Letters pubs.acs.org/acschemicalbiology
Fluorescent RNA Labeling Using Self-Alkylating Ribozymes Ashwani K. Sharma,† Joshua J. Plant,‡ Alexandra E. Rangel,† Kirsten N. Meek,† April J. Anamisis,† Julie Hollien,*,‡ and Jennifer M. Heemstra*,† †
Department of Chemistry and the Center for Cell and Genome Science and ‡Department of Biology and the Center for Cell and Genome Science, University of Utah, Salt Lake City, Utah 84112, United States S Supporting Information *
ABSTRACT: The ability to fluorescently label specific RNA sequences is of significant utility for both in vitro and live cell applications. Currently, most RNA labeling methods utilize RNAnucleic acid or RNA-protein molecular recognition. However, in the search for improved RNA labeling methods, harnessing the smallmolecule recognition capabilities of RNA is rapidly emerging as a promising alternative. Along these lines, we propose a novel strategy in which a ribozyme acts to promote self-alkylation with a fluorophore, providing a robust, covalent linkage between the RNA and the fluorophore. Here we describe the selection and characterization of ribozymes that promote self-labeling with fluorescein iodoacetamide (FIA). Kinetic studies reveal a secondorder rate constant that is on par with those of other reactions used for biomolecular labeling. Additionally, we demonstrate that labeling is specific to the ribozyme sequences, as FIA does not react nonspecifically with RNA.
F
less likely to perturb RNA behavior.6,7 A number of RNA aptamers have been reported in which the aptamer sequence binds to a fluorogenic small molecule, causing a dramatic increase in fluorescence intensity.5,8−13 In 2011, Jaffrey and coworkers reported that an RNA aptamer for an analogue of the GFP fluorophore could be attached to the 5S rRNA, enabling fluorescence-based visualization of this RNA in living cells (Figure 1b).14,15 This aptamer has since been utilized to visualize small molecule metabolites in bacterial cells and for in vitro labeling of RNA.16−18 The introduction of this method represented a significant advance in RNA imaging technology, but the noncovalent nature of the RNA-small molecule interaction can limit the imaging sensitivity and scope of potential applications for this method. We envision an alternative approach to fluorescent RNA labeling in which a ribozyme is used as the recognition element. The ribozyme catalyzes self-alkylation with a fluorophore bearing an electrophilic reactive group. This provides a robust, covalent linkage between the RNA and the fluorophore, which is anticipated to negate the problem of signal loss from RNAfluorophore dissociation (Figure 1c). Interestingly, covalent labeling approaches have proven to be very successful in protein labeling19 but have yet to be adapted in a general manner for RNA labeling. In the search for effective alkylation chemistry, we were inspired by a report from Wilson and Szostak describing RNA self-alkylation with biotin iodoacetamide.20 This system was not designed to be used for RNA imaging, however, as biotin cannot be directly observed spectroscopically and the selectivity of these ribozymes was not established. We instead chose fluorescein iodoacetamide
luorescent RNA labeling both in vitro and in living cells forms the basis for numerous applications in biotechnology and cellular biology. A number of elegant RNA labeling methods have been reported, and these primarily rely on binding of a fluorescent protein or fluorescently labeled oligonucleotide to the RNA of interest (Figure 1a).1−4 These
Figure 1. RNA labeling using (a) RNA-binding proteins, (b) aptamer and fluorogenic small molecule, and (c) fluorescently self-alkylating ribozyme. In all examples, the appropriate recognition element is fused to the RNA of interest to enable transcript-specific labeling.
methods enable highly efficient labeling of RNA but can suffer from challenges including high background fluorescence, probe instability, and the propensity to disrupt native RNA structure or function. Harnessing the small-molecule recognition properties of RNA has long been viewed as a promising alternative approach for the development of RNA labeling methods,5 as small-molecule fluorophores benefit from excellent quantum yield and functional diversity and owing to their small size are © 2014 American Chemical Society
Received: March 19, 2014 Accepted: June 4, 2014 Published: June 4, 2014 1680
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Figure 2. (a) IP-SELEX to generate self-labeling ribozyme. A DNA library is PCR amplified and then transcribed into an RNA library. The RNA library is allowed to react with FIA, and then active sequences are pulled down using an anti-fluorescein antibody. PBS = primer binding site. (b) Denaturing PAGE showing fluorescein labeling of RNA from SELEX rounds.
(FIA, Figure 2a) as the substrate, as fluorescein can be imaged directly and has excellent optical properties, being brighter than most fluorescent proteins.21 Here we report selection and characterization of the first ribozymes capable of fluorescent self-labeling and demonstrate that the labeling reaction proceeds with excellent selectivity for both the FIA substrate and the ribozyme sequence. In the process of selecting for our fluorescently self-alkylating ribozymes, we implemented an immunoprecipitation SELEX (IP-SELEX) process, in which “hit” sequences are pulled down using magnetic beads functionalized with an antibody for the fluorescein label. This provides a convenient and highly selective method for separating ribozymes from the overwhelming majority of inactive sequences.22 Additionally, reverse transcription of the ribozymes is carried out on the beads, obviating the need for an elution step. As shown in Figure 2a, a starting DNA library was synthesized having a 116 nt random region (N116) flanked by two primer binding sites. We amplified this DNA by PCR using a forward primer containing the T7 promoter sequence and converted the products into the corresponding RNA library via T7 in vitro transcription. We then incubated the RNA library with 200 μM FIA (initial incubation time was 8 h but was reduced to 15 min over iterative SELEX cycles to drive the selection toward ribozymes having fast reaction rates). The labeling reaction was quenched with 2-mercaptoethanol, and the RNA was purified by ethanol precipitation. To isolate RNA sequences that had been successfully labeled, we incubated the total RNA pool with magnetic beads prefunctionalized with an anti-fluorescein antibody. Unbound RNA was removed by washing, and bound RNAs were reverse transcribed and PCR amplified, resulting in a double-stranded DNA library that could be subjected to a subsequent round of selection. We evaluated the overall reactivity of the RNA library after rounds 4, 7, 10, 11, and 12. In these experiments, we incubated the library with 100 μM FIA for 15 min, ethanol precipitated the RNA to remove excess FIA, and examined the products
using denaturing PAGE followed by fluorescence imaging. The same quantity of RNA was loaded into each lane of the gel, such that the intensity of the fluorescein band is representative of reaction efficiency. Importantly, the denaturing conditions of the gel disrupt any noncovalent RNA-fluorophore interactions, ensuring that fluorescence is observed only when the FIA is covalently tethered to the RNA sequence. Using this analysis, labeling efficiency appeared to plateau at round 12 (Figure 2b). Thus, selection cycles were stopped, and the RNA library was amplified by reverse transcription-PCR, inserted into a TOPO cloning vector, and amplified in E. coli. We sequenced 20 individual clones from the round 12 RNA library. This revealed four different types of ribozyme sequences, each represented by several highly related or identical clones (Supplementary Figure 1). We then used PCR and in vitro transcription to generate representative ribozymes from each of the 4 groups and compared them using the denaturing PAGE assay described above (Figure 3a, lanes 1−4). All four sequences showed self-labeling activity, but sequences 1 and 5 appeared to be the most reactive. Importantly, control RNA sequences including the biotin selfalkylating ribozyme and total cellular RNA do not show observable reaction with FIA (Figure 3a, lane 5 and Supplementary Figure 2), indicating that FIA reacts only with the selected ribozyme sequences and does not label RNA molecules nonspecifically. The PAGE assay described above is useful for determining whether RNA sequences show self-labeling activity with FIA and can provide insight into the relative reactivity of these ribozyme sequences. However, this assay does not provide quantitative information regarding reaction yields. Thus, to quantify reaction yields, we turned to HPLC. Both reacted and unreacted RNA elute together as a single peak, but the relative peak areas (A) for absorbance at 260 nm (RNA) and 495 nm (fluorescein) can be used in conjunction with the molar absorptivity (ε) values for the RNA and fluorescein to calculate reaction yield according to eq 1: 1681
dx.doi.org/10.1021/cb5002119 | ACS Chem. Biol. 2014, 9, 1680−1684
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30 min. These data verified that sequences 1 and 5 show the fastest reaction kinetics, and thus we selected these two sequences for further analysis. To evaluate ribozymes 1 and 5, we first sought to determine whether the sequences could be truncated without impacting reactivity. Using denaturing PAGE and HPLC, we evaluated sequences having 18 nt removed from either the 5′ or 3′ terminus (Supplementary Table 1, Figure 3c,d). For ribozyme 1, shortening the sequence from either terminus to give 1F1R or 1FR1 resulted in a dramatic decrease in reactivity. For ribozyme 5, truncation at the 5′ terminus to give 5F1R also resulted in a dramatic decrease in reactivity. However, we were able to remove 18 nt from the 3′ terminus of ribozyme 5 to give 5FR1 without impacting reactivity. Further truncation at the 3′ terminus to give 5FR2 resulted in a decrease in reactivity, and thus sequence 5FR1 was selected for further study. To test the ability of the ribozymes to function when fused to an RNA of interest, we inserted each ribozyme into the 3′ untranslated region (UTR) of an RNA containing 432 nucleotides of the coding sequence for the fluorescent protein mCherry. As observed in Figure 3e, we observe labeling for both of these ribozyme-RNA fusions compared with the mCherry RNA alone as a control. We also sought to evaluate the ability of the ribozymes to undergo labeling in a complex biological sample, as the thiols present in proteins and glutathione may outcompete the ribozyme for labeling with FIA. We were pleased to observe that labeling of the ribozymes proceeded in cell lysate with efficiencies similar to those observed in buffer (Supplementary Figure 3). To characterize the kinetics of the self-labeling reaction, we carried out labeling reactions under pseudo-first-order conditions using a large excess of FIA. Ribozymes 1 and 5FR1 were each reacted with FIA, then quenched, purified, and analyzed by HPLC as described above. For each concentration of FIA, we fit data from a series of time points to the integrated rate law [A] = [A]0 e−kt to give the pseudo-first-order rate constant (kobs) for that reaction (Figure 4a). We then calculated the second order rate constant (k2) for each reaction using the equation k2 = kobs /[FIA]. The values of k2 were averaged over the region for which kobs vs [FIA] was linear (10−50 μM for 1 and 10−200 μM for 5FR1), providing k2 values of 100 ± 8 and
Figure 3. (a) Denaturing PAGE showing fluorescein labeling of ribozyme sequences from SELEX using 10 μM (top) or 100 μM (bottom) FIA. (b) Labeling yields determined by HPLC for ribozyme sequences from SELEX. (c) Denaturing PAGE showing fluorescein labeling of truncated ribozyme sequences using 10 μM (top) or 100 μM (bottom) FIA. (d) Labeling yields determined by HPLC for truncated ribozyme sequences. Conditions: 4 μM RNA in selection buffer, 30 min, 37 °C. (e) Labeling of ribozymes 1 and 5FR1 when inserted into the 3′ UTR of an RNA encoding a portion of the mCherry protein. Conditions: 400 nM RNA in phosphate-buffered saline (8.1 mM Na2HPO4, 1.5 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, 0.90 mM CaCl2, 0.49 mM MgCl2), 200 μM FIA, 1 h, 37 °C.
⎡ A ·ε ⎤ % yield = ⎢ 495 260 ⎥ × 100 ⎣ A 260 ·ε495 ⎦
(1) Figure 4. (a) Pseudo-first-order rate constant vs [FIA] for ribozymes 1 and 5FR1. Solid lines show curve fitting to eq 3. (b) Kinetic and thermodynamic parameters for self-labeling of ribozymes 1 and 5FR1.
Figure 3b shows the ligation yield for each of the ribozyme sequences after reaction with either 10 μM or 100 μM FIA for 1682
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77 ± 12 M−1 s−1 for ribozymes 1 and 5FR1, respectively. Encouragingly, these rate constants are on par with those of other reactions that have been used successfully for intracellular biomolecule labeling.23,24 We hypothesize that ribozymes 1 and 5FR1 bind to FIA, increasing effective molarity and thus promoting the selflabeling reaction. The corresponding reaction mechanism is represented by eq 2 in which R is the ribozyme, F is FIA, P is labeled product, Ka is the association constant between the ribozyme and FIA, and ku is the unimolecular rate constant for conversion of ribozyme-FIA complex into labeled product. Ka
ku
R + F ⇌ R·F → P
observed almost no labeling of ribozymes 1 and 5FR1 with BFIA (Supplementary Figure 22). The yields for the BFIA reactions were too small to measure by HPLC, but quantification of the fluorescence bands in the gel enabled calculation of approximate FIA:BFIA selectivities of 160:1 and 280:1 for ribozymes 1 and 5FR1, respectively. In summary, we describe here the selection and characterization of ribozymes capable of promoting self-alkylation with an electrophilic fluorescein analogue, FIA. Kinetic studies reveal a second-order rate constant of 100 ± 8 M−1 s−1 for the most reactive ribozyme sequence, and our studies reveal that the ribozymes function at least in part by binding to FIA, thus increasing effective molarity. Investigation of the substrate selectivity of the ribozymes shows that they have high selectivity for fluorescein vs BODIPY FL iodoacetamide. Additionally, we demonstrate that labeling is specific to the ribozyme sequences, as FIA does not react nonspecifically with control RNA. In advancing our self-alkylating ribozymes toward use in cellular RNA labeling, we plan to wash excess fluorophore out of the cells after labeling. In the case of FIA, the high reactivity of the iodoacetamide group may lead to nonspecific labeling of other biomolecules, generating unwanted background signal. However, the ribozymes reported here function at least in part by binding to the fluorescein and promoting labeling via increased effective molarity. Thus, we anticipate that these ribozymes will be compatible with alternative fluorescein electrophiles having slightly attenuated reactivity. Additionally, our IP-SELEX methodology is broadly applicable for selecting ribozymes that self-alkylate with structurally diverse small molecule electrophiles, enabling background fluorescence to be minimized by replacing fluorescein with a fluorogenic small molecule. Experiments are underway in our laboratories to investigate cellular RNA labeling using these modified approaches. Together, the research reported here introduces fluorescently self-labeling ribozymes, demonstrates their utility for in vitro RNA labeling, and lays the foundation for the development of new methods for labeling and imaging RNA in living cells.
(2)
If our hypothesized mechanism is correct, then the data in Figure 4a should fit to eq 3:25 kobs =
k uK a[F] K a[F] + 1
(3)
Using OriginPro software, we fit our kinetic data to eq 3 (Figure 4a, solid lines) and obtained R-squared values of 0.972 and 0.989 for fitting of data from ribozymes 1 and 5FR1, respectively. The kinetic and thermodynamic parameters obtained from the curve fitting are shown in Figure 4b. These data demonstrate that the ribozymes do in fact bind to FIA, but the high micromolar Kd values observed reflect weaker affinity than is typical for RNA-small molecule interactions.26,27 This is likely due in large part to the fact that our reactions are carried out at 37 °C, whereas RNA-small molecule binding is usually measured at room temperature. However, two additional potential causes are (1) the concentration of FIA was held constant at 200 μM throughout the SELEX cycles, providing no selection pressure to achieve a low Kd value, or (2) in the immunoprecipitation step of our SELEX method, the antibody and RNA compete for binding to the fluorescein, and the sequences that survive selection are those for which fluorescein binds more efficiently to the antibody than to the RNA. Interestingly, the relatively weak affinity observed suggests that the ribozymes do not function simply by binding to FIA, as previously reported fluorescein aptamers have superior affinity for fluorescein28 but do not show detectable reaction with FIA (Supplementary Figure 20). Thus, it is likely that our ribozymes either bind FIA in a conformation that places the iodoacetamide in close proximity to a nucleophilic group, or specific residues in the ribozyme participate in activating the electrophile for nucleophilic attack. We were also curious to gain insight into the site at which the FIA reacts with the ribozyme. We chose 5FR1 for these studies, as it is the shorter and thus more practically useful ribozyme. The ribozyme was reacted with FIA, biotinylated at the 3′ terminus, and then subjected to alkaline hydrolysis. Sequences having the 3′ terminus intact were pulled down using streptavidin beads and were analyzed by PAGE (Supplementary Figure 21). These studies revealed that the FIA reacts within the 40 nt closest to the 5′ terminus of the ribozyme. Our data in Figure 3a,b demonstrate that labeling with FIA is selective to our ribozyme sequences, as the control sequence shows no observable labeling. However, we were also curious to investigate whether labeling of the ribozymes is specific to FIA in comparison with other iodoacetamides. To test this, we reacted ribozymes 1 and 5FR1 with 100 μM FIA or BODIPY FL iodoacetamide (BFIA) for 30 min. Encouragingly, we
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METHODS
IP SELEX. A dsDNA library (0.4−1 μg, see Supporting Information for sequence) was transcribed in a 20 μL reaction for 5 h at 37 °C using an AmpliScribe T7-Flash transcription kit (Epicentre Biotechnologies), followed by DNase treatment for 1 h at 37 °C. The resulting RNA was precipitated using sodium acetate/ethanol. The precipitated RNA library was dissolved in 500 μL of selection buffer (100 mM LiCl, 10 mM Na-HEPES, 5 mM MgCl2, pH 7.4) and coincubated with 200 μM 5-(iodoacetamido)fluorescein (Aldrich) at 37 °C. The incubation time was 8 h for the initial round of SELEX and was gradually shortened to 15 min over subsequent rounds of SELEX to increase the stringency of selection. The reaction was quenched by the addition of 100 mM β-mercaptoethanol, 5 mM EDTA, 0.3 M NaCl, and 50 μg of yeast tRNA (Ambion). After 15 min of quenching, the reaction was precipitated using sodium acetate/ethanol. Twenty microliters of antibody-functionalized magnetic beads (suspended in PBS) was placed on a magnetic separation stand to enable removal of the supernatant. The precipitated RNA was resuspended in 100 μL of PBS and added to the antibodyfunctionalized magnetic beads. The mixture was incubated at RT for 3 h with mild shaking. The unbound RNA was removed by washing the beads three times with 100 μL of wash buffer (1 M NaCl, 10 mM Na-HEPES, 5 mM EDTA, pH 7.4) and then three times with 100 μL of water. The RNA-bound beads were subjected to reverse transcription in a 25 μL reaction using 1 μL (200 units) of M-MLV 1683
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reverse transcriptase (Life Technologies). The reaction was incubated at 37 °C for 1 h, then diluted into ThermoPol buffer (New England Biolabs), and subjected to 34 rounds of PCR in a 500 μL reaction using the thermocycling protocol and primers described in the Supporting Information. The resulting dsDNA was precipitated using sodium acetate/ethanol. The dsDNA was separated on a 2% agarose gel at 70 V for 45 min, and the desired band was cut from the gel and purified using a Zymoclean Gel DNA Recovery Kit (Zymo Research). The resulting dsDNA was used as a template for in vitro transcription for the next round of SELEX. After 12 rounds of SELEX, the dsDNA was cloned into a TOPO vector using a TOPO TA cloning kit (Life Technologies) and amplified in E. coli. Plasmid DNA from 20 individual colonies was recovered using a MiniPrep kit (Qiagen) and was sequenced at the University of Utah DNA sequencing core facility.
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(11) Wiesmayr, A., and Jäschke, A. (2011) Isolation and characterization of fluorescence-enhancing RNA tags. Bioorg. Med. Chem. 19, 1041−1047. (12) Lee, J., Lee, K. H., Jeon, J., Dragulescu-Andrasi, A., Xiao, F., and Rao, J. (2010) Combining SELEX screening and rational design to develop light-up fluorophore-RNA aptamer pairs for RNA tagging. ACS Chem. Biol. 5, 1065−1074. (13) Sunbul, M., and Jäschke, A. (2013) Contact-mediated quenching for RNA imaging in bacteria with a fluorophore-binding aptamer. Angew. Chem., Int. Ed. 52, 13401−13404. (14) Paige, J. S., Wu, K. Y., and Jaffrey, S. R. (2011) RNA mimics of green fluorescent protein. Science 333, 642−646. (15) Strack, R. L., Disney, M. D., and Jaffrey, S. R. (2013) A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat-containing RNA. Nat. Methods 10, 1219−1224. (16) Paige, J. S., Nguyen-Duc, T., Song, W., and Jaffrey, S. R. (2012) Fluorescence imaging of cellular metabolites with RNA. Science 335, 1194. (17) Kellenberger, C. A., WIlson, S. C., Sales-Lee, J., and Hammond, M. C. (2013) RNA-based fluorescent biosensors for live cell imaging of second messengers cyclic di-GMP and cyclic AMP-GMP. J. Am. Chem. Soc. 135, 4906−4909. (18) Höfer, K., Langejürgen, L. V., and Jäschke, A. (2013) Universal aptamer-based real-time monitoring of enzymatic RNA synthesis. J. Am. Chem. Soc. 135, 13692−13694. (19) Gronemeyer, T., Godin, G., and Johnsson, K. (2005) Adding value to fusion proteins through covalent labelling. Curr. Opin. Biotechnol. 16, 453−458. (20) Wilson, C., and Szostak, J. W. (1995) In vitro evolution of a selfalkylating ribozyme. Nature 374, 777−782. (21) Sjöback, R., Nygren, J., and Kubista, M. (1995) Absorption and fluorescence properties of fluorescein. Spectrochim. Acta, Part A 51, L7−L21. (22) Silverman, S. K. (2008) Catalytic DNA (deoxyribozymes) for synthetic applications-current abilities and future prospects. Chem. Commun., 3467−3485. (23) Debets, M. F., van, B. S. S., Schoffelen, S., Rutjes, F. P. J. T., van Hest, J. C. M., and van Delft, F. L. (2010) Aza-dibenzocyclooctynes for fast and efficient enzyme PEGylation via copper-free (3 + 2) cycloaddition. Chem. Commun., 97−99. (24) Yao, J. Z., Uttamapinant, C., Poloukhtine, A. A., Baskin, J. M., Codelli, J. A., Sletten, E. M., Bertozzi, C. R., Popik, V. V., and Ting, A. Y. (2012) Fluorophore targeting to cellular proteins via enzymemediated azide ligation and strain-promoted cycloaddition. J. Am. Chem. Soc. 134, 3720−3728. (25) Heemstra, J. M., and Moore, J. S. (2004) Enhanced methylation rate within a foldable molecular receptor. J. Org. Chem. 69, 9234− 9237. (26) Famulok, M. (1999) Oligonucleotide aptamers that recognize small molecules. Curr. Opin. Struct. Biol. 9, 324−329. (27) Osborne, S. E., and Ellington, A. D. (1997) Nucleic acid selection and the challenge of combinatorial chemistry. Chem. Rev. 97, 349−370. (28) Holeman, L. A., Robinson, S. L., Szostak, J. W., and Wilson, C. (1998) Isolation and characterization of fluorophore-binding RNA aptamers. Folding Des. 3, 423−431.
ASSOCIATED CONTENT
S Supporting Information *
Complete sequence data, tabulated self-labeling yields, secondary structure and reaction site analysis, and kinetic curve fitting data. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank M. Oney for help with plasmid construction. This work was supported by a University of Utah Funding Incentive Seed Grant to J.H. and J.M.H.
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REFERENCES
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