MicroRNA detection in biologically relevant media using a split aptamer platform

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Abstract

MicroRNA (miRNA)-based intercellular communication has been implicated in many functional and dysfunctional biological processes. This has raised interest in the potential use of miRNAs as biomarkers for diagnosis and prognosis. Though the list of clinically significant miRNA biomarkers is expanding, it remains challenging to adapt current chemical tools to investigate miRNAs in complex environments native to cells and tissues. We describe here a methodology for rapidly developing aptamer-based fluorescent biosensors that can specifically detect miRNAs in biologically relevant media (10–30% v/v), including medium collected from cultured HeLa cells, human serum, and human plasma. This methodology involves the semi-rational design of the hybridization between DNA oligonucleotides and the miRNA target to build a pool of potential aptamers, and the screening of this pool for high signal-to-background ratio and target specificity. The DNA oligonucleotides are readily available and require no chemical modification, rendering these chemical tools highly adaptable to any novel and niche miRNA target. Following this approach, we developed sensors that detect distinct oncogenic miRNA targets (miR-19b, miR-21, and miR-92a) at concentrations as low as 5 nM without amplification and are selective against single-nucleotide mutants. This work provides a systematic approach toward the development of miRNA biosensors that are easily accessible and can perform in biological environments with minimal sample handling.

Introduction

MicroRNAs (miRNAs) are ca. 22 nucleotide-long noncoding RNA oligomers that contribute to post-transcriptional regulation of gene expression, typically via facilitation of degradation of complementary messenger RNAs in a process known as RNA silencing.1 Recently, intercellular communication employing miRNAs as signalling molecules has acquired much attention due to its role in the regulation of several biological processes,2 including cell homeostasis, cell fate specification, and embryonic development.[3], [4] When the dysregulation of these processes results in disease, miRNAs can serve as disease markers. Tumour cells secrete miRNAs to facilitate cancer progression and metastasis by altering gene expression in neighbouring cells.[5], [6] The bodily fluids of cancer patients typically show elevated concentrations of certain miRNAs.7 These observations have suggested the potential for the use of miRNAs as biomarkers for cancer diagnosis and prognosis[8], [9], [10] and stimulated recent developments in miRNA diagnostics.[11], [12], [13], [14], [15], [16], [17], [18], [19].

As the critical roles of miRNAs continue to be illuminated, there is a growing need for investigative platforms that are easily accessible and configurable for the detection of different miRNA targets in biological environments.20 However, the sequence space of miRNAs is large. The human transcriptome alone contains over 2600 miRNA sequences and over 48,000 have been identified in all organisms studied.21 Independent approaches for the development of unique tools for each miRNA sequence is inadequate to address this large sequence space. Systematic approaches to cost-effectively and rapidly produce molecular tools for sequence-specific miRNA detection can greatly increase the accessibility of investigative platforms to study miRNAs of interest.

One technology with significant potential for RNA detection and cancer diagnosis is aptamer-based biosensing.22 Aptamers, which are oligonucleotides that exhibit high binding affinity toward a specific ligand,23 can produce fluorescence without covalent labelling and can be reconstituted to specifically hybridize with a nucleic acid target.[24], [25] Furthermore, the ability of aptamers to attain high sequence specificity at room temperature26 provides an advantage over molecular beacon sensors, which require elevated temperatures to differentiate nucleic acid mutants.27 A promising aptamer-based nucleic acid-sensing platform is composed of a split form of the malachite green aptamer (MGA),26 an in vitro-selected, light-up RNA aptamer to which malachite green (MG) tightly binds28 and enhances its fluorescence.29 In this split aptamer design, MGA was separated into two strands that must come together to form the MG-binding motif. Each aptamer strand contains a 7-nt region complementary to a 14-nt target sequence. Hybridization of the aptamer strands with a single-stranded DNA oligomer containing the target sequence stabilizes the folding of the MG-binding motif, facilitating binding of MG and thus increasing its quantum yield.

Despite their potential, aptamer-based detection tools have yet to meet certain requirements for biological application as miRNA biosensors. First, the system must hybridize only with specific sequences of ca. 22-nt, ensuring that the sensor is specific to the miRNA target against the vast sequence space found in transcriptome.21 This is challenging, because as the length of the hybridizing region increases, so too does the strength of the hybridization and the tolerance of mismatches between the sequences. Secondly, these biosensors must operate in the complex environments native to cells and tissues. These environments contain a multitude of confounding factors, including non-target oligonucleotides and a cocktail of biomolecules that may interfere with the ligand-aptamer interaction.30 Finally, it is critical to design the aptamer platform with no chemical modification to allow accessibility and configurability toward the detection of any novel or niche miRNA target. Methods that can produce a wide array of miRNA sensors that overcome these challenges can be viable options for investigating miRNAs in biological environments.

Here we report a systematic methodology for producing split aptamer biosensors that can detect miRNA targets in various biologically relevant media, including cell medium (CM), which was collected from cultured HeLa cells, human serum (HS), and human plasma (HP) (Fig. 1).

The biosensors produced via this methodology require minimal sample handling and can detect miRNA with single-nucleotide specificity. The split aptamer strands are composed of DNA, which has a superior intrinsic biostability against nucleolytic degradation compared to native RNA.[31], [32] Furthermore, unlike the structurally complex split aptamers that use chemical modifications,33 the aptamer designs accessed through this methodology are composed of the natural polymer of DNA, rendering the production of biosensors for new targets rapidly accessible and cost-effective.

Our approach of biosensor development focuses on the optimization of the miRNA-interacting region to maximize stability and signal generation in the presence of the miRNA target, while minimizing stability and background signal in the absence of it. This methodology has three steps: (1) using semi-rational design to construct a pool of potential biosensors with diverse structural characteristics, (2) screening this pool against a specific miRNA target to identify designs with high signal-to-background ratios, and (3) screening the identified designs against the single-nucleotide mutants of the target miRNA to select aptamer designs with high sequence specificity. The semi-rational design of potential biosensors narrows the sequence space to a manageable list, of which further screening can rapidly produce high-fidelity biosensors.

We demonstrated the flexibility of this methodology by designing biosensors for three miRNA targets with relevance to dysfunctional cellular physiology: miR-19b, miR-21, and miR-92a. MiR-19b and miR-92a are part of the miR-17/92 cluster—a group of related miRNA genes that plays a regulatory role in the metabolic reprogramming of lymphoma cells and promotes Myc-dependent tumour growth.34 Consistently elevated levels of these two miRNAs are correlated with silencing of critical tumour suppressor genes.[34], [35] The third target, miR-21, downregulates tumour-suppressing apoptotic proteins, and its overexpression is observed in breast, ovarian, colorectal, gastric, prostate, and lung cancers.36 All three of these miRNAs were detected in the bodily fluid of patients with diffuse B-cell lymphoma at mean expression levels 10–16-fold higher than those of healthy individuals.7 Important to the development of aptamer-based biosensors, these miRNA targets all have different chemical features—secondary structures and hybridization—that potentially challenge the design of a miRNA biosensor.

All three sensors developed by this methodology displayed good single-nucleotide specificity for their targets in both buffer and biologically relevant media. At 2 µM miRNA concentration, an ca. 20-fold signal enhancement in both buffer and 10% (v/v) HS, and a 10-fold signal enhancement in 30% (v/v) HS were reached. Without amplification, the sensors could detect miRNAs at low nanomolar levels at room temperature within one hour in both buffer and the biological media.

Section snippets

Split aptamer design

As a template for our aptamer designs, we devised a multi-stranded complex (Fig. 1) formed from two DNA strands (St1 and St2) and the target miRNA strand. The miRNA sequences (Table S1) were analyzed with the Vienna RNA Websuite for secondary structure and hybridization.37 MiR-19b has a strong secondary structure, miR-21 can self-dimerize, and miR-92a has the highest hybridization energy, including a 7-nt stretch of C and G nucleotides (Fig. S1). The St1:St2:miRNA complex possesses a

Conclusions

Accurate detection of specific miRNAs remains challenging due in part to the sequence similarity in the transcriptome and other confounding factors within biological environments. We herein presented a methodology for developing accessible miRNA biosensors that can perform in biologically relevant media, including the medium collected from cultured HeLa cells, human serum, and human plasma. This methodology generated split aptamer designs displaying good levels of target sequence specificity

Materials

The synthesis of dapoxyl sulfonate and dapoxyl sulfonyl fluoride were described in the Supplementary Data. 2-amino-4′-dimethylaminoacetophenone hydrochloride was purchased from Combi Blocks. 4-(fluorosulfonyl)benzoyl chloride, pyridine, methylene chloride, chloroform, sodium hydroxide, sodium carbonate (all anhydrous), HPLC grade acetonitrile, ACS grade acetone, auramine O (AO, 85% dye content), benzoic acid, bovine serum albumin (BSA), formamide, ethylenediaminetetraacetic acid, urea, and

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

We acknowledge Dr. M. Rhia L. Stone for preparation of HeLa cell media. We thank Prof. Zheng Shi and Shilong Yang for providing us with the HeLa cell line, and Dr. Venu Gopal Vandavasi for helpful discussions. We thank Dr. Alexei Ermakov for assistance with HRMS analyses. We also thank Dr. Stone and Hakan Guven for careful reading of the manuscript. This work was supported by the US National Institutes of Health / NIBIB Trailblazer Award (EB029548), American Cancer Society, Institutional

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    These authors contributed equally.

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