![]() This method is powerful and as demonstrated can successfully detect, quantify, and differentiate between multiple BRCA1 splice variants with single copy sensitivity, paving the way for single cell quantitative genetic profiling in the long-term. Two nanoparticle-conjugated probes were attached to the BRCA1 mRNA target in a sequence-specific manner, resulting in a spectral shift of the signal due to dimer formation. This method was used to detect and quantify BRCA1 mRNA splice variants both in vitro and in vivo. Here, we present an elegant plasmonic nanoparticle network structure-based approach, which generates a plasmon-coupled dimer capable of detecting single mRNA variants. However, achieving the sensitivity required to detect and quantify single molecules in living cells is challenging because of the inability to discriminate specific signals from the background noise due to the intracellular environment. Hence, nanoplasmonic dimer-based approaches using dark-field microscopy are powerful tools for detecting molecules such as DNA 30 and protein 31, 32. 28, 29 In particular, when a dimer with less than 3 nm of inter-particle spacing is formed as a result of two probes binding to a single target molecule, the signal intensity of the single dimer is strong enough to allow intracellular imaging due to local field enhancement. To this end, the plasmon resonance of noble metal nanoparticles in the visible range has been utilized to detect particle structures 20, 25– 27 or nanoparticle dimer formation. 18– 22 Relative to individual GNPs, network structures formed by target molecule binding display enhanced surface-enhanced Raman scattering signal (SERS) 23 and Rayleigh scattering 24, making them ideal candidates for single molecule probes. 14 During interaction with photons, the plasmonic cross section generates a 10- to 10 4-fold stronger signal than fluorophores or quantum dots, while still retaining well-defined optical characteristics. Gold nanoparticle (GNP) conjugated DNA probes hybridized to target molecules are of significant interest owing to their strong plasmonic signals and probe stability, which enable intracellular single particle detection 16, 17 without notable degradation. Therefore, detection and quantification of mRNA splicing variants in living cells are currently challenging. ![]() Fluorescence 5– 8, molecular beacons 9, Förster resonance energy transfer (FRET) 10, 11, and fluorescently labeled modified metal nanoparticles 12– 14 have also been used to detect mRNA in living cells however, these methods lack the flexibility to detect small gene sequence variations 15. ![]() Northern blots 3 and reverse transcription polymerase chain reaction (RT-PCR) 4 are commonly used to detect specific mRNA transcripts however, these methods require millions of cells and are not applicable to real-time studies of live single cells. However, the regulation, localization, and relative abundance of BRCA1 splicing transcripts in different cell types, as well as the contribution of the translated protein isoforms to cell growth, are poorly characterized. Breast cancer susceptibility gene 1 (BRCA1) plays a significant role in formation of sporadic tumors in the absence of germline mutations 1, 2 and is used as an indicator of malignant breast cancer. ![]() Although their expression level is generally low, mRNA splice variants are critical for cell growth and development defects in the expression of specific isoforms can lead to abnormal gene expression, cellular dysfunction, and disease. Our study provides valuable insights on RNA and its transport in living cells, which has the potential to enhance our understanding of cellular protein complex, pharmacogenomics, genetic diagnosis, and gene therapies.Īlternative transcripts enable generation of multiple mRNA isoforms from single genes more than 90% of human genes undergo alternative splicing. The spatial and temporal distribution of three selected splice variants of the breast cancer susceptibility gene, BRCA1 were monitored at single copy resolution by measuring the hybridization dynamics of nanoplasmonic antennas targeting complementary mRNA sequences in live cells. Here, we report a spectroscopic strategy for quantitative imaging of mRNA splice variants in living cells, using nanoplasmonic dimer antennas. Errors in RNA splicing have been known to correlate with different diseases however, a key limitation is the lack of technologies for live cell monitoring and quantification to understand the process of alternative splicing. Alternative mRNA splicing is a fundamental process of gene regulation via the precise control of the post-transcriptional step that occurs before mRNA translation.
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