Intracellular RNA localization, dynamics, and functions determine contemporary understanding of gene regulation and the cell cycle. Meanwhile, inaccurate mRNA translation may cause gene-associated diseases or cancer. Precise control of localization and quantification of RNA targets in living cells are key points for the study of RNA processing and advanced translational medicine. However, RNA imaging in cells often requires signal-amplification methodologies for low-abundance sequence targets. Dye-loaded polymeric nanoparticles demonstrated excellent fluorescence properties, which brightness overcomes quantum dots in between 1-2 orders of magnitude. “DNANanoProbes” project is dedicated to bright polymeric nanoparticles decorated with DNA for intracellular imaging of nucleic acids. The proposed nanoparticles-based assay potentially enhances the sensitivity of the RNA detection compared to classical hybridization probes.
Nowadays, the value of modern diagnostic methods for high-throughput screening became as highly demanded as never before. RNA diagnostics (e.g. qPCR, LAMP, FISH, NGS) in daily life associated with infectious, orphan, neurodegenerative diseases, biopsy profiling for tumor tissues and pregnancy monitoring. Unfortunately, most RNA analyses today manipulate with fixed cells or cellular lysates, while a significant part of RNA may be lost or degraded upon extraction and separation, especially for small RNAs and low-abundant targets. Fluorescent nanosensors proposed in the project are a new type of platform for rapid RNA testing with quantitate fluorescence response not exploiting enzymatic or hybridization amplification schemes. Manipulation with living cells provides a unique opportunity for RNA sensing with minimal disruption of cellular membrane maintaining the normal circle of life cells. These advantages provide remarkable improvement for the sensitivity of direct RNA quantification along with an insight into RNA intracellular transport, RNA-protein complex organization and gene regulation.
The current project was dedicated to the creation of DNA-nanosensors based on dye-loaded polymeric nanoparticles (NPs), decorated with oligonucleotides that target sequences of interest in cells. The fundamental value of the proposed technology is high sensitivity in comparison to classical DNA probes due to high fluorescence brightness of polymer NPs encapsulated with organic fluorophores. The implementation of this strategy is associated with the development of the nanosensors from scratch, including: i) synthesis of new fluorescent dyes with improved brightness and tuned emission spectrum; ii) rational design and assembly of DNA-decorated nanoparticles encapsulated with fluorescent dyes; iii) application of the DNA-nanoprobes in living cells.
