Detection of Tumor-Associated Membrane Receptors on Extracellular Vesicles from Non-Small Cell Lung Cancer Patients via Immuno-PCR.

Precision cancer medicine for non-small-cell lung cancer (NSCLC) has increased patient survival. Nevertheless, targeted agents towards tumor-associated membrane receptors only result in partial remission for a limited time, calling for approaches which allow longitudinal treatment monitoring. Rebiopsy of tumors in the lung is challenging, and metastatic lesions may have heterogeneous signaling. One way ahead is to use liquid biopsies such as circulating tumor DNA or small extracellular vesicles (sEVs) secreted by the tumor into blood or other body fluids. Herein, an immuno-PCR-based detection of the tumor-associated membrane receptors EGFR, HER2, and IGF-1R on CD9-positive sEVs from NSCLC cells and pleural effusion fluid (PE) of NSCLC patients is developed utilizing DNA conjugates of antibody mimetics and affibodies, as detection agents. Results on sEVs purified from culture media of NSCLC cells treated with anti-EGFR siRNA, showed that the reduction of EGFR expression can be detected via immuno-PCR. Protein profiling of sEVs from NSCLC patient PE samples revealed the capacity to monitor EGFR, HER2, and IGF-1R with the immuno-PCR method. We detected a significantly higher EGFR level in sEVs derived from a PE sample of a patient with an EGFR-driven NSCLC adenocarcinoma than in sEVs from PE samples of non-EGFR driven adenocarcinoma patients or in samples from patients with benign lung disease. In summary, we have developed a diagnostic method for sEVs in liquid biopsies of cancer patients which may be used for longitudinal treatment monitoring to detect emerging bypassing resistance mechanisms in a noninvasive way.

Antibody detection by agglutination-PCR (ADAP) enables early diagnosis of HIV infection by oral fluid analysis.

Oral fluid (OF) is a highly effective substrate for population-based HIV screening efforts, as it is noninfectious and significantly easier to collect than blood. However, anti-HIV antibodies are found at far lower concentrations in OF compared with blood, leading to poor sensitivity and a longer period of time from infection to detection threshold. Thus, despite its inherent advantages in sample collection, OF is not widely used for population screening. Here we report the development of an HIV OF assay based on Antibody Detection by Agglutination-PCR (ADAP) technology. This assay is 1,000-10,000 times more analytically sensitive than clinical enzyme-linked immunoassays (EIAs), displaying both 100% clinical sensitivity and 100% specificity for detecting HIV antibodies within OF samples. We show that the enhanced analytical sensitivity enables this assay to correctly identify HIV-infected individuals otherwise missed by current OF assays. We envision that the attributes of this improved HIV OF assay can increase testing rates of at-risk individuals while enabling diagnosis and treatment at an earlier time point.

DNA-Specific Biosensors Based on Intramolecular ß-Lactamase-Inhibitor Complex Formation.

Synthetic protein switches that sequence-specifically respond to oligonucleotide-based input triggers provide valuable tools for the readout of oligonucleotide-based biomolecular systems and networks. Here, we discuss a highly modular approach to reversibly control the DNA-directed assembly and disassembly of a complex between TEM1-ß-lactamase and its inhibitor protein BLIP. By conjugating each protein to a unique handle oligonucleotide, the enzyme-inhibitor pair is noncovalently assembled upon the addition of a complementary ssDNA template strand, resulting in inhibition of enzyme activity. Hybridization of an input-oligonucleotide that is complementary to a target recognition sequence in the ssDNA template strand results in the formation of a rigid dsDNA helix that mechanically disrupts the enzyme-inhibitor complex, hereby restoring enzyme activity. Following this noncovalent approach allowed straightforward tuning of the ssDNA template recognition sequence and target oligonucleotide lengths with only a single set of oligonucleotide-functionalized enzyme and inhibitor domains. Using a fluorescent substrate, as little as 10 pM target oligonucleotide resulted in a distinguishable increase in enzyme activity.

Spatial Organization of Enzyme Cascade on a DNA Origami Nanostructure.

Self-assembled DNA nanostructures hold great promise to organize multi-enzyme systems with the precise control of the geometric arrangements. Enzymes modified with single-stranded DNA anchors are assembled onto the DNA origami tiles by hybridizing with the corresponding complementary strands displayed on the surface of the DNA nanostructures. Here, we describe a protocol of assembling a two-enzyme cascade on a discrete, rectangular DNA origami tile, where the distance between enzymes is precisely controlled for investigating the distance-dependent cascade activities.