Ashley R. Connolly

Calculating the lower critical solution temperature (LCST) of a thermoresponsive polymer is an important measure of its physical properties. Here, the LCST of poly(N-isopropylacrylamide) (pNIPAM) was determined by measuring the scattered light intensity in a real-time thermocycler. This method allowed for the investigation of low concentration (μg mL⁻¹) samples in low volumes (μL). Subsequently, the critical micelle temperatures (CMTs) and concentrations (CMCs) were simultaneously recorded in 2 h for over 220 samples of the thermoresponsive copolymers of NIPAM with N-hydroxymethyl acrylamide (HMAM) (pNIPAM-co-HMAM (2.5–12.5 mol% HMAM)). This high-throughput approach paves the way to rapidly ‘map’ the physicochemical properties of low abundance polymers with statistical accuracy.

Core microRNA (miRNA) sequences exist as populations of variants called isomiRs made up of different lengths and nucleotide compositions. In particular, the short sequences of miRNA make single-base isomiR mismatches very difficult to be discriminated. Non-specific hybridizations often arise when DNA probe-miRNA target hybridization is the primary, or initial, mode of detection. These errors then become exacerbated through subsequent amplification steps. Here, we present the design of DNA probes modified with poly-guanine (PG) tracts that were induced to form G-quadruplexes (G4) for hi-fidelity discrimination of miRNA core target sequence from single-base mismatched isomiRs. We demonstrate that, when compared to unmodified probes, this G4 'gate-keeping' function within the G4-modified probes enables more stringent hybridization of complementary core miRNA target transcripts while limiting non-specific hybridizations. This increased discriminatory power of the G4-modified probes over unmodified probes is maintained even after further reverse transcriptase extension of probe-target hybrids. Enzymatic extension also enhanced the clarity and sensitivity of readouts and allows different isomiRs to be distinguished from one another via the relative positions of the mismatches.

A synthetic DNA oligonucleotide has been programmed to function as a biological circuit to detect 5′-IsomiRs. The circuit consists of two integrated DNA switches. The first is “activated” when a DNA probe is enzymatically modified by a reverse transcriptase that incorporates nucleotides complementary to the 5′-region of a microRNA (miRNA). Addition of the correct number and sequence of nucleotides enables the probe to assemble into an asymmetric DNA hairpin. The reconfigured hairpin probe then primes an internal polymerisation reaction, resulting in the synthesis of a symmetrical DNA hairpin. This activates the second switch, which then initiates the amplification of reverse-transcribed miRNA through a continuous cycle of DNA nicking and polymerisation. The DNA circuit enables sensitive and rapid detection of femtomoles of a miRNA transcript under isothermal conditions.

Monitoring DNA methylation can be a useful biomarker for disease diagnosis and prognosis. However, monitoring the methylation status of a specific cytosine biomarker is often confounded by heterogeneous peripheral DNA methylation. To address this issue, molecular inversion probes were designed with inosine strategically positioned to complement suspected DNA methylation sites. This enabled the methylation status of a specific cytosine to be accurately measured with a high level of specificity, irrespective of adjacent epigenetic modifications.

Somatic mutations in DNA can serve as cancer specific biomarkers and are increasingly being used to direct treatment. However, they can be difficult to detect in tissue biopsies because there is often only a minimal amount of sample and the mutations are often masked by the presence of wild type alleles from nontumor material in the sample. To facilitate the sensitive and specific analysis of DNA mutations in tissues, a multiplex assay capable of detecting nucleotide changes in less than 150 cells was developed. The assay extends the application of molecular inversion probes to enable sensitive discrimination and quantification of nucleotide mutations that are present in less than 0.1% of a cell population. The assay was characterized by detecting selected mutations in the KRAS gene, which has been implicated in up to 25% of all cancers. These mutations were detected in a single multiplex assay by incorporating the rapid flow cytometric readout of multiplexable DNA biosensors.

This protocol describes a new and rapid isothermal reaction process designed to amplify and detect a specific DNA sequence in purified DNA extracted from cultured cells. The protocol uses a DNA nanomachine that comprises two molecular switches that function in concert to isothermally amplify and detect a DNA target. First, a molecular beacon detection switch is 'activated' only if a DNA target sequence is present. A DNA primer and DNA polymerase are used to lock the beacon in an activated conformation. Second, an amplification and signal-transduction switch is initiated following successful activation. A nicking endonuclease and the DNA polymerase are used to replicate the DNA target. Both switches operate simultaneously at 40 °C in a single reaction to rapidly generate multiple copies of the DNA target in a cyclic polymerization reaction. This protocol enables femtomole amounts of a DNA target to be reproducibly amplified and detected in <40 min. We demonstrate the successful use of this protocol in assays containing synthetic DNA components and purified DNA extracted from biological samples.

The location and level of DNA methylation within a genome is emerging as an important biomarker for cancer diagnosis. Despite its potential, it is difficult to comprehensively analyze the epialleles that are often found in a biological sample. Therefore, an assay utilizing molecular inversion probes was designed and used to expose and quantify epialleles in heterogeneously methylated bisulphite treated genomic DNA. Different CpG dinucleotides were able to be rapidly quantified with high resolution, sensitivity and specificity over a large dynamic range using rapid flow cytometric readout of multiplexable microbead DNA biosensors.