ABSTRACT
Tracking the viral progression of SARS-CoV-2 in COVID-19 infected body tissues is an emerging need of the current pandemic. Imaging at near infrared second biological window (NIR-II) offers striking benefits over the other technologies to explore deep-tissue information. Here we design, synthesise and characterise a molecular probe that selectively targets the N-gene of SARS-CoV-2. Highly specific antisense oligonucleotides (ASOs) were conjugated to lead sulfide quantum dots using a UV-triggered thiol-ene click chemistry for the recognition of viral RNA. Our ex vivo imaging studies demonstrated that the probe exhibits aggregation induced NIR-II emission only in presence of SARS-CoV-2 RNA which can be attributed to the efficient hybridisation of the ASOs with their target RNA strands.
Subject(s)
COVID-19/diagnosis , COVID-19/virology , Fluorescent Dyes/chemistry , Oligonucleotides, Antisense/chemistry , Quantum Dots/chemistry , SARS-CoV-2/isolation & purification , Spectroscopy, Near-Infrared/methods , Animals , COVID-19/diagnostic imaging , COVID-19/metabolism , Click Chemistry/methods , Fluorescent Dyes/chemical synthesis , Humans , Lung/diagnostic imaging , Lung/metabolism , Lung/virology , Metal Nanoparticles/chemistry , Mice , Mice, Inbred BALB C , Models, Animal , SARS-CoV-2/genetics , SARS-CoV-2/metabolismABSTRACT
Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that releases Ca2+ from acidic organelles through the activation of two-pore channels (TPCs) to regulate endolysosomal trafficking events. NAADP action is mediated by NAADP-binding protein(s) of unknown identity that confer NAADP sensitivity to TPCs. Here, we used a "clickable" NAADP-based photoprobe to isolate human NAADP-binding proteins and identified Jupiter microtubule-associated homolog 2 (JPT2) as a TPC accessory protein required for endogenous NAADP-evoked Ca2+ signaling. JPT2 was also required for the translocation of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus through the endolysosomal system. Thus, JPT2 is a component of the NAADP receptor complex that is essential for TPC-dependent Ca2+ signaling and control of coronaviral entry.
Subject(s)
COVID-19/metabolism , COVID-19/virology , Calcium Signaling/physiology , Microtubule-Associated Proteins/metabolism , NADP/analogs & derivatives , SARS-CoV-2/physiology , Affinity Labels , Animals , Calcium Channels/metabolism , Carrier Proteins/metabolism , Click Chemistry/methods , Gene Knockdown Techniques , HEK293 Cells , Humans , Microtubule-Associated Proteins/antagonists & inhibitors , Microtubule-Associated Proteins/genetics , NADP/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Second Messenger Systems/physiology , Transcriptome , Virus InternalizationABSTRACT
The reliable detection of transcription events through the quantification of the corresponding mRNA is of paramount importance for the diagnostics of infections and diseases. The quantification and localization analysis of the transcripts of a particular gene allows disease states to be characterized more directly compared to an analysis on the transcriptome wide level. This is particularly needed for the early detection of virus infections as now required for emergent viral diseases, e. g. Covid-19. In situ mRNA analysis, however, is a formidable challenge and currently performed with sets of single-fluorophore-containing oligonucleotide probes that hybridize to the mRNA in question. Often a large number of probe strands (>30) are required to get a reliable signal. The more oligonucleotide probes are used, however, the higher the potential off-target binding effects that create background noise. Here, we used click chemistry and alkyne-modified DNA oligonucleotides to prepare multiple-fluorophore-containing probes. We found that these multiple-dye probes allow reliable detection and direct visualization of mRNA with only a very small number (5-10) of probe strands. The new method enabled the inâ situ detection of viral transcripts as early as 4 hours after infection.