Amplification-free detection of circulating microRNA biomarkers from body fluids based on fluorogenic oligonucleotide-templated reaction between engineered peptide nucleic acid probes: application to prostate cancer diagnosis

Highly abundant in cells, microRNAs (or miRs) play a key role as regulators of gene expression. A proportion of them are also detectable in biofluids making them ideal noninvasive biomarkers for pathologies in which miR levels are aberrantly expressed, such as cancer. Peptide nucleic acids (PNAs) are engineered uncharged oligonucleotide analogues capable of hybridizing to complementary nucleic acids with high affinity and high specificity. Herein, novel PNA-based fluorogenic biosensors have been designed and synthesized that target miR biomarkers for prostate cancer (PCa). The sensing strategy is based on oligonucleotide-templated reactions where the only miR of interest serves as a matrix to catalyze an otherwise highly unfavorable fluorogenic reaction. Validated in vitro using synthetic RNAs, these newly developed biosensors were then shown to detect endogenous concentrations of miR in human blood samples without the need for any amplification step and with minimal sample processing. This low-cost, quantitative, and versatile sensing technology has been technically validated using gold-standard RT-qPCR. Compared to RT-qPCR however, this enzyme-free, isothermal blood test is amenable to incorporation into low-cost portable devices and could therefore be suitable for widespread public screening

Oligonucleotide-templated lateral flow assays for amplification-free sensing of circulating microRNAs

Herein we demonstrate the first example of oligonucleotide-templated reaction (OTR) performed on paper, using lateral flow to capture and concentrate specific nucleic acid biomarkers on a test line. Quantitative analysis, using a low-cost benchtop fluorescence reader showed very high specificity down to the single nucleotide level and proved sensitive enough for amplification-free, on-chip, detection of endogenous concentrations of miR-150-5p, a recently identified predictive blood biomarker for preterm birth

Hydrogel-coated microneedle arrays for minimally invasive sampling and sensing of specific circulating nucleic acids from skin interstitial fluid

Minimally invasive technologies that can sample and detect cell-free nucleic acid biomarkers from liquid biopsies have recently emerged as clinically useful for early diagnosis of a broad range of pathologies, including cancer. Although blood has so far been the most commonly interrogated bodily fluid, skin interstitial fluid has been mostly overlooked despite containing the same broad variety of molecular biomarkers originating from cells and surrounding blood capillaries. Emerging technologies to sample this fluid in a pain-free and minimally-invasive manner often take the form of microneedle patches. Herein, we developed microneedles that are coated with an alginate–peptide nucleic acid hybrid material for sequence-specific sampling, isolation, and detection of nucleic acid biomarkers from skin interstitial fluid. Characterized by fast sampling kinetics and large sampling capacity (∼6.5 μL in 2 min), this platform technology also enables the detection of specific nucleic acid biomarkers either on the patch itself or in solution after light-triggered release from the hydrogel. Considering the emergence of cell-free nucleic acids in bodily fluids as clinically informative biomarkers, platform technologies that can detect them in an automated and minimally invasive fashion have great potential for personalized diagnosis and longitudinal monitoring of patient-specific disease progression

Lateral Flow Test (LFT) detects cell-free microRNAs predictive of preterm birth directly from human plasma

Despite extensive research toward the development of point-of-care nucleic acid tests (POC NATs) for the detection of microRNAs (miRs) from liquid biopsies, major hurdles remain including the strict requirement for extensive off-chip sample preprocessing. Herein, a nucleic acid lateral flow test (NALFT) is reported on that enables the direct detection of endogenous miRs from as little as 3 μL of plasma without the requirement for any enzyme-catalyzed target amplification or complex miR extraction steps. This is achieved through integration of a denaturing hydrogel composite material onto the LFT, allowing for near-instantaneous on-chip release of miRs from their carriers (extracellular vesicles or transport proteins) prior to detection. This next-generation LFT is sensitive enough to detect endogenous concentrations of miR-150-5p, a predictive biomarker for preterm birth (PTB) found deregulated in maternal blood from as early as 12th week of pregnancy. Herein, a key step is represented toward a first bedside test for risk-stratification during pregnancy by predicting true outcome at a very early stage. More generally, the universal and versatile nature of this novel sample preprocessing platform can further improve the robustness of existing NALFTs and facilitate their application at the POC

The Drosophila Gap Gene Network Is Composed of Two Parallel Toggle Switches

Drosophila “gap” genes provide the first response to maternal gradients in the early fly embryo. Gap genes are expressed in a series of broad bands across the embryo during first hours of development. The gene network controlling the gap gene expression patterns includes inputs from maternal gradients and mutual repression between the gap genes themselves. In this study we propose a modular design for the gap gene network, involving two relatively independent network domains. The core of each network domain includes a toggle switch corresponding to a pair of mutually repressive gap genes, operated in space by maternal inputs. The toggle switches present in the gap network are evocative of the phage lambda switch, but they are operated positionally (in space) by the maternal gradients, so the synthesis rates for the competing components change along the embryo anterior-posterior axis. Dynamic model, constructed based on the proposed principle, with elements of fractional site occupancy, required 5–7 parameters to fit quantitative spatial expression data for gap gradients. The identified model solutions (parameter combinations) reproduced major dynamic features of the gap gradient system and explained gap expression in a variety of segmentation mutants

Platforms for bioorthogonal oligonucleotide-templated reactions: Translating Concepts into devices

The exponential improvements made in DNA sequencing technologies, together with the rapidly declining associated costs, has increasingly led to the expansion of the field of personalised genomic medicine. Changes in the sequence or copy number of specific deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules represent key signatures for the diagnosis, prognosis, classification and monitoring of a broad range of pathologies, most notably cancer. Technologies that can detect these changes require analytical tools that can detect DNA or RNA with high sensitivity and high specificity. Sensing based on bioorthogonal oligonucleotide-templated reactions (OTRs) has been recognised as an elegant strategy that satisfies these criteria and was successfully used for the quantitative detection of nucleic acids both in vitro and in vivo. Herein, we will focus on recent efforts to implement bioorthogonal OTRs into clinically useful biosensors using probes immobilised on or embedded in customised materials and platforms