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Branched Hybridization Chain Reaction (bHCR) Circuit for Ultrasensitive Localizable Imaging of mRNA in Living Cells Lan Liu, Jin-Wen Liu, Han Wu, Xiang-Nan Wang, Ru-Qin Yu, and Jian-hui Jiang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04848 • Publication Date (Web): 04 Jan 2018 Downloaded from http://pubs.acs.org on January 4, 2018
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Analytical Chemistry
Branched Hybridization Chain Reaction (bHCR) Circuit for Ultrasensitive Localizable Imaging of mRNA in Living Cells Lan Liu, Jin-Wen Liu, Han Wu, Xiang-Nan Wang, Ru-Qin Yu*, Jian-Hui Jiang* Institute of Chemical Biology and Nanomedicine, State Key Laboratory of Chemo/ Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, P. R. China * Corresponding Author: Fax: +86-731-88821916; E-mail:
[email protected];
[email protected] ABSTRACT: Hybridization chain reaction (HCR) circuits are valuable approaches to monitor low-abundance mRNA, and current HCR is still subjected to issues such as limited amplification efficiency, compromised localization resolution or complicated designs. We report a novel branched HCR (bHCR) circuit for efficient signal-amplified imaging of mRNA in living cells. The bHCR can be realized using a simplified design by hierarchically coupling two HCR circuits with two split initiator fragments of the secondary HCR circuit incorporated in the probes for the primary HCR circuit. The bHCR circuit enables to generate a hyperbranched assembly seeded from a single target initiator, affording the potential for localizing single target molecules in live cells. In vitro assays show that bHCR offers very high amplification efficiency and specificity in single mismatch discrimination with a detection limit of 500 fM. Live cell studies reveal that bHCR displays intense fluorescence spots indicating mRNA localization in living cells with improved contrast. The bHCR method can provide a useful platform for low-abundance biomarker detection and imaging for cell biology and diagnostics.
Messenger RNAs (mRNAs) play a fundamental role in conveying the genetic blueprint encoded by DNA to proteins. Abnormalities of mRNA expression could be useful biomarkers for various diseases such as cancer.1 Hybridization based probes allowing directly detection and imaging of endogenous mRNA in living cells afford a valuable approach for study of mRNA biology and diagnosis of related diseases.2 Molecular beacons (MBs)3,4 and spherical nucleic acids (SNAs)5,6 are the most common probes for mRNA detection in living cells. These probes convert the mRNA hybridization events into fluorescence signals in an equivalent reaction ratio. The lack of signal amplification in these probes limits their utility for expression analysis of low-abundance mRNA. While expression of mRNA is heterogeneous, failures in monitoring the low-abundance subpopulations could be impediments to understanding the functions of these mRNA and early assessment of the associated diseases.7 Nucleic acid amplification provides a direct solution to expression analysis of low-abundance mRNA.8,9 Recent development of nucleic acid circuits, such as hybridization chain reaction (HCR),10-12 cascade hybridization reaction,13 catalyzed hairpin assembly (CHA),14 and entropy driven catalysis,15 has enabled programming of the kinetically controlled assembly of DNA modules, which creates an efficient approach for non-enzymatic amplification. An obvious advantage of such isothermal, enzyme-free amplification is the applicability to highly sensitive nucleic acid based sensors for signal amplified imaging in living cells. Motivated by this rationale, others14,16,17 and our group18 have developed
sensitive imaging approaches for visualizing RNAs in living cells. Nonetheless, current non-enzymatic amplification circuits are realized in chain-like reactions, only conferring limited efficiency in signal amplification and compromised resolution for localization. To address this issue, new HCR circuits with nonlinear or branched reactions have been developed.19-22 We have demonstrated a branched HCR design that is realized using multi-step consecutive reactions for visualization of single mRNA molecule in fixed cells or tissues.19 However, the multiple reaction and washing steps preclude its applications in living cells. Other dendritic or hyperbranched HCR circuits, though allowing generating a hyperbranched assembly in a single reaction,20-23 still require complicated designs using multiple-hairpin probes or multiple probes. Moreover, these nonlinear HCR circuits have been largely unexplored for mRNA imaging. Herein, we report a novel branched HCR (bHCR) circuit that allows formation of a hyperbranched assembly of probes in a single reaction using a simplified design for efficient signal-amplified imaging of mRNA in living cells. This bHCR strategy relies on a hierarchical coupling design of two HCR circuits in a single reaction, as illustrated in Scheme 1. A primary linear HCR circuit is designed using two hairpin probes H1 and H2, which are triggered by target mRNA to form the backbone chain of the hyperbranched assembly. A distinct design of probes H1 and H2 is that two split initiator fragments of the secondary HCR are incorporated in the loop region of H1 and the toehold region of H2, respectively. When the backbone assembly forms in response to target mRNA,
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these split initiator fragments are drawn into close proximity, activating the secondary HCR circuit and enabling branched growth of chain-like assembly between hairpin probes H3 and H4. The hierarchical coupling of the primary and the secondary HCR circuits is able to generate a hyperbranched, brush-like assembly with a single mRNA target as the initiator. In order to light up the mRNA target, the probe F-H3 is designed to have a fluorophore FAM and a quencher BHQ1 in the stem region of H3. This design allows the probe to exhibit a low fluorescence background in its folded, hairpin state while deliver enhanced fluorescence in the hyperbranched assembly in which H3 is extended by hybridization with H4. When the probes H1, H2, H3 and H4 are transfected into cells, a hyperbranched, highly-fluorescent assembly of these probes is generated seeded from each single mRNA target, enabling high-contrast and spatially localizable imaging of the mRNA target. Hence, our design can provide a useful platform for highly sensitive and localizable imaging of intracellular mRNA, especially for the low-abundance subpopulations.
with lower concentration of the initiators, suggested high ratio of probes to initiator gave larger HCR assembly. No new bands appeared when the initiator was absent. These results evidenced successful construction of the primary and secondary HCR circuits. After confirming two individual HCR circuits, we examined bHCR using gel electrophoresis (Figure 1a). After incubating 250 nM target T with 500 nM H1, 500 nM H2, 1 μM H3 and 1 μM H4, we obtained many bright bands with size around 10,000 bp (lane 4). In contrast, the linear HCR products obtained by incubating 250 nM T with 500 nM H1, 500 nM H2 (lane 1) or incubating 500 nM T2 with 1 μM H3 and 1 μM H4 (lane 2) gave much smaller sizes. Incubation of 250 nM T with 500 nM H1 and 500 nM H2 (lane 3) and 1 μM H3 also gave smaller-size bands as linear HCR. These observations manifested that coupling of two individual HCR circuits afforded larger assembly of the probes, validating the proposed bHCR design. In the absence of target T, there were some low-fluorescence bands (lane 5), which was attributed to marginal system leakage from imperfectly annealed hairpins.
Scheme 1. Working principle of bHCR for mRNA imaging.
To demonstrate the principle of our bHCR strategy for mRNA imaging in living cells, we chose survivin mRNA, an important biomarker overexpressed in many malignant tumors,24 as the model target. Based on bioinformatic calculation of the secondary structure of the mRNA, a target region of 24 nucleotides with no predicted hairpin structure was selected. Accordingly, the hairpin probes for the primary HCR circuit H1 and H2 and for the secondary circuit H3 and H4 were designed. To investigate the feasibility of these probes, the primary and secondary HCR circuits were firstly analyzed using agarose gel electrophoresis. Gel images showed that bright ladder-like bands characteristic for HCR circuits were obtained when 1 μM target RNA sequence T was incubating with 1 μM H1 and 1 μM H2, or 1 μM secondary initiator sequence T2 was incubating with 1 μM H3 and 1 μM H4 (Figure S-1). The results verified HCR assembly of H1 and H2 or H3 and H4 seeded by target RNA sequence or secondary initiator sequence, respectively. We also observed brighter bands with larger molecular weights
Figure 1. (a) Gel electrophoresis analysis of bHCR. lane 1, 500 nM H1 and 500 nM H2 with 250 nM T; lane 2, 1 μM H3 and 1 μM H4 with 500 nM T2; lane 3, 500 nM H1, 500 nM H2, 1 μM H3 with 250 nM T; lane 4, 500 nM H1, 500 nM H2, 1 μM H3 and 1 μM H4 with 250 nM T; lane 5, 500 nM H1, 500 nM H2, 1 μM H3 and 1 μM H4. (b-f) AFM images of the assembly products in primary HCR circuit (b), seconadry HCR circuit (c), bHCR system in the presence of T (d) and absence of T (e), (f) enlarged details of (d). Scale bar: 200 nm.
To further confirm the bHCR system, we direct visualized the morphology of the assembly products with AFM imaging. When only incubated H1, H2, H3 and H4, we obtained tiny spots with heights of ~1.5 nm (Figure 1e), which were ascribed to the hairpin probes failing to be assembled in the absence of target RNA. In contrast, linear
structures were found when T was incubated with H1 and H2, or T2 was incubated with H3 and H4 (Figure 1b, 1c), suggesting linear polymers generated in HCR. Many branched structures were found in the bHCR system upon incubating target T with H1, H2, H3 and H4 (Figure 1e, 1f and S-2). The bHCR products were polydisperse both in sizes and branching efficiency because of the random assembly of the hairpins, which, therefore, also generated a few smaller linear polymers. These images gave clear
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Analytical Chemistry evidences for branched assembly in the bHCR system. This finding was consistent with the extended bands of varying molecular weights in the gel images.
Figure 2. (a) Fluorescence spectral responses for mixtures of 200 nM H1 and 200 nM H2 (blue), 200 nM F-H1 and 200 nM H2 with 2 nM T (green), 200 nM H1 and 200 nM H2, 200 nM F-H3 and 200 nM H4 (black), 200 nM H1, 200 nM H2, 200 nM F-H3 and 200 nM H4 with 2 nM T (pink), or with 2 nM one-base mismatched T (Mu-T) (red). (b) Fluorescence responses of bHCR system to T of varying concentrations. (c) Calibration curves of fluorescence intensities versus target concentrations. Insert shows the linear relationship between fluorescence intensities and target concentrations in the linear range. (d) Histograms of signal-to-background (STB) ratios of bHCR system (blue) and linear system (red) at different target concentrations.
The bHCR system was further investigated using fluorescence spectroscopy for in vitro detection of target sequence. Incubation of 200 nM H1, 200 nM H2, 200 nM F-H3 and 200 nM H4 for 3 h at 37 °C only gave a weak fluorescence peak (Figure 2a), indicating low fluorescence background and marginal leakage for the bHCR system. Appreciable fluorescence activation was observed when 2 nM target sequence T was incubated with 200 nM H1, 200 nM H2, 200 nM F-H3 and 200 nM H4, and the signal-to-background ratio was ~8 fold. This observation implied high-contrast activation of the bHCR system in response to the target. Interestingly, incubation of 200 nM F-H1 and 200 nM H2 with 2 nM T merely showed slight increase of the fluorescence peak, and the signal-to-background ratio (4.4 fold) was much smaller than that obtained in the bHCR system. This result clearly demonstrated the higher amplification efficiency of bHCR than conventional linear HCR. Selectivity analysis of the bHCR systems was examined using one-base mutation sequence of the target. The obtained fluorescence intensity exhibited little enhancement to the background, implying that the bHCR system afforded very high selectivity with the ability to discriminating single-base mutations. The fluorescence peaks were also found to be dynamically dependent upon the concentrations of target sequence in a three-decade range from 1 pM to 2 nM (Figure 2b). A quasi-linear correlation was obtained for
peak intensities at 523 nm to target concentrations ranging from 1 pM to 0.8 nM (Figure 2c). The detection limit was estimated as low as 500 fM, which was much better than current dendritic or hyperbranched HCR circuits.20-23 A further comparison was performed between the bHCR system and conventional linear HCR circuit consisting of hairpins F-H1 and H2 and the corresponding initiator T. It was found that the fluorescence background of the linear HCR system was 2-fold lower than that of the bHCR circuit. The result was sensible as the system leakage was also amplified by the hierarchical coupling of two HCR circuits. The fluorescence responses of the linear HCR system were also dynamically correlated to target concentrations, and the peak fluorescence intensities showed linear correlation to target concentrations in the range from 20 pM to 5 nM with a detection limit of 10 pM (Figure S-3). The results revealed that the bHCR system offered higher sensitivity than the linear HCR assay. At different target concentrations, the bHCR system was found to consistently give higher signal-to-background ratios than the linear HCR (Figure 2d). These data testified that bHCR provided much higher amplification efficiency than the linear HCR system, indicating the successful branched growth of the brush-like secondary HCR polymers along the primary HCR assemblies. In addition, time-dependent measurements showed that the peak intensities increased with increasing reaction time, and at a lower concentration the reaction required prolonged time to achieved saturated responses (Figure S-4).
Figure 3. Fluorescence images for (a) HeLa cells transfected with H1, H2, F-H3 and H4, (b) C166 cells transfected with H1, H2 and F-H3 H4, (c) HeLa cells transfected with F-H1 and H2. (1) Fluorescence; (2) merged with DIC. Scale bar: 20 μm.
Next, we explored the ability of bHCR for imaging of mRNA in living cells. When HeLa cells were transfected with the hairpin probes H1, H2, F-H3 and H4 using Lipofectamine 3000, very intense green fluorescent spots was observed in the cells(Figure 3a). A control experiment using C166 cells with no expression of target mRNA25 revealed that almost no fluorescent spots appeared in the cells (Figure 3b). This result evidenced that these bright fluorescent spots found in HeLa cells were specific to target mRNA, implying that fluorescent spots were ascribed to the hyperbranched assembly of hairpin probes
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seeded from target mRNA. Furthermore, we transfected HeLa cells using only two probes F-H1 and H2 in the linear HCR system. As anticipated, the image only showed fewer and much weak fluorescent spots in the cells. The average fluorescence intensity of the spots in the bHCR circuit were found to be ~5 fold enhancement as compared to that for the linear HCR system. Further flow cytometry assay of these three types of cells also revealed that bHCR gave much higher fluorescence signals than linear HCR (Figure S-5). These findings clearly proved that bHCR had the promise in affording higher amplification efficiency and better sensitivity in live cell imaging relative to linear HCR. The bHCR circuits was further applied to fluorescence imaging of different cell lines, HeLa, HepG-2 and C166. It was found that HeLa cells showed much more fluorescence spots than HepG2 cells (Figure S-6), indicating higher expression of survivin mRNA in HeLa cells than HepG2 cells. This result was consistent with the finding reported previously.25 With HeLa cells pretreated using 5 nM or 10 nM YM155, an imidazolium-based compound specifically repressing survivin mRNA expression,26 followed by transfection of the probes, we also found quite a few intense fluorescent spots in the cells treated using 5 nM YM155 and much less fluorescent spots appeared in the cells treated using 5 nM YM155(Figure S-7). These results confirmed the knock-down effect of YM155 in regulating survivin mRNA. To validate the results, quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis of the expression of survivin mRNA in these cells was performed (Figure S-8). The results revealed that the number of intense fluorescent spots in the cells increased with increasing expression levels of target mRNA in the cells. This result verified the ability of the bHCR circuit for quantitative imaging mRNA expression in living cells. In conclusion, we successfully demonstrated a novel bHCR circuit for ultrasensitive imaging of mRNA in living cells. This bHCR strategy exploited a hierarchical coupling of two HCR circuits in a single reaction, and could be developed using a simplified design by incorporating two split initiator fragments in the probes in the primary HCR circuit. The bHCR circuit enabled to generated a hyperbranched assembly seeded from a single target initiator, which provided the possibility of localizing single target molecules in live cell imaging applications. The result revealed that bHCR generated highly branchch structures with high molecular weights in response to target sequence with a detection limit of 500 fM and high specificity in single mismatch discrimination. The bHCR system was also demonstrated to display intense fluorescence spots indicating the localization of target mRNA in living cells. Collectively, this bHCR strategy can provide a powerful platform for low-abundance biomarker detection and imaging for cell biology and clinical diagnostics.
ASSOCIATED CONTENT Supporting Information
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The supporting information is available free of charge on the ACS Publication website at http://pubs.acs.org. Experimental methods including materials and instruments, gel electrophoresis analysis, AFM imaging, fluorescence measurements, cell culture and fluorescence imaging, flow cytometry assay and RT-PCR assay (PDF)
AUTHOR INFORMATION Corresponding Author
[email protected];
[email protected] Notes The authors declare no competing financial interest.
ACKNOWLEDGMENT This work was supported by NSFC (21527810, 21521063).
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