Labeling of prokaryotic mRNA in live cells using fluorescentin situhybridization of transcript-annealing molecular beacons (FISH-TAMB)

Abstract

High-throughput sequencing and cellular imaging have expanded our knowledge of microbial diversity and expression of cellular activity. However, it remains challenging to characterize low-abundance, slow-growing microorganisms that play key roles in biogeochemical cycling. With the goal of isolating transcriptionally active cells of these microorganisms from environmental samples, we developed fluorescent in situ hybridization of transcript-annealing molecular beacons (FISH-TAMB) to label living prokaryotic cells. FISH-TAMB utilizes polyarginine cell-penetrating peptides to deliver molecular beacons across cell walls and membranes. Target cells are fluorescently labeled via hybridization between molecular beacons and messenger RNA of targeted functional genes. FISH-TAMB’s target specificity and deliverance into both bacterial and archaeal cells were demonstrated by labeling intracellular methyl-coenzyme M reductase A (mcrA) transcripts expressed by Escherichia coli mcrA+, Methanosarcina barkeri, and a methanogenic enrichment of deep continental fracture fluid. Growth curve analysis supported sustained cellular viability following FISH-TAMB treatment. Flow cytometry and confocal microscopy detected labeled single cells and single cells in aggregates with unlabeled cells. As FISH-TAMB is amenable to target any functional gene of interest, when coupled with cell sorting, imaging, and sequencing techniques, FISH-TAMB will enable characterization of key uncharacterized rare biosphere microorganisms and of the syntrophically activated metabolic pathways between physically associated microorganisms.

Recent developments and future directions of deep biosphere research

“…在90℃以下, 醋酸的合成往往是由微生 物完成, 但是在高于90℃, 醋酸往往由热裂解产生, 因此深地微生物的最高承受温度可能是由生物维持 代谢所需要的能量与化学反应能提供的能量相对大 小所决定的 [38] [47] . [106] 和荧光原位杂交技术(BONCAT-FISH) [107,108] 为我们研 究微生物的功能与代谢提供了最新的技术支撑. 高 精度质谱仪, 包括测定微生物脂类单体同位素的质 谱仪也将成为定量研究深部生物量的一项重要技术.…”

MicroRNA Expression Profiles Analysis in Sperm Reveals hsa-mir-191 is an Auspicious Omen of in Vitro Fertilization

“…Recently, the FISH-Flow technique reported by Riccardo Arrigucci et al makes it possible to analyze single cells by flow cytometry and RNA probes[22]. Rachel L. Harris et al's FISH-TAMB technology further implements labeling of mRNA in live cells[23].If these technologies can be combined with highly sensitive miRNA-191 probe[24], it will enable to help us implement the miRNA-191 probe as a sorting label for sperm selection.Merck Millipore's SmartFlare TM has provided some commercial methods for flow detection of live cellular RNA, which has inspired us to further apply this concept to assisted reproductive technologies and assess the safety and effectiveness of miRNA-191 probe.…”