Reverse Transcriptase Inhibition Disrupts Repeat Element Life Cycle in Colorectal Cancer.

Altered RNA expression of repetitive sequences and retrotransposition are frequently seen in colorectal cancer, implicating a functional importance of repeat activity in cancer progression. We show the nucleoside reverse transcriptase inhibitor 3TC targets activities of these repeat elements in colorectal cancer preclinical models with a preferential effect in p53-mutant cell lines linked with direct binding of p53 to repeat elements. We translate these findings to a human phase II trial of single-agent 3TC treatment in metastatic colorectal cancer with demonstration of clinical benefit in 9 of 32 patients. Analysis of 3TC effects on colorectal cancer tumorspheres demonstrates accumulation of immunogenic RNA:DNA hybrids linked with induction of interferon response genes and DNA damage response. Epigenetic and DNA-damaging agents induce repeat RNAs and have enhanced cytotoxicity with 3TC. These findings identify a vulnerability in colorectal cancer by targeting the viral mimicry of repeat elements. SIGNIFICANCE: Colorectal cancers express abundant repeat elements that have a viral-like life cycle that can be therapeutically targeted with nucleoside reverse transcriptase inhibitors (NRTI) commonly used for viral diseases. NRTIs induce DNA damage and interferon response that provide a new anticancer therapeutic strategy. This article is highlighted in the In This Issue feature, p. 1397.

Simplified Digital Enzyme-Linked Immunosorbent Assay Using Tyramide Signal Amplification and Fibrin Hydrogels.

Many protein biomarkers occur at very low concentrations in biofluids like blood and saliva, and ultrasensitive detection methods are required in order to measure them. Approaches such as digital enzyme-linked immunosorbent assays (ELISA) and single molecule arrays (Simoa) have been developed to accurately quantitate protein concentrations as low as attomolar levels. Although these techniques are being implemented in research and clinical laboratories to develop ultrasensitive clinical diagnostic assays, the size and cost of the instruments required to run these digital assays have precluded them from being implemented into point-of-care diagnostic formats. Here, we report the development of a simplified digital ELISA format that is more amenable to point-of-care technologies, referred to as catalyzed reporter deposition digital ELISA (CARD-dELISA). On-bead signal generation using the CARD tyramide signal amplification technique is combined with bead immobilization in fibrin hydrogels for single molecule counting in a simplified workflow format. CARD-dELISA allows for ultrasensitive protein detection (IL-6: ∼1 fM) with a dynamic range similar to the conventional Simoa assay. We use CARD-dELISA to measure IL-6 in saliva samples and show good agreement with conventional Simoa.

Transcription Factor Based Small-Molecule Sensing with a Rapid Cell Phone Enabled Fluorescent Bead Assay.

Recently, allosteric transcription factors (TFs) were identified as a novel class of biorecognition elements for in vitro sensing, whereby an indicator of the differential binding affinity between a TF and its cognate DNA exhibits dose-dependent responsivity to an analyte. Described is a modular bead-based biosensor design that can be applied to such TF-DNA-analyte systems. DNA-functionalized beads enable efficient mixing and spatial separation, while TF-labeled semiconductor quantum dots serve as bright fluorescent indicators of the TF-DNA bound (on bead) and unbound states. The prototype sensor for derivatives of the antibiotic tetracycline exhibits nanomolar sensitivity with visual detection of bead fluorescence. Facile changes to the sensor enable sensor response tuning without necessitating changes to the biomolecular affinities. Assay components self-assemble, and readout by eye or digital camera is possible within 5 minutes of analyte addition, making sensor use facile, rapid, and instrument-free.

Ultrasensitive Detection of Attomolar Protein Concentrations by Dropcast Single Molecule Assays.

Measurements of very low levels of biomolecules, including proteins and nucleic acids, remain a critical challenge in many clinical diagnostic applications due to insufficient sensitivity. While digital measurement methods such as Single Molecule Arrays (Simoa), or digital ELISA, have made significant advances in sensitivity, there are still many potential disease biomarkers that exist in accessible biofluids at levels below the detection limits of these techniques. To overcome this barrier, we have developed a simple strategy for single molecule counting, dropcast single molecule assays (dSimoa), that enables more target molecules to be counted through increased sampling efficiency and with a simpler workflow. In this approach, beads are simply dropcast onto a microscope slide and dried into a monolayer film for digital signal readout. The dSimoa platform achieves attomolar limits of detection, with an up to 25-fold improvement in sensitivity over Simoa, the current state of the art for ultrasensitive protein detection. Furthermore, due to its simple readout process and improved cost-effectiveness compared to existing digital bioassays, dSimoa increases amenability to integration into point-of-care platforms. As an illustration of the potential utility of dSimoa, we demonstrate its ability to measure previously undetectable levels of Brachyury, a tissue biomarker for chordoma, in plasma samples. With its significantly enhanced sensitivity and simplicity, dSimoa can pave the way toward the discovery of new biomarkers for early disease diagnosis and improved health outcomes.

Single Molecule Protein Detection with Attomolar Sensitivity Using Droplet Digital Enzyme-Linked Immunosorbent Assay.

Many proteins are present at low concentrations in biological samples, and therefore, techniques for ultrasensitive protein detection are necessary. To overcome challenges with sensitivity, the digital enzyme-linked immunosorbent assay (ELISA) was developed, which is 1000× more sensitive than conventional ELISA and allows sub-femtomolar protein detection. However, this sensitivity is still not sufficient to measure many proteins in various biological samples, thereby limiting our ability to detect and discover biomarkers. To overcome this limitation, we developed droplet digital ELISA (ddELISA), a simple approach for detecting low protein levels using digital ELISA and droplet microfluidics. ddELISA achieves maximal sensitivity by improving the sampling efficiency and counting more target molecules. ddELISA can detect proteins in the low attomolar range and is up to 25-fold more sensitive than digital ELISA using Single Molecule Arrays (Simoa), the current gold standard tool for ultrasensitive protein detection. Using ddELISA, we measured the LINE1/ORF1 protein, a potential cancer biomarker that has not been previously measured in serum. Additionally, due to the simplicity of our device design, ddELISA is promising for point-of-care applications. Thus, ddELISA will facilitate the discovery of biomarkers that have never been measured before for various clinical applications.

Single-molecule analysis of nucleic acid biomarkers - A review.

Nucleic acids are important biomarkers for disease detection, monitoring, and treatment. Advances in technologies for nucleic acid analysis have enabled discovery and clinical implementation of nucleic acid biomarkers. However, challenges remain with technologies for nucleic acid analysis, thereby limiting the use of nucleic acid biomarkers in certain contexts. Here, we review single-molecule technologies for nucleic acid analysis that can be used to overcome these challenges. We first discuss the various types of nucleic acid biomarkers important for clinical applications and conventional technologies for nucleic acid analysis. We then discuss technologies for single-molecule in vitro and in situ analysis of nucleic acid biomarkers. Finally, we discuss other ultra-sensitive techniques for nucleic acid biomarker detection.