if spinach quenching can be achieved by exchanging

our

previously

used

organic

protein-dyes

for

biological

fluorophores. Therefore, we produced fusion proteins of PP7∆FG with the red fluorescent

protein

mCherry

(mCherry-PP7

and

PP7-mCherry),

which

comprises spectral characteristics similar to the organic labels (Fig. S5). We ACS Paragon Plus Environment

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purified and tested the fusion proteins for binding and quenching together with the spinach-pp7 complex (Fig. 3). Linkers between both proteins derived from cloning overhangs (linker sequence: ENLYFQG) and ensured a flexible orientation of mCherry to spinach. Complex formation was also verified via REMSAs in which each fusion protein was titrated against spinach-pp7. Both mCherry tagged PP7 proteins bound in a concentration dependent manner similar to unlabelled PP7∆FG, while mCherry alone did not show any binding (Fig. 3a). Interestingly, only the N-terminally tagged fusion protein was able to quench spinach-pp7 (~20%), whereas the C-terminally fused PP7-mCherry did not quench significantly in comparison to controls (Fig. 3b). Due to an overall low energy transfer from the donor and potential additional radiationless relaxations, an acceptor increase could not be observed (Fig. S6). To analyse the RNA-protein interaction in a quantitative manner, we titrated mCherry-PP7 to spinach-pp7 RNA. Based on the fluorescence intensity, a KD of 140 nM (Fig. 3c, Tab. S4) was determined. The lower affinity compared to a previously reported value of 1.6 nM obtained by REMSA, may result from the addition of the mCherry label or be due to varied experimental conditions (e.g. buffer, ion strength).31 Then we tested the reversibility of the system by adding increasing amounts of untagged PP7∆FG to the preformed complex, thereby competing with mCherry-PP7. Fluorescence could be restored up to 95% compared to the RNA-aptamer fluorescence alone (Fig. 3d). Overall, the increased complex size dropped the spinach quenching efficiency as expected, due to larger distances of both fluorophores. Nevertheless, we successfully utilized mCherry as

a

biological

fluorophore

to

determine

an

quantitatively.


RNA-protein

interaction


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Finally, we were interested, if spinach quenching is possible in a more complex environment (cellular lysate). For that purpose, the spinach-pp7 RNA was expressed in BL21(DE3) cells and lysates were supplemented with DFHBI (final concentration 1.3 µM) as well as mCherry or mCherry-PP7 (66 µM). Knowing that fluorescence intensity and quenching efficiency highly depend on the spectral setting, influences of the excitation wavelength as well as bandwidths was investigated (Fig. S7). To ensure minimal acceptor (quencher) fluorescence bleed through, we chose the following settings: λEx = 425 nm, BWEx and BWEm = 5 nm. Using this setup, emission spectra (465 – 700 nm) were recorded (Fig. 4a).

The lysate (grey) shows only spinach-pp7

fluorescence. Upon addition of mCherry-PP7 protein, we observe a significant decrease of donor fluorescence (QE = 20%) (Fig. 4b), which is in line with the behaviour of the purified components (Fig. 3c). Importantly, addition of the mCherry protein only (lacking the PP7 fusion element) does not result in a reduction of donor fluorescence (Fig. 4b). In both mCherry containing samples, due to residual bleed through, some mCherry fluorescence is observed (maximum at 615 nm). In line with the results above, we do not observe FRETbased increase of acceptor fluorescence (Fig. 4a, S8). In summary, our data show

the

usage

of

the

spinach

aptamer in

RNA-protein

measurements for the first time, even under lysate conditions.


interaction

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Conclusions

Investigations of RNA interactions are of crucial interest, but simple homogenous methods such as FRET are rare. For FRET the RNA of interest needs a fluorescent tag. The spinach aptamer is a genetically encodable RNA label, binding to the DFHBI chromophore and thereby activating its fluorescence. Here we describe how spinach is quenched upon interaction with biomolecules such as nucleic acids or proteins. Binding induces proximity based radiationless energy transfer to acceptor dyes like quenchers and red fluorophores. We could monitor and quantify the specific hybridization to complementary DNA probes. Also, direct RNA-protein interactions can be observed and analysed for their binding affinity. Additionally, we successfully substitute chemical dyes for genetically encodable fluorescent proteins such as mCherry and could show that donor quenching can be detected in lysates. This setup can possibly be used under in cellulo conditions, if better fluorescent RNA tags as well as more suitable FRET pairing fluorescent proteins are developed. Optimizing the overall signal strength and FRET efficiency, these systems might increase acceptor fluorescence which is preferred in some cases over donor quenching. Methods Experimental procedures and methods are described in detail in the Supporting Information. Associated Content Supporting Information Available: This material is available free of charge via the Internet. Author Information ACS Paragon Plus Environment


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Corresponding Author [email protected] Acknowledgments L. R., S. K. and S. H. are grateful for support from AstraZeneca, Bayer CropScience, Bayer HealthCare, Boehringer Ingelheim, Merck KGaA, and the Max Planck Society. We thank D. Schade and D. Längle for providing DFHBI and T. N. Grossmann for critical reading of the manuscript. We also thank S. Koch for technical support. Supporting Information The

Supporting

Information

is

available

online

free

of

charge

at

http://pubs.acs.org. Supplemental Methods: RNA hybridizing oligonucleotides, Cloning of RNA and protein constructs, RNA in vitro transcription, Protein expression, purification and

labelling,

REMSAs,

Quenching

and

competition

measurements,

Fluorescence analysis of E. coli expressed spinach-pp7 lysates. Supplemental Figures: Spinach∆15 and labeled DNA probes, Emission spectra spinach∆15 and 5’-TMR binding probe, Spinach-pp7 binding to PP7-protein mutants, Emission spectra analysis of PP7-N93C w/o spinach-pp7, Spectral properties of spinach-DFHBI and mCherry protein, Emmission spectra analysis of mCherry tagged PP7 protein w/o spinach-pp7, Optimization of plate readeer settings, Spectroscopic analysis of spinach-pp7 expressing E. coli lysates. Supplement Tables: Oligonucleotide details used in hybridization assay, Spectroscopical properties of the used fluorophores, Spectroscopical and biochemical properties of the used spinach constructs, Spectroscopical and biochemical properties of the used labeled PP7 proteins. ACS Paragon Plus Environment

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Figure 1: Quenching properties of spinach RNA. a) Schematic overview of the analysis of RNA-DNA interaction by using spinach∆15 RNA (green) and complementary ssDNA probes (red) labelled on their 5’- or 3’-end with organic dyes (for details: Tab. S1). Resonance energy transfer might occur upon



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binding due to close proximity of the fluorophores (black arrow). b) REMSA of spinach∆15 (250 nM) and TMR labelled ssDNA probes (750 nM). Upper panel (green channel at 560 nm) shows green fluorescent emission of spinach∆15 RNA. Lower panel (red channel at 700 nm) shows signal of TMR labelled ssDNA probes. c) Proximity-related quenching of spinach∆15 with labelled DNA probes. Different ssDNA probes were added to spinach∆15 in presence of DFHBI. Spinach∆15 fluorescence was detected at 506 nm. Data is normalized to spinach∆15 fluorescence with addition of an unlabelled but binding probe. *=p≤0.05, **=p≤0.01, ***=p≤0.001. Data shown as mean ±SEM of triplicates. d) 5’-TMR labelled DNA was titrated to spinach∆15. Data was normalized to spinach∆15 fluorescence. Data shown as mean ±SEM of triplicates. e) Complementary, unlabelled DNA competes with 5’-TMR labelled DNA for binding to spinach∆15. Data shown as mean ±SEM of triplicates. TMR = 5-Carboxytetramethylrhodamine, JOE = 6-Carboxy-4',5'-Dichloro-2',7'Dimethoxyfluorescein, RHOR = Rhodamine Red, Cy3 = Cyanine3 and u. b.= unlabeled binding.


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Figure 2: Detection and analysis of RNA:protein interactions via spinach quenching. a) Structural model of one possible spatial orientation of PP7protein:pp7-spinach was generated by arranging crystal structures of PP7-pp7 (light grey and grey surface: PP7∆FG dimer, black: pp7-RNA, pdb: 2qux and spinach-DFBHI

(green: spinach-RNA,

black:

DFHBI,

pdb:

4ts2) using

nucleotides 36G and 43C (spinach) and 1G and 25C (pp7-RNA) for superimpositioning (Coot34 and Pymol35)). Protein labelling with TF3 was shown (two red spheres). Distance to spinach chromophore DFHBI is measured (Pymol35) and indicated accordingly (red line). b) Binding of spinachpp7 and labelled PP7-protein dimer via REMSA. Spinach-pp7 (250 nM) was mixed with different concentrations of PP7-N93C-TF3 protein (0–12.5 µM, 1:1 dilution) and analysed electrophoretically. Fluorescence was detected at 520 nm for spinach-pp7 fluorescence and at 620 nm for TF3-labelled proteins. c) Upon addition of TF3-labelled cysteine-mutants spinach-pp7 fluorescence is quenched significantly. *=p≤0.05, **=p≤0.01, ***=p≤0.001. Data shown as mean ±SEM of triplicates. d) PP7 protein variants are titrated against spinachpp7. PP7-N93C-TF3 shows a concentration dependent quenching effect of spinach-pp7. Data shown as mean ±SEM of triplicates. e) Reversibility of complex formation was shown by competition with unlabelled PP7∆FG-protein. Data shown as mean ±SEM of triplicates.


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Figure 3: Genetically encodable quenching system for the detection and analysis of RNA-protein interactions. a) REMSA of PP7∆FG and mCherry PP7-fusion variants (0–1.6 µM, 1:1 dilution) with spinach-pp7 (250 nM). Detection of spinach fluorescence at 520 nm. b) Quenching of spinach-pp7 upon addition of mCherry labelled PP7 variants. *=p≤0.05, **=p≤0.01, ***=p≤0.001. Data shown as mean ±SEM of triplicates. c) Increasing concentrations of mCherry-PP7, mCherry and PP7∆FG were titrated against spinach-pp7. Data shown as mean ±SEM of triplicates. d) Quenching of spinach-pp7 by mCherry-PP7 is reversible via addition of unlabelled PP7∆FG. Data shown as mean ± SEM of triplicates. n. s. = not significant.



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Figure 4: Quenching of spinach-pp7 from overexpressing E.coli BL21(DE3) cell lysates. a) Emission spectra of lysates from spinach-pp7 overexpressing E. coli BL21(DE3) cell supplemented with 1.3 µM DFHBI after addition of either mCherry-PP7 or mCherry (66 µM). λEx = 425 nm. b) Quantification of donor quenching effect of samples shown in a) at spinach-pp7 peak emission wavelength (dashed line, λEm = 505). Data shown as mean ±SEM of triplicates. *=p≤0.05, **=p≤0.01, ***=p≤0.001. n. s. = not significant.


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+

Quenching readout

red labeled spinach-pp7PP7 protein RNA


Using a Specific RNAâProtein Interaction To Quench the Fluorescent

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Using a Specific RNAâProtein Interaction To Quench the Fluorescent