Advancements in cellular imaging and high throughput sequencing have significantly increased our knowledge of microbial phylogenetic and metabolic diversity. However, it remains difficult to characterize cellular activities of most microbial species, especially low-abundance, slow-growing microorganisms. One such way to identify the taxonomy of single cells from environmental samples is through fluorescence in situ hybridization (FISH), which involves the use of fluorescent oligonucleotide linear probes targeting the universally conserved 16S ribosomal RNA (rRNA) gene. However, 16S rRNA-based identification requires prior knowledge of the target organisms’ phylogenies and provides no direct evidence of the target organisms’ metabolic roles. FISH methods targeting messenger RNA (mRNA) in prokaryotes have been developed to relate functional gene expression within organisms to their metabolic contributions to biogeochemical cycling (e.g. mRNA-FISH and its derivatives). However, these methods are performed on fixed (i.e. dead) cells, offering only a snapshot of past activity in labeled cells based solely on the single target gene being expressed. It would be advantageous to use a method that targets metabolically active cells that exhibit certain specific functions, and meanwhile maintains their viability. To our knowledge such a method has not yet been reported.
The technology relates to fluorescent in situ hybridization of transcript-annealing molecular beacons (FISH-TAMB), a method that utilizes cell-penetrating peptides bound to molecular beacons to enter living, metabolically active Bacteria and Archaea and fluorescently label target messenger RNA (mRNA). FISH-TAMB can be applied to target mRNA of any functional gene of interest without discriminating against low abundance or uncharacterized species, and does not require cellular fixation, permeabilization buffer treatments, or secondary signal amplification. FISH-TAMB’s chief advantage over traditional FISH methods is that it can be applied to living cells and not impact their viability. As many microbial communities researched by environmental microbiologists come from environments that are not easily accessible for resampling, the ability to maintain living biomass post analysis offers opportunities to further cultivate and characterize microorganisms that would otherwise be destined for biomedical waste. FISH-TAMB is a versatile addition to the molecular ecologist’s toolkit, with widespread application potential in the field of microbiology.
• Research tool for labs
• Diagnostic tool for active pathogenic microbial infections
• Detecting undescribed bacterial pathogens
• Diagnostic tool for wastewater treatment plants, bioremediation sites, water quality assessments, and monitoring infrastructure stability
• Can label active cells in complex, multi-species microbial aggregates
• Can identify active metabolic functions in cells without discriminating against unknown taxonomies (i.e. 16S rRNA -based phylogeny) and without prior cell fixation step
• Sensitive to real-time transcriptional activity
• Time efficient compared to traditional FISH methodologies
• Cost effective
Intellectual Property (and Development or Technology) Status
• Patent protection is pending.
• Princeton is currently seeking commercial partners for further development and commercialization of this technology
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