Fluorescence Lifetime Imaging of Nanoflares for mRNA Detection in Living Cells

Abstract

The expression level of tumor-related mRNA can reveal significant information about tumor progression and prognosis, so specific mRNA in cells provides an important approach for biological and disease studies. Here, fluorescence lifetime imaging of nanoflares in living cells was first employed to detect specific intracellular mRNA. We characterized the lifetime changes of the prepared nanoflares before and after the treatment of target mRNA and also compared the results with those of fluorescence intensity-based measurements both intracellularly and extracellularly. The nanoflares released the cy5-modified oligonucleotides and bound to the targets, resulting in a fluorescence lifetime lengthening. This work puts forward another dimension of detecting specific mRNA in cells and can also open new ways for detection of many other biomolecules.

An integrated flow cytometry-based platform for isolation and molecular characterization of circulating tumor single cells and clusters

“…A significant advantage of this methodology is its versatility in terms of the biomarkers that can be assessed due to a large repertoire of commercially available antibodies and fluorescent probes. Other detection techniques such as RNA probes 55 and molecular beacons that bind intracellular RNA transcripts 56 as well as aptamers that can be generated to recognize virtually any cell surface marker 57 , 58 , can also be easily adapted for this approach. Further, cell sorters offer considerable flexibility in modes of collection including different sized tubes, multi-well plates, or even microscope slides depending on the downstream application.…”

Optical Diagnostic Based on Functionalized Gold Nanoparticles

“…Therefore, the identification and characterization of disease-related mRNA in cells is of great significance for early diagnosis and treatment of numerous diseases (e.g., cancer). However, the conventional approaches—such as reverse transcription polymerase chain reaction (RT-PCR), Northern-blots, and microarray analysis—are limited by the requirement for groups of cells and incapable of analysis of single cells in real time [102,103]. Oligonucleotide-functionalized Au NPs have high intracellular delivery efficiency by endocytosis, high stability, and high signal-to- background ratios for mRNA detection in living cells [104,105].…”

“…Oligonucleotide-functionalized Au NPs have high intracellular delivery efficiency by endocytosis, high stability, and high signal-to- background ratios for mRNA detection in living cells [104,105]. Nanoflare, a new class of probe, has exhibited great promise in intracellular mRNA detection as well as labeling and isolating specific cells since its first appearance in 2007 [102,103,105,106,107,108]. A typical nanoflare is a spherical nucleic acid Au NP conjugates consisting of densely packed recognition oligonucleotides with complementary sequences to the target mRNA, many of which are hybridized to the so-called flare oligonucleotides with fluorophore labeling.…”

Recent advances in high-performance fluorescent and bioluminescent RNA imaging probes

“…The probable cellular uptake mechanism is that they target class A scavenger receptors and then enter cells through endocytosis via a lipid-raft-dependent, caveolae-mediated pathway. 136 (4) Enabling additional imaging modality (fluorescence lifetime imaging) due to lengthening of the fluorescence lifetime of Nano-MBs upon binding of target RNAs 137 . On the other hand, it is worth mentioning that in some cases, GNP-Nano-MBs also lead to mRNA downregulation.…”