David W. Wegman

Direct Quantitative Analysis of Multiple miRNAs (DQAMmiR)

MicroRNAs (miRNAs) are short RNA molecules (18–25 nucleotides) that were recently proven to play an important role in the regulation of cellular processes, and their abnormal expression is associated with pathologies such as cancer. A change in the cellular status is typically associated with a simultaneous change in the level of several miRNAs. For example, abnormal expression of two miRNAs was found to be indicative of colorectal cancer in humans. Therefore, both the study of the biological role of miRNA and the use of miRNA for informative disease diagnostics require accurate quantitative analysis of multiple miRNAs. Most methods of miRNA detection are indirect (e.g. PCR, microarrays, SPR, next generation sequencing, etc.), that is, they require chemical or enzymatic modifications of miRNA prior to the analysis. Not only do these modifications make the analysis more complex and timeconsuming but they also reduce the accuracy of the method owing to different efficiencies for modifications of different miRNAs. There are a few direct methods that do not require any modification of the target miRNA. Northern blotting does not require any modifications, however, the method can be tedious and although it can be quantitative its sensitivity is limited. Signal-amplifying ribozymes, in situ hybridization, bioluminescence detection, and two-probe single-molecule fluorescence are other direct miRNA detection methods however, the first two methods are only semi-quantitative while the latter two can hardly be used for multiple miRNAs. Cheng et al. used rolling-circle amplification (RCA), which does not require modification of the miRNA to detect low concentrations of miRNA and can be run in parallel; however, the process is tedious taking over 8 h and the amplification step can potentially lead to biases in quantitation. Thus, there is currently no method for direct quantitative analysis of multiple miRNAs. Herein we report the first direct quantitative analysis of multiple miRNAs (DQAMmiR). DQAMmiR uses miRNAs directly, without any modification, and accurately determines concentrations of multiple miRNAs without the need for calibration curves. This approach was achieved using a capillary-electrophoresisbased hybridization assay with an ideologically simple combination of two well-known separation-enhancement approaches: 1) drag tags on the DNA probes, and 2) single strand DNA binding protein (SSB) in the buffer. In this proof-of-principle work, we developed DQAMmiR for three miRNAs (mir21, 125b, 145) known to be deregulated in breast cancer. DQAMmiR opens the opportunity for simple, fast, and quantitative fingerprinting of up to several tens of miRNAs in basic research and clinical applications. The availability of suitable commercial instruments for DQAMmiR makes the method practical for a large community of researchers. We based DQAMmiR upon a classical hybridization approach, in which an excess of labeled DNA probes are bound to their complementary miRNA targets. Electrophoresis can be used to efficiently separate oligonucleotides, but simultaneously separating the hybrids from each other and from the unbound probes is challenging and so far has not been achieved. We solved the separation problem through a combination of two well-known mobility-shift approaches: 1) drag tags on the probes and 2) single strand DNA binding (SSB) protein in the buffer. This hypothetical approach is illustrated in Figure 1, in which the miRNAs and their complimentary ssDNA probes are shown as short lines of the same color, drag tags are shown as parachutes, fluorescent labels are shown as small green circles, and SSB is shown as a large black circle. In the hybridization step, an excess of the probes is mixed with the miRNAs, thus leading to all miRNAs being hybridized but with some probes left unbound to miRNA. A short plug of the hybridization mixture is introduced into a capillary prefilled with an SSBcontaining buffer. SSB binds all ssDNA probes but does not bind the double-stranded miRNA–DNA hybrid. When an electric field is applied, all SSB-bound probes move faster than all the hybrids (SSB works as a propellant). Different drag tags make different hybrids move with different velocities. SSB-bound probes, however, can move with similar velocities if the drag tags are small with respect to SSB. In such a case, a fluorescent detector at the end of the capillary generates separate signals for the hybrids and a cumulative signal (one peak or multiple peaks) for the excess of the probes. The amounts of the different miRNAs are finally determined from integrated signals (peak areas in the graph) by a simple mathematical approach. We reserve the term of direct quantitative analysis of multiple miRNAs and its abbreviation of DQAMmiR for the specific approach described above. To experimentally test the viability of our hypothetical DQAMmiR, we decided to use three miRNAs known to be deregulated in breast cancer: mir21 (5’-UAGCUUAUCAGA CUGAUGUUGA-3’), mir125b (5’-UCCCUGAGACCCUAACUU GUGA-3’), and mir145 (5’-GUCCAGUUUUCCCAGGAAUCCC U-3’). Three ssDNA probes were designed and all are labeled with Alexa 488 at the 5 end; the 3 end was reserved for drag tags. To separate the three hybrids we needed only two probes modified with drag [*] D. W. Wegman, Prof. S. N. Krylov Department of Chemistry, York University 4700 Keele Street, Toronto, Ontario M3J 1P3 (Canada) E-mail: skrylov@yorku.ca

Detection of a Thousand Copies of miRNA without Enrichment or Modification

We report direct quantitative analysis of multiple miRNAs with a detection limit of 1000 copies without miRNA enrichment or modification. A 300-fold improvement over the previously published detection limit was achieved by combining capillary electrophoresis with confocal time-resolved fluorescence detection through an embedded capillary interface. The method was used to determine levels of three miRNA biomarkers of breast cancer (miRNA 21, 125b, 145) in a human breast cancer cell line (MCF-7). A 30 pL volume of the cell lysate with approximately a material content of a single cell was sampled for the analysis. MiRNA 21, which is up-regulated in breast cancer, was detected at a level of approximately 12 thousand copies per cells. MiRNAs 125b and 145, which are down-regulated in breast cancer, were below the 1000-copy detection limit. This sensitive method may facilitate the analysis of miRNA in fine-needle-biopsy samples and even in single cells without enrichment or modification of miRNA. Advantageously, the instrumental setup developed here can be reproduced by others as it requires no sophisticated custom-made parts.

Dynamic combinatorial mass spectrometry leads to inhibitors of a 2-oxoglutarate-dependent nucleic acid demethylase.

2-Oxoglutarate-dependent nucleic acid demethylases are of biological interest because of their roles in nucleic acid repair and modification. Although some of these enzymes are linked to physiology, their regulatory roles are unclear. Hence, there is a desire to develop selective inhibitors for them; we report studies on AlkB, which reveal it as being amenable to selective inhibition by small molecules. Dynamic combinatorial chemistry linked to mass spectrometric analyses (DCMS) led to the identification of lead compounds, one of which was analyzed by crystallography. Subsequent structure-guided studies led to the identification of inhibitors of improved potency, some of which were shown to be selective over two other 2OG oxygenases. The work further validates the use of the DCMS method and will help to enable the development of inhibitors of nucleic acid modifying 2OG oxygenases both for use as functional probes and, in the longer term, for potential therapeutic use.

Universal Drag Tag for Direct Quantitative Analysis of Multiple MicroRNAs

Using microRNA (miRNA) as molecular markers of diseases requires a method for accurate measurement of multiple miRNAs in biological samples. Direct quantitative analysis of multiple miRNAs (DQAMmiR) has been recently developed based on a classical hybridization approach. In DQAMmiR, miRNAs are hybridized with excess fluorescently labeled complementary DNA probes. Capillary electrophoresis (CE) is used to separate the unreacted probes from the hybrids and the hybrids from each other. The challenging separation is achieved by using two types of mobility modifiers. Single-strand DNA binding protein (SSB) is added to the running buffer to bind and shift the single-stranded unreacted probes from the double-stranded hybrids. Different drag tags are built into the probes to introduce significant differential mobility between their respective hybrids. For the method to be practical it requires a universal extendable drag tag. Polymers are a logical choice for making extendable drag tags. Our recent theoretical work suggested that short peptides could provide a sufficient mobility shift to facilitate DQAMmiR. Here, we experimentally confirm this prediction in the analysis of five miRNAs: mir10b, mir21, mir125b, mir145, and mir155. We conjugated four fluorescently labeled DNA molecules with peptides of 5, 10, 15, or 20 neutral amino acids in length; the fifth probe was peptide-free. The peptide tags showed no interference with SSB binding to the probes and facilitated separation of the five hybrids. The mobilities of the five hybrids were used to refine the previously suggested theory. The analysis was performed in both a pure buffer and in cell lysate. Our analysis of the experimental data suggests that using DNA-peptide probes can readily facilitate simultaneous analysis of more than 10 miRNAs.

Highly-Sensitive Amplification-Free Analysis of Multiple miRNAs by Capillary Electrophoresis

Sets of deregulated microRNAs (miRNAs), termed miRNA signatures, are promising biomarkers for cancer. Validation of miRNA signatures requires a technique that is accurate, sensitive, capable of detecting multiple miRNAs, fast, robust, and not cost-prohibitive. Direct quantitative analysis of multiple miRNAs (DQAMmiR) is a capillary electrophoresis (CE)-based hybridization assay that was suggested as a methodological platform for validation and clinical use of miRNA signatures. While satisfying the other requirements, DQAMmiR is not sufficiently sensitive to detect low-abundance miRNAs. Here, we solve this problem by combining DQAMmiR with the preconcentration technique, isotachophoresis (ITP). The sensitivity improved 100 times (to 1 pM) allowing us to detect low-abundance miRNAs in an RNA extract. Importantly, ITP-DQAMmiR can be performed in a fully automated mode using a commercial CE instrument making it suitable for practical applications.

Achieving Single-Nucleotide Specificity in Direct Quantitative Analysis of Multiple MicroRNAs (DQAMmiR)

Direct quantitative analysis of multiple miRNAs (DQAMmiR) utilizes CE with fluorescence detection for fast, accurate, and sensitive quantitation of multiple miRNAs. Here we report on achieving single-nucleotide specificity and, thus, overcoming a principle obstacle on the way of DQAMmiR becoming a practical miRNA analysis tool. In general, sequence specificity is reached by raising the temperature to the level at which the probe-miRNA hybrids with mismatches melt while the matches remain intact. This elevated temperature is used as the hybridization temperature. Practical implementation of this apparently trivial approach in DQAMmiR has two major challenges. First, melting temperatures of all mismatched hybrids should be similar to each other and should not reach the melting temperature of any of the matched hybrids. Second, the elevated hybridization temperature should not deteriorate CE separation of the hybrids from the excess probes and the hybrids from each other. The second problem is further complicated by the reliance of separation in DQAMmiR on single-strand DNA binding protein (SSB) whose native structure and binding properties may be drastically affected by the elevated temperature. These problems were solved by two approaches. First, locked nucleic acid (LNA) bases were incorporated into the probes to normalize the melting temperatures of all target miRNA hybrids allowing for a single hybridization temperature; binding of SSB was not affected by LNA bases. Second, a dual-temperature CE was developed in which separation started with a high capillary temperature required for proper hybridization and continued at a low capillary temperature required for quality electrophoretic separation of the hybrids from excess probes and the hybrids from each other. The developed approach was sufficiently robust to allow its integration with sample preconcentration by isotachophoresis to achieve a limit of detection below 10 pM.

Making DNA hybridization assays in capillary electrophoresis quantitative.

A single-stranded DNA-binding protein (SSB) has been recently proven to facilitate highly efficient separation of the excess hybridization probe from the probe-target hybrid in gel-free capillary electrophoresis. SSB added to the electrophoresis run buffer binds the single-stranded DNA probe but does not bind the double stranded DNA-DNA or DNA-RNA hybrid. As a result, SSB changes the electrophoretic mobility of the probe but does not affect the mobility of the hybrid. If the probe is labeled fluorescently, real-time sensitive detection can be facilitated. While the concept of SSB-mediated hybridization analysis has been proven in principle, the question of how to make such analysis quantitative without building a calibration curve remains open. Here, we propose a general approach for making SSB-mediated analysis quantitative. This approach takes into account such phenomena as (i) the potential influence of the probe-target hybridization and probe-SSB binding on the quantum yield of the fluorescent label and (ii) the potential dissociation of the hybrid by SSB. The proposed approach was used to study the quenching and the dissociation phenomena experimentally. We proved for the first time that SSB does not detectably dissociate the probe-target hybrid, which significantly simplifies the analysis.

Accurate MicroRNA Analysis in Crude Cell Lysate by Capillary Electrophoresis-Based Hybridization Assay in Comparison with Quantitative Reverse Transcription-Polymerase Chain Reaction

Accurate quantitation of microRNA (miRNA) in tissue samples is required for validation and clinical use of miRNA-based disease biomarkers. Since sample processing, such as RNA extraction, introduces undesirable biases, it is advantageous to measure miRNA in a crude cell lysate. Here, we report on accurate miRNA quantitation in crude cell lysate by a CE-based hybridization assay termed direct quantitative analysis of multiple miRNAs (DQAMmiR). Accuracy and precision of miRNA quantitation were determined for miRNA samples in a crude cell lysate, RNA extract from the lysate, and a pure buffer. The results showed that the measurements were matrix-independent with inaccuracies of below 13% from true values and relative standard deviations of below 11% from the mean values in a miRNA concentration range of 2 orders of magnitude. We compared DQAMmiR-derived results with those obtained by a benchmark miRNA-quantitation method-quantitative reverse transcription-polymerase chain reaction (qRT-PCR). qRT-PCR-based measurements revealed multifold inaccuracies and relative standard deviations of up to 70% in crude cell lysate. Robustness of DQAMmiR to changes in sample matrix makes it a perfect candidate for validation and clinical use of miRNA-based disease biomarkers.