Simple Isothermal Strategy for Multiplexed, Rapid, Sensitive, and

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Simple isothermal strategy for multiplexed, rapid, sensitive and accurate miRNA detection Eugene J.H. Wee, and Matt Trau ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.6b00105 • Publication Date (Web): 05 Apr 2016 Downloaded from http://pubs.acs.org on April 13, 2016

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Simple isothermal strategy for multiplexed, rapid, sensitive and accurate miRNA detection Eugene J. H. Wee,*[a] and Matt Trau*[a][b] [a] Center for Personalized NanoMedicine, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD 4072, Australia [b] School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia

KEYWORDS: miRNA, recombinase polymerase amplification, PBCV-1 DNA ligase, SSB, miRPA

ABSTRACT: MicroRNAs (miRNA) are potential biomarkers. Current preferred miRNA detection methods rely on various PCR-based approaches. While effective, such methods are typically tedious, slow and require expensive equipment. Hence, faster and simpler miRNA detection approaches are still needed. Herein, we describe miRPA: a novel combination of recombinase polymerase amplification (RPA) with PBCV-1 DNA ligase for simple, specific, rapid and multiplexed isothermal miRNA detection. MiRPA is sensitive to picogram levels of total RNA input (or ~40 copies/pg) and can discriminate between closely related miRNAs. MiRPA was applied to cell lines and was subsequently validated with a commercial qPCR method. Potential clinical application was also demonstrated by detecting miRNAs in urine derived RNA. This is the first application of RPA for rapid isothermal miRNA detection and could have wide applications as a miRNA sensor in both research and in the clinic.

MiRNAs are short (~20 bases) non-coding RNAs and are a relatively new class of biomarkers for diseases such as cancer.1-4 Currently, the detection of miRNAs is popularly detected through a suite of PCR-based methodologies such quantitative PCR (qPCR) and sequencing. However, due to their short lengths, native miRNAs require some form of in vitro “lengthening” manipulation for compatibility with PCR. Strategies to ready miRNAs as templates for PCR include reverse transcriptase based methods (polyA tailing/oligo dT and stemloop primers)5, 6 and T4 RNA/DNA ligase based methods7, 8 with the former being the more popular approach. While useful, such methods are slow to perform (hours to days) due to limiting enzyme kinetics. Moreover, limitations in both speed and costs are further confounded by the need for expensive qPCR platforms/ DNA sequencers and thus may not be suitable for routine diagnostics. Hence, there is still a need for simpler, faster miRNA sensing strategies to aid the use of miRNAs in clinic and for advancing miRNA research. Isothermal DNA amplification methods have been developed as an attractive alternative to PCR approaches. For example, rolling circle9 and EXPAR10 amplificationS have been adapted for detecting miRNAs isothermally. While these methods potentially avoid the limitations of PCR, they however lack the specificity of PCR as they are very prone to template-independent (non-specific)

amplification11 and therefore may have limited applications in diagnostics. Recently, a new isothermal amplification method, Recombinase Polymerase Amplification (RPA),12 was described and had since been successfully adapted for a wide variety of rapid (15-20 minutes) point-of-care applications in detecting cancer biomarkers, viral, bacterial and fungal diseases.13, 14 With its PCR-like sensitivity and specificity, RPA could be a promising rapid isothermal detection strategy for miRNA detection. However, as with PCR-based methods, the short miRNA lengths poses a major challenge for adapting RPA for miRNA applications. To the best of our knowledge, no RPA-based miRNA detection approach has been described in the literature. RNA-templated ligation is traditionally performed with T4 DNA ligase and more recently, the T4 RNA ligase 2 and have been used for both messenger RNA (mRNA) and miRNA detection.7, 8, 15, 16 While effective, the T4 enzymes have several limitations including inefficient reactions and the need for expensive DNA probe modifications.15, 16 In contrast, the PBCV-1 DNA ligase16 avoids these limitations with efficient RNA-templated DNA ligation and thus is an excellent candidate for newer, faster assays. However, the relavtively low fidelity (high off-target ligations) of PBCV-1 DNA ligase identified by previous studies16 needs to be addressed in order for it to useful. To the best of our knowledge, no miRNA applications have

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Figure 1. Conceptual schematic of the miRPA method.

ods pertaining to clinical samples were carried out in accordance with approved guidelines.

been described with PBCV-1 DNA ligase. A ligation approach also has the advantage of barcoding multiple RNA species of interest in a library for downstream analysis. By using probes encoded with endogenous sequences/barcodes that also serve as primer binding sites to “lengthen” the miRNA sequence thus addressing the primer length requirements for RPA.12 By combining the advantages of both PBCV-1 DNA ligase and RPA, one may potentially be able to rapidly detect miRNA at PCR-like sensitivity with minimal equipment. Herein, we describe for the first time, the miRNA-RPA (miRPA) assay (Fig 1) which combined the use of the PBCV-1 DNA ligase with RPA to detect miRNAs. We first solved the fidelity issue of the PBCV-1 DNA which was key in enabling miRPA. From an end user perspective, miRPA had PCR-like analytical performance in detecting miRNA but at faster assay speeds and with simpler equipment. We envisage miRPA could have wide applications in miRNA research and to enable simple miRNA detection in the clinic.

Experimental Section RNA isolation The Direct-zol RNA miniprep kit (Zymo Research) was used to isolate total RNA from MCF7 and MDA-MB-231 cell lines (ATCC) according to the manufacturer’s protocol. Briefly, TRIzol reagent (Life Technologies) was used to lyse cells, centrifuged to pellet cell debris and the supernatant was used directly in the Direct-Zol purification protocol. Total RNA was then eluted in RNase-free water and quantified with spectrometry (NanoDrop). Prior to the collection of urine specimens, ethics approval was obtained from The University of Queensland Institutional Human Research Ethics Committee (Approval No. 2004000047) and informed consent was obtained from all subjects prior to sample collection. Meth-

Four 1.5 mL frozen aliquots of archival urine specimens were used in this study. To extract RNA, the ZR Urine RNA isolation kit (Zymo Research) was used as instructed by the manufacturer, eluted in 15 µL of RNase-free water and quantified with spectrometry (NanoDrop). 10 ng of urine total RNA was then used for miRPA. miRPA protocol Total RNA (amounts were as stated in the text) with 10 nM of each probe were adjusted to 8 µL in water and briefly heated to 85°C for 2 minutes and placed on ice immediately. 2 µL of ligation mix which consisted of 5x SplintR buffer, 25 units of PBCV-1 DNA ligase (SplintR, NEB) and 65 ng of ET SSB (NEB) and incubated at 37°C for 10 minutes. 1 µL of ligation product was then used in qRPA which was based on the TwistAmp Basic kit (TwistDX) with some modifications. Briefly, each 12.5 µL RPA reaction contained 250 nM of primers, 2 nM of SYTO9 (Life Technologies) and 14 mM magnesium acetate. qRPA was then performed on a Life Technologies 7500 qPCR machine set to a constant 37°C for 15 minutes with fluorescence acquired every 30 seconds. The fluorescence level of the no RNA control at 15 minutes was used to determine cutoff thresholds for reaction times. This approach was adopted to account for non-RNA mediated ligation events. Expression levels were estimated with an approach analogous to the ΔΔCt17 method used in qPCR. Essentially, Ct values were replaced by the ratio of threshold reaction time to doubling time. To generate a titration curve, the threshold reaction time was plotted against the log value of the RNA input. RPA products were also evaluated using standard gel electrophoresis. All oligonucleotides were purchased from Integrated DNA Technologies (IDT) and sequences are provided in Table S1 of supporting information.

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miScript qPCR The Qiagen miScript qPCR miRNA assay kits were used as instructed by the manufacturer. Briefly 10 ng of total RNA was polyadenylated and reverse transcribed in a 10 µL reaction to generate a cDNA library. This was then diluted 10-fold with water and 1µL of the diluted cDNA then used in 15 µL qPCR reactions on a Life Technologies 7500 qPCR machine using cycling conditions recommended by the manufacturer. Expression levels were then estimated using the standard ΔΔCt method.17

Results and Discussion

miRNA dependent ligation and amplification To realise miRPA, we attempted to detect miR200a, a member of the miR200 family that is implicated in cancer metastasis,4, 18-20 in two model breast cancer cell lines: MCF7 (high miR200a) and MDA-MB-231 (low miR200a). MiR200a Assay results were then assessed with simple gel electrophoresis (Fig 2a). As expected, a modestly higher amplification was detected in MCF7 compared to MDA-MB-231 (10 ng total RNA each), the no RNA control (representing non-specific ligation events) and RPA water controls (representing ligationindependent RPA amplification). These promising initial results suggested a miRNA dependent ligation and amplification. Improved PBCV-1 ligase stringency with SSB While initial results were promising, miRPA performance was not ideal due to high levels of targetindependent ligation (Fig 2a). This was consistent with recent studies evaluating the effectiveness of PBCV-1 DNA ligase for mRNA detection.15, 21 These miRNA independent ligations could be due to (1) non-specific probe hybridizations which, in turn, resulted in non-specific ligations and (2) an over sensitive RPA amplification. To modulate an over sensitive RPA, the manufacturer of RPA suggested reducing primers lengths (FAQ section of manufacturer’s manual). As expected, reducing primer lengths to 26 bases resulted in a better signal to noise (Fig 2A). However, levels of off target ligations remained high and thus needed to be addressed to enable a robust assay.

Figure 2. miRNA dependent ligation and amplification. (A) Gel images of RPA amplicons reflecting assay performance as a function of primer length and the presence of SSB enzyme. (B) Typical qRPA data as fluorescence accumulated over time with the stated RNA inputs. Main: miR200a. Insert: RNU6b RNA loading control. Replicates are represented as solid and broken lines. Total RNA from MCF7 and MDA-MB-231 were used with no RNA and water controls.

The miRPA method The miRPA assay exploited the rapid, specific and efficient ligation of adjacent DNA probes on a miRNA splint by the PBCV-1 DNA ligase. These probes had barcoded handles on the ends that served both to facilitate multiplexed generation of an RPA-amplifiable miRNA library and as priming sites for RPA. Only correctly hybridized probes on cognate miRNA targets were ligated and able to be subsequently amplified via RPA. Finally, as a proofof-concept, we used the double stranded DNA interacting fluorescent dye (SYTO9) in a real time quantitative RPA (qRPA) assay as a convenient means of measuring the amount of miRNA present in the sample. From RNA extraction to final results, miRPA could be completed in approximately 30 minutes.

Single stranded DNA binding proteins (SSB) have been used to improve PCR stringency possibly by sterically preventing polymerization of mismatched primers and while increasing PCR efficiency by stabilizing any single stranded DNA substrates.22, 23 So we hypothesize that the same mechanisms could be used to improve ligation specificity and efficiency. Indeed, with the addition of SSB, both ligation stringency and RPA yield improved (Fig 2a). To the best of our knowledge, this is the first description of SSBs being used to improve ligation performance. Since measuring the levels of miRNA expression was desirable, we used, as a proof-of-concept, SYTO9, a double stranded DNA specific intercalating fluorescent dye to enable a “qPCR-like” quantification system with RPA i.e, qRPA (Fig 2b). Reflecting the end-point gel electrophoresis data, high miR200a expressing MCF7 accumulated florescence significantly earlier than MDAMB-231, no RNA controls and RPA water controls. RNU6b was used as a loading control of RNA input, which as expected showed similar expresssion levels for both cell lines. Low sample requirements

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Next, we evaluated the sample requirements of the miRPA assay. To this end, total RNA from high miR200a expressing MCF7 was titrated and assayed (Fig 3a, S1). miR200a from as little as 100 pg of total RNA (~amol levels24 or ~40 copies/pg as estimated by qPCR) could still be detected over the no RNA controls thus demonstrating the low sample requirements of miRPA. This level of sensitivity was at least comparable to qPCR-based methods. From the slope of the titration graph, we inferred that every 10-fold change in miRNA amount resulted in an approximate 2.21 minute difference in reaction time, i.e., the time needed to acheive a detectable signal

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miR200a, miR200b, miR200c, miR141 and miR429, is one typical example. As such, miRNA assays able to distinguish between highly similar species are highly desirable. The fidelity of DNA ligases to mismatches and low melting temperature of short probes25, 26 may facilitate stringency against closely related miRNA species. To this end, we challenged our miR200a assay with other members of the miR200 family (Fig 3b). As expected, the assay was highly specific to miR200a. Only against miR141, which was the closest member with only a 2 base difference, had an appreciable signal with an estimated 30% assay efficiency relative to the cognate target. This high stringency against closely related miRNAs (Fig 3b), at least for small RNA

Figure 3. Limit of detection and specificity. (A) Titration plot of miRPA assay over a range of total RNA inputs. (B) Specificity of miRPA assay against closely related miRNA species. Sequence differences are highlighted in grey. Dotted line represents the ligation junction. Error bars represent standard error of 3 biological replicates

Therefore, using the general equation for an exponential amplification system:

At = A02t/τ

--------------------------(1)

Where t is the reaction time, At is amplification (florescence intensity) at time t and A0 is the initial amount of miRNA. The doubling time, τ, was thus estimated to be 39.8 seconds, consistent with previous reports on RPA.12 This value of τ was then used in subsequent analysis.

Figure 4. miRPA validation and application on clinical specimens. Expression profiles of miR200a, miR15 and RNU6b in MCF7 and MDA-MB-231 cells using (A) miRPA or (B) miScript. (C) Detecting miR200b and miR200c in MCF7, MDA-MB-231 and four urine derived samples. Error bars represent standard error of 3 biological replicates for cell lines and standard deviation of 3 technical replicates for urine specimens.

Stringency against closely related miRNA species

applications, further underscored the low off-target ligation events by the PBCV-1 DNA ligase and SSB combination.

A consequence of the short length of miRNA, is the highly similar sequences of closely related mature miRNAs. The miR200 family which comprises of

Another contributing factor for miRPA’s good selectively against highly similar miRNAs was that short4

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probes (~10 bases for target recognition, Table S1) were employed for the ligation step. This approach potentially conferred several benefits such as lowered melting temperatures and thus allowed for more stringent hybridization at the optimal ligation temperature (37°C). In addition, any destabilization as a result of mismatches might potentially be enhanced with short sequences. Lastly, DNA ligases are also very sensitive to mismatches up to 10 bases from the 3’OH ligating end25, 26 and therefore a PBCV-1 DNA ligase-base assay could be a useful and rapid strategy in discriminating closely related miRNAs and possibly isomiRs. Detecting multiple miRNAs in cell lines Satisfied that miRPA could potentially be useful as a miRNA detection tool, we turned our attention to more complex biological problems such as profiling multiple miRNAs in cells. As a proof-of-concept, we first generated a triplex ligation library (miR200a, miR15 and RNU6B) with 10 ng total RNA from MCF7 and MDA-MB-231 breast cancer cells and used qRPA to estimate the expression levels of the RNA species (Fig 4a and 4b). Expression of miR200a was higher in MCF7 while miR15 and RNU6B were higher in MDA-MB-231. Expression levels were then validated with a standard commercial qPCR kit with 10 ng of total RNA and similar expression profiles were observed thus underscoring the accuracy of the miRPA assay. RNU6B is a small nucleolar RNA that is thought to be ubiqutiously expressed in most cells lines and hence is commonly used as a normalizing control for RNA input27. However, recent studies27 have indicated that RNU6B expression is highly variable between cell lines and our data was consitent with the literature.

of mRNAs.29 Consistent with the literature, MCF7 had higher levels of miR200b and miR200c compared to MDAMB231, and thus confirming the analytical performance of the assay. For the urine samples, all four had higher levels of miR200b expression than miR200c. However, due to limiting amounts of RNA recovered, an independent method could not be performed on the urine samples. Nonetheless, since assay performance was confirmed with cell line controls, the data generated from urine RNA was likely correct and suggests a possible application with liquid biopsies. Nonetheless the data presented in this study, suggests that miRPA may be a viable and simpler alternative to standard qPCR methods but with significantly faster assay times.

Conclusion In conclusion, miRPA is a viable alternative to current qPCR methods with comparable accuracy, similar or better sensitivity yet requiring only a fraction of the time to complete (approximately 30 minutes compared to 2 hours for miScript qPCR). With portable flurometers, we envision that miRPA could be a platform strategy for simiple and fast miRNA sensors and could have broad applications in both research and clinic.

ASSOCIATED CONTENT Supporting Information Available:The following files are available free of charge. Supplementary info. The file contains a Table of oligonucleotides used in this study and raw data for Fig 2A.

AUTHOR INFORMATION Corresponding Author

While both methods produced similar expression profiles, there were differences in the estimated absolute levels of miRNA. Using miR200a as an example, miRPA measured 10-fold overexpression in MCF7 compared to MDA-MB-231. In contrast, miScript measured a 20-fold overexpression. One likely reason could be that different approaches used to “ready” miRNA targets can lead to subtle (or dramatic) differences in quantification.28 For instance, the ligation probes used in miRPA unavoidably contained a GA motif at the ligation junction- a potentially inefficient nucleotide combination for PBCV-1 DNA ligase.16 On the other hand, the highly optimized miScript system which was based on a polyadenylation/reverse transcription method may be less sensitive to sequence-dependent modulation.

E-mail: [email protected], [email protected]

Author Contributions All authors conceived and wrote the manuscript. EJHW performed all experiments.

Funding Sources We gratefully acknowledge funding received from the National Breast Cancer Foundation of Australia (CG-08-07 and CG-12-07).

ACKNOWLEDGMENT We thank Robert (Frank) Gardiner for providing clinical urine specimens.

Detecting multiple miRNAs in urine specimens

REFERENCES

Finally to demonstrate a potential diagnostic application, the method was successfully extended to detect miR200b and miR200c in 4 random urine specimens from prostate cancer patients and 2 cell lines of known expression profiles4, 18-20 to control for assay performance (Fig 4c). miRNA levels were normalized to total RNA input which was estimated using the conserved ALU sequences

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