Letter Cite This: ACS Sens. XXXX, XXX, XXX−XXX
pubs.acs.org/acssensors
A Mirror Image Fluorogenic Aptamer Sensor for Live-Cell Imaging of MicroRNAs Wenrui Zhong and Jonathan T. Sczepanski* Department of Chemistry, Texas A&M University, College Station, Texas 77842, United States
ACS Sens. Downloaded from pubs.acs.org by IMPERIAL COLLEGE LONDON on 03/16/19. For personal use only.
S Supporting Information *
ABSTRACT: Development of biocompatible tools for intracellular imaging of RNA expression remains a central challenge. Herein, we report the use of heterochiral strand-displacement to sequence-specifically interface endogenous D-miRNAs with an LRNA version of the fluorogenic aptamer Mango III, thereby generating a novel class of biocompatible miRNA sensors. Fluorescence activation of the sensor is achieved through the displacement of an achiral blocking strand from the L-Mango aptamer by the D-RNA target. In contrast to D-Mango, we show that the L-Mango sensor retains full functionality in serum, enabling a light-up fluorescence response to the target. Importantly, we employ a self-delivering version of the L-Mango sensor to image the expression of microRNA-155 in living cells, representing the first time L-oligonucleotides have been interfaced with a living system. Overall, this work provides a new paradigm for the development of biocompatible hybridization-based sensors for live-cell imaging of RNAs and greatly expands the utility of fluorogenic aptamers for cellular applications. KEYWORDS: fluorogenic aptamer, mango aptamer, L-RNA, microRNA imaging, peptide nucleic acid, heterochiral strand-displacement, hybridization probe
M
susceptible to off-target interactions with cellular components.16,17 Therefore, L-nucleic acid-based molecular probes are expected to have dramatically improved intracellular performance, reliability, and utility compared to those composed of the native stereoisomer. Despite these advantages, however, L-oligonucleotides are incapable of forming contiguous WC base pairs with native D-nucleic acids,18,19 which until now has limited their usefulness in the design of hybridization-based probes for imaging endogenous RNAs. In order to overcome this limitation, we recently reported a novel strand-displacement methodology for sequence-specifically interfacing oligonucleotides of opposite stereochemistry.20 Our approach takes advantage of peptide nucleic acid (PNA), which unlike native oligonucleotides, has no inherent chirality. As a result, PNA hybridizes to both DNA and RNA irrespective of stereochemistry. On the basis of this property, we have shown that PNA can serve as an intermediary allowing D- and L-oligonucleotides to be interfaced in a sequencespecific manner. Using this approach, we now report the first attempt to image RNA in live cells using a programmable, hybridization-based sensor composed of bio-orthogonal Lnucleic acids. As depicted in Figure 1a, the sensor (L-pM) consists of a heteroduplex between an achiral PNA strand (P1) and a fluorogenic aptamer composed of L-RNA (L-M). Fluorogenic
icroRNAs (miRNAs) are a large family of short, noncoding RNAs that play a critical role in posttranscriptional regulation of gene expression.1,2 Moreover, aberrant miRNA expression is associated with a wide range of human diseases, including cancer.3,4 Consequently, significant efforts have been made to image miRNA expression in living cells and organisms, which not only provides unparalleled insight into the biological functions and dynamics of these important molecules, but also holds great promise for early disease detection. Owing to the straightforward programmability of Watson−Crick (WC) base-pairing rules, current strategies for imaging endogenous miRNAs mostly rely on the use of hybridization-based probes, such as molecular beacons,5−8 binary probes,9−11 and molecular switches,12−14 all of which are composed of nucleic acids. Despite their success for imaging miRNA in living cells, nucleic acid-based probes still suffer from two key limitations when applied to biological environments: rapid nuclease degradation and unintended interactions with endogenous macromolecules, both of which adversely affect performance and/or sensitivity of the probe.15 Thus, development of biocompatible nucleic acid-based probes for live-cell miRNA imaging remains an unmet need. In principle, many of the disadvantages associated with the use of nucleic acids in live cells can be overcome by simply inverting the stereochemistry of the sugar backbone. Indeed, L(deoxy)ribose-based nucleic acids (L-DNA and L-RNA), which are synthetic enantiomers of natural D-nucleotides, are intrinsically resistant to nuclease degradation and less © XXXX American Chemical Society
Received: February 2, 2019 Accepted: March 7, 2019 Published: March 7, 2019 A
DOI: 10.1021/acssensors.9b00252 ACS Sens. XXXX, XXX, XXX−XXX
Letter
ACS Sensors
Figure 1. Design of a mirror-image fluorogenic aptamer sensor for detection of miRNAs in live cells. (a) Schematic illustration of the heterochiral strand-displacement sensor mechanism. Oligonucleotides are depicted as lines with the half arrow denoting the 3′ end (or C-terminus for PNA), and an asterisk indicating complementarity. D-DNA (black), L-DNA (blue), and PNA (green) are distinguished by color throughout the text. (b) Sequences of oligonucleotides described in this work. The core TO binding domain of the Mango III aptamer is highlighted in orange and the closing stem domain (3/3*) is highlighted in blue. The PNA binding site for each Mango III variant (M-1−4) is indicated by bold lettering. (c) Optimization of the signal-to-background ratio based on different sequence designs. All reactions contained either 0 or 400 nM P1, 400 nM of the indicated aptamer, 400 nM TO dye, 100 mM KCl, 0.5 mM MgCl2, and 10 mM HEPES (pH 7.4) and were carried out at 37 °C for 10 min. All fluorescence values were normalized to wild-type Mango III (M-0).
S1a). In the absence of the PNA blocking strand, both D- and activated the fluorescence of TO to a similar extent, confirming that L-RNA versions of fluorogenic aptamers retain their activity (Figures 2a and S1b). Likewise, in the presence of
aptamers are engineered to bind nonfluorescent small molecule dyes, resulting in strong fluorescent activation.21,22 We chose the recently identified Mango III aptamer as the model system for this study due to its bright fluorescent signal, high binding affinity toward its target dye (thiazole orange; TO), and small size relative to other fluorogenic aptamers (e.g., Spinach and Broccoli).23 Importantly, TO dyes are achiral, implying that they can be bound and activated by both D- and L-versions of Mango III. Throughout the current study we utilized a biotinylated version of TO (TO1-biotin), which we refer to simply as TO. In the absence of the D-RNA input (D-IN), folding of a critical stem domain (3/3*) in the aptamer is blocked by the bound PNA strand (Figure 1a). Because the toehold domain (1*) resides on the achiral PNA, D-IN can still bind to the sensor (via 1 and 1*) and displace the incumbent L-Mango III aptamer (L-M) from the PNA blocking strand. This enables proper folding of the aptamer, which in turn activates an enhanced fluorescent signal by binding TO. We designed the sensor based on the sequence of the PNA blocking strand (P1), which was itself complementary to the intended intracellular target, miRNA-155 (D-IN; Figure 1b). MiRNA-155 is a prototypical oncogenic miRNA associated with the development and invasiveness of various types of malignancies.24 Based on the sequence of miRNA-155, the sequence of the closing stem domain of Mango III (3/3*) required significant changes compared to the parent aptamer (M-0; Figure 1b). However, previous biochemical and structural studies have suggested that the sequence of this stem could be varied, as long as complementarity was maintained.23 Based on this design consideration, we prepared several D-RNA versions of the Mango III aptamer (M-1−4), varying the length of domains 2 and 3, as well as the position of the PNA binding site relative to the 5′ end of the aptamer. We initially examined the ability of the PNA strand (P1) to prevent folding of the aptamer in the presence of TO (Figure 1c). We found that D-M-4 (Tm ∼ 60 °C25) exhibited the greatest signal-to-background ratio in the presence of the PNA (>70-fold), and was therefore used in all subsequent studies. Next, we synthesized the L-RNA version of M-4 (L-M-4), which exhibited mirror image symmetry with D-M-4 when measured by circular dichroism (CD) spectroscopy (Figure
L-M-4
Figure 2. In vitro characterization of the pM-4 sensor. (a) Fluorescence activation of pM-4 under various conditions. All reactions contained 400 nM of the indicated aptamer complex (M4 or pM-4), 0 or 400 nM D-IN, 400 nM TO dye, 100 mM KCl, 0.5 mM MgCl2, and 10 mM HEPES (pH 7.4) and were carried out at 37 °C for 20 min. The presence or absence of 10% FBS is indicated by shading. For reference, the fluorescence of TO alone in the presence (+) or absence (−) of 10% FBS is also shown. (b) Fluorescence monitoring (Mango) of L-pM-4 activation in the absence of (solid line) or presence of (dashed line) 10% FBS. Reaction conditions were identical to those described in (a). Fluorescence values in both (a) and (b) were normalized to the intensity of the unblocked Mango aptamer (D- or L-M-4). The asterisk indicates use of a DNA version of the input (D-IN).
the PNA blocking strand, both P1:M-4 complexes (D- and LpM-4) were unable to activate TO fluorescence, indicating that the achiral PNA strand hybridized efficiently to both D- and LRNA versions of the aptamer. Importantly, treatment of both D- and L-pM-4 complexes with an equimolar amount of D-IN RNA resulted in the recovery of approximately 55% of the fluorescent signal observed for the unblocked aptamers within 20 min (Figure 2a). Proper operation of the sensor in the presence of D-IN was further validated by native gel electrophoresis (Figure S2a). Even in the presence of excess D-IN, we were unable to fully activate the fluorescence signal of B
DOI: 10.1021/acssensors.9b00252 ACS Sens. XXXX, XXX, XXX−XXX
Letter
ACS Sensors the pM-4 sensor relative to free M-4 aptamer (Figure S2b). Given the unique mechanism of strand-displacement, which requires helical inversion through domains 2 and 3, it is possible that a fraction of the M-4 aptamer remains improperly folded upon release of the PNA blocking strand. Nevertheless, the >50-fold fluorescence enhancement upon the addition of D-IN (i.e., miRNA-155) to D- and L-pM-4 was deemed more than sufficient for subsequent intracellular studies. In contrast, D-IN failed to activate the sensor when the sequence of the toehold domain (1*) on the PNA blocking strand was scrambled (Figure S2b). Furthermore, only a minor fluorescence signal was detected for both D- and L-pM-4 complexes in the presence of excess nuclear RNAs (200-fold) relative to wild-type HeLa cells (Figure S4). For perspective, the expressions level of miRNA-155 in HeLa155 cells was found to be ∼30-fold higher than in MDA-MB-231 breast cancer cells known to have elevated levels of miRNA-155 (Figure S4).32 As shown in Figure 4a, the Mango fluorescence signal was notably brighter in HeLa155 cells compared to wild-type HeLa cells, consistent with overexpression of miRNA-155 in HeLa155 cells. Ratiometric quantification of these data, which was carried out by dividing the averaged fluorescence intensities of Mango and Cy5 (FMango/FCy5), revealed that ∼2-fold more L-pM-4.chol sensors were activated within HeLa155 cells as compared to wild-type HeLa cells (Figure 4b). This result was further confirmed by flow cytometry (Figure 4c). In contrast, when we scrambled the sequence of the of the toehold domain (1*) on the PNA blocking strand, no significant difference in fluorescence was observed between wild-type and HeLa155 cells (L-pM-4.chol.S; Figure 4a). A similar result was obtained using a version of the sensor having a defunct Mango aptamer (L-pM-4.chol.M; Figures 4a and S2b). Together, these results indicated that the L-pM-4.chol sensor successfully activated a fluorescent signal in response to elevated miRNA-155 expression levels in live cells. We note that treatment of wild-type HeLa cells with TO alone resulted in a fluorescence signal that was nearly equivalent to the same cells treated with L-pM-4.chol (Figure 4c), suggesting that the TO dye itself was responsible for the majority of background fluorescence in the absence of miRNA-155 expression. Indeed, nonspecific fluorescence in cells has been previously observed for TO dyes.21 We also confirmed that the background was not L-M-4.chol
C
DOI: 10.1021/acssensors.9b00252 ACS Sens. XXXX, XXX, XXX−XXX
Letter
ACS Sensors
major advances in the area of intercellular biosensing, which we anticipate will have a far-reaching impact on how nucleic acid-based sensors are designed and implemented.
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssensors.9b00252. Materials and methods with associated characterization and imaging (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Jonathan T. Sczepanski: 0000-0002-9275-2597 Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS The authors are grateful to Dr. Jean-Philippe Pellois for insightful suggestions and comments. This work was supported by the Cancer Prevention and Research Institute of Texas (RR150038) and The Welch Foundation (A1909).
■
REFERENCES
(1) Bartel, D. P. MicroRNAs: Target recognition and regulatory function. Cell 2009, 136, 215−233. (2) Wilson, R. C.; Doudna, J. A. Molecular mechanisms of RNA interference. Annu. Rev. Biophys. 2013, 42, 217−239. (3) Croce, C. M. Causes and consequences of microRNA dysregulation in cancer. Nat. Rev. Genet. 2009, 10, 704−714. (4) Iorio, M. V.; Croce, C. M. MicroRNA Dysregulation in Cancer: Diagnostics, monitoring and therapeutics. A comprehensive review. EMBO Mol. Med. 2012, 4, 143−159. (5) Yao, Q.; Zhang, A. M.; Ma, H.; Lin, S.; Wang, X. X.; Sun, J. J.; Chen, Z. T. Novel molecular beacons to monitor microRNAs in nonsmall-cell lung cancer. Mol. Cell. Probes 2012, 26, 182−187. (6) Ko, H. Y.; Lee, J.; Joo, J. Y.; Lee, Y. S.; Heo, H.; Ko, J. J.; Kim, S. A color-tunable molecular beacon to sense miRNA-9 expression during neurogenesis. Sci. Rep. 2015, DOI: 10.1038/srep04626. (7) Catrina, I. E.; Marras, S. A. E.; Bratu, D. P. Tiny molecular beacons: LNA/2′-O-methyl RNA chimeric probes for imaging dynamic mRNA processes in living cells. ACS Chem. Biol. 2012, 7, 1586−1595. (8) Cheglakov, Z.; Cronin, T. M.; He, C.; Weizmann, Y. Live Cell MicroRNA Imaging Using Cascade Hybridization Reaction. J. Am. Chem. Soc. 2015, 137, 6116−6119. (9) Sando, S.; Abe, H.; Kool, E. T. Quenched auto-ligating DNAs: Multicolor identification of nucleic acids at single nucleotide resolution. J. Am. Chem. Soc. 2004, 126, 1081−1087. (10) Wu, H.; Alexander, S. C.; Jin, S.; Devaraj, N. K. A Bioorthogonal near-infrared fluorogenic probe for mRNA detection. J. Am. Chem. Soc. 2016, 138, 11429−11432. (11) Holtzer, L.; Oleinich, I.; Anzola, M.; Lindberg, E.; Sadhu, K. K.; Gonzalez-Gaitan, M.; Winssinger, N. Nucleic acid templated chemical reaction in a live vertebrate. ACS Cent. Sci. 2016, 2, 394−400. (12) Hartig, J. S.; Grüne, I.; Najafi-Shoushtari, S. H.; Famulok, M. Sequence-specific detection of microRNAs by signal-amplifying ribozymes. J. Am. Chem. Soc. 2004, 126, 722−723. (13) Ying, Z. M.; Wu, Z.; Tu, B.; Tan, W.; Jiang, J. H. Genetically encoded fluorescent RNA sensor for ratiometric imaging of microRNA in living tumor cells. J. Am. Chem. Soc. 2017, 139, 9779−9782.
Figure 4. Detection of miRNA-155 in HeLa cells. (a) Representative fluorescence microscopy images of different HeLa cell lines following a 12 h incubation with either L-pM-4.chol or control sensors L-pM4.chol.S (scrambled toehold) and L-pM-4.chol.M (defunct Mango aptamer). Scale bar: 100 μm. (b) Mean fluorescence intensities (FMango/FCy5) in the above cell lines. Error bars represent the standard deviation from at least six images obtained from two separate experiments. (c) Flow cytometry histogram of HeLa cells treated with either L-pM-4.chol or TO alone (the black line indicates the mean value).
due to accumulation of TO in dead cells (Figure S5). In the future, it will be important to further optimize the imaging approach reported herein, possibly through the use of alternative dyes or aptamer−dye pairs, in order to increase the sensitivity of the sensor for detection for low copy number RNAs in living cells. In summary, we have developed a novel fluorogenic aptamer-based sensor composed of L-RNA. The sensor exhibited excellent stability in both serum and living cells, where it was successfully employed to image the expression of miRNA-155. We note that, in its current form, the sensor cannot be used to track the intracellular location of its RNA targets. In the future, it may be useful to redesign the sensor for this purpose. Importantly, this is the first time an endogenously expressed nucleic acid has been sequence-specifically interfaced with a synthetic L-oligonucleotide in a living system. Thus, this study provides a critical starting point for interfacing more complex L-oligonucleotide-based circuits with living cells and organisms for exciting applications in bioengineering, synthetic biology, and clinical diagnostics. Moreover, our results show that fluorogenic aptamers composed of L-RNA are compatible with complex biological environments and live-cell imaging, thereby greatly expanding the utility of this important class of bioimaging tools. Taken together, this study signifies several D
DOI: 10.1021/acssensors.9b00252 ACS Sens. XXXX, XXX, XXX−XXX
Letter
ACS Sensors (14) Deng, R.; Tang, L.; Tian, Q.; Wang, Y.; Lin, L.; Li, J. Toeholdinitiated rolling circle amplification for visualizing individual microRNAs in situ in single cells. Angew. Chem., Int. Ed. 2014, 53, 2389− 2393. (15) Chen, Y. J.; Groves, B.; Muscat, R. A.; Seelig, G. DNA nanotechnology from the test tube to the cell. Nat. Nanotechnol. 2015, 10, 748−760. (16) Ashley, G. W. Modeling, synthesis, and hybridization properties of (L)-ribonucleic acid. J. Am. Chem. Soc. 1992, 114, 9732−9736. (17) Urata, H.; Ogura, E.; Shinohara, K.; Ueda, Y.; Akagi, M. Synthesis and properties of mirror-image DNA. Nucleic Acids Res. 1992, 20, 3325−3332. (18) Hoehlig, K.; Bethge, L.; Klussmann, S. Stereospecificity of oligonucleotide interactions revisited: No evidence for heterochiral hybridization and ribozyme/DNAzyme activity. PLoS One 2015, DOI: 10.1371/journal.pone.0115328. (19) Szabat, M.; Gudanis, D.; Kotkowiak, W.; Gdaniec, Z.; Kierzek, R.; Pasternak, A. Thermodynamic features of structural motifs formed by β-L-RNA. PLoS One 2016, DOI: 10.1371/journal.pone.0149478. (20) Kabza, A. M.; Young, B. E.; Sczepanski, J. T. Heterochiral DNA strand-displacement circuits. J. Am. Chem. Soc. 2017, 139, 17715− 17718. (21) Paige, J. S.; Wu, K. Y.; Jaffrey, S. R. RNA mimics of green fluorescent protein. Science 2011, 333, 642−646. (22) You, M.; Jaffrey, S. R. Structure and mechanism of RNA mimics of green fluorescent protein. Annu. Rev. Biophys. 2015, 44, 187−206. (23) Autour, A.; C. Y. Jeng, S.; D. Cawte, A.; Abdolahzadeh, A.; Galli, A.; Panchapakesan, S. S. S.; Rueda, D.; Ryckelynck, M.; Unrau, P. J. Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells. Nat. Commun. 2018, DOI: 10.1038/ s41467-018-02993-8. (24) Higgs, G.; Slack, F. The multiple roles of microRNA-155 in oncogenesis. J. Clin. Bioinf. 2013, 3, 17. (25) Giesen, U.; Kleider, W.; Berding, C.; Geiger, A.; Orum, H.; Nielsen, P. E. A formula for thermal stability (Tm) prediction of PNA/DNA duplexes. Nucleic Acids Res. 1998, 26, 5004−5006. (26) Klussmann, S.; Nolte, A.; Bald, R.; Erdmann, V. A.; Fürste, J. P. Mirror-image RNA that binds D-adenosine. Nat. Biotechnol. 1996, 14, 1112−1115. (27) Dowdy, S. F. Overcoming cellular barriers for RNA therapeutics. Nat. Biotechnol. 2017, 35, 222−229. (28) Krutzfeldt, J.; Rajewsky, N.; Braich, R.; Rajeev, K. G.; Tuschl, T.; Manoharan, M.; Stoffel, M. Silencing of microRNAs in vivo with ’Antagomirs’. Nature 2005, 438, 685−689. (29) Wolfrum, C.; Shi, S.; Jayaprakash, K. N.; Jayaraman, M.; Wang, G.; Pandey, R. K.; Rajeev, K. G.; Nakayama, T.; Charrise, K.; Ndungo, E. M.; Zimmermann, T.; Koteliansky, V.; Manoharan, M.; Stoffel, M. Mechanisms and optimization of in vivo delivery of lipophilic siRNAs. Nat. Biotechnol. 2007, 25, 1149−1157. (30) Lee, C. H.; Lee, S. H.; Kim, J. H.; Noh, Y. H.; Noh, G. J.; Lee, S. W. Pharmacokinetics of a cholesterol-conjugated aptamer against the hepatitis C virus (HCV) NS5B protein. Mol. Ther. Nucleic Acids 2015, DOI: 10.1038/mtna.2015.30. (31) Zhou, J.; Li, H.; Li, S.; Zaia, J.; Rossi, J. J. Novel dual inhibitory function aptamer−siRNA delivery system for HIV-1 therapy. Mol. Ther. 2008, 16, 1481−1489. (32) Volinia, S.; Calin, G. A.; Liu, C. G.; Ambs, S.; Cimmino, A.; Petrocca, F.; Visone, R.; Iorio, M. V.; Roldo, C.; Ferracin, M.; Prueitt, R. L.; Yanaihara, N.; Lanza, G.; Scarpa, A.; Vecchione, A.; Negrini, M.; Harris, C. C.; Croce, C. M. A MicroRNA Expression Signature of Human Solid Tumors Defines Cancer Gene Targets. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 2257−2261.
E
DOI: 10.1021/acssensors.9b00252 ACS Sens. XXXX, XXX, XXX−XXX
