Paired Capture and FISH Detection of Individual Virions Enable Cell-Free Determination of Infectious Titers.

Early detection of viruses can prevent the uncontrolled spread of viral infections. Determination of viral infectivity is also critical for determining the dosage of gene therapies, including vector-based vaccines, CAR T-cell therapies, and CRISPR therapeutics. In both cases, for viral pathogens and viral vector delivery vehicles, fast and accurate measurement of infectious titers is desirable. The most common methods for virus detection are antigen-based (rapid but not sensitive) and polymerase chain reaction (PCR)-based (sensitive but not rapid). Current viral titration methods heavily rely on cultured cells, which introduces variability within labs and between labs. Thus, it is highly desirable to directly determine the infectious titer without using cells. Here, we report the development of a direct, fast, and sensitive assay for virus detection (dubbed rapid capture fluorescence in situ hybridization (FISH) or rapture FISH) and cell-free determination of infectious titers. Importantly, we demonstrate that the virions captured are "infectious," thus serving as a more consistent proxy of infectious titers. This assay is unique because it first captures viruses bearing an intact coat protein using an aptamer and then detects genomes directly in individual virions using fluorescence in situ hybridization (FISH); thus, it is selective for infectious particles (i.e., positive for coat proteins and positive for genomes).

Click chemistry-based amplification and detection of endogenous RNA and DNA molecules in situ using clampFISH probes.

Direct labeling and measurement of gene expression in single cells show the tremendous variability otherwise hidden in bulk measurements. Single-molecule RNA fluorescence in situ hybridization (FISH) has become a mainstay in laboratories worldwide for measuring gene expression with precision. However, this method remains relatively low throughput because the total fluorescent signal produced is weak and requires long exposure times and high magnification microscopy, which limits the total number of cells sampled in each image. As such, it is experimentally difficult and time-consuming to sample a large enough population of cells to visualize and quantify specific gene expression of rare cells directly. Several FISH-based tools were recently developed that retain single-molecule sensitivity and specificity while greatly amplifying the fluorescent signal, thus making FISH-based analysis possible using standard microscopes with low magnification objectives. These tools have also enabled the detection of smaller and more specific targets like splice junctions or single nucleotide polymorphisms. Here we will describe one such tool, clampFISH, an oligonucleotide-based fluorescence amplification strategy for visualizing genomic loci and individual RNA transcripts in fixed cells. ClampFISH maintains specificity while amplifying fluorescent signals, making it amenable to high throughput assays such as low magnification microscopy, spatial transcriptomics, and flow sorting. The clampFISH technique involves probing the target RNA or DNA using a series of C-shaped oligonucleotide probes, each with a 3' azide and a 5' alkyne. Hybridization of the probe with the target nucleic acid brings the azide and the alkyne in close proximity, allowing for ligation via bioorthogonal click chemistry (CuAAC). As a result, the probe forms a closed loop around the target sequence, thus enabling stringent washes to remove nonspecific binding in further rounds of amplification and retention of signal throughout liquid handling steps. Iterative rounds of hybridization with C-shaped, fluorescently labeled probes exponentially amplify the fluorescent signal. ClampFISH is simple to implement and expands the utility of in situ hybridization for multiple high throughput techniques such as low magnification microscopy, flow cytometry, and sorting based on RNA expression levels.