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Design of light-harvesting DNA-nanoprobes with ratiometric signal amplification for fluorescence imaging of live cells.

Periodic Reporting for period 1 - DNANanoProbes (Design of light-harvesting DNA-nanoprobes with ratiometric signal amplification for fluorescence imaging of live cells.)

Reporting period: 2019-05-15 to 2021-05-14

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.
The body of the project aimed at development of DNA-nanosensors for ratiometric RNA imaging. The concept includes formation of biocompatible fluorescent nanoparticles – “the core” of nanosensor, covered with oligonucleotides capturing target sequences – “the shell”. Further, the nanosensors are planned to apply for intracellular RNA imaging in living cells.
First, we synthesized a series of cationic hydrophobic dyes of different excitation/emission spectra to alter photophysical properties of future nanoparticles. These dyes were ion-exchanged with hydrophobic tetraphenylborate derivatives as a counterion. Next, the fluorescent ion pairs were applied in polymeric PMMA nanoparticles preparation with different concentrations of encapsulated dyes. Thus, we selected a green-yellow emitting rhodamine, demonstrating excellent fluorescence properties: high QY (37%) at 250 mM loading with respect to the polymer. Further, for the first time, we demonstrated successful application of poorly emissive styryl pyrydinium dyes with tetraphenylborates for encapsulation into polymer NPs. These styryl pyridinium dyes drastically enhance fluorescence QY in polymeric matrix reaching approx. QY of 40% at 500 mM dye-loading, featuring high fluorescence brightness (50-fold brighter than QDdot-605) and good photostability without detectable blinking. The phenomenon of QY enhancement for poorly emissive dyes in presence of sterically hindered hydrophobic counterion was coined as ionic aggregation-induced emission – iAIE.
To make a step towards biocompatibility of fluorescent nanoparticles we proposed a new class of bulky hydrophobic counterions derived from C-acyl barbiturates. By tuning their structure related to enol acidity and overall hydrophobicity, it became possible to create a much less toxic organic counterion – BarPh2 compare to standard perfluorinated tetraphenylborate – F5-TPB. The new counterions in PLGA NPs loaded with octadecyl rhodamine B demonstrated similar emitting performance as F5-TPB in terms of preventing ACQ effect and leakage.
Within the project, we also proposed the design of DNA probes labeled with intercalating dyes: thiazole orange and quinolone blue as fluorescent acceptors for the DNA-nanosensors. Since oligonucleotide probes labeled with both dyes demonstrated low QY enhancement upon hybridization with miR21 or miR141 targets, dimeric fluorescent labels featuring H-aggregation were synthesized and applied for nanosensor development.
Finally, we aimed to develop a safe and robust delivery method of DNA-decorated nanoparticles into the cytosol living cells. For this purpose, we prepared fluorescent polymeric NPs covered by (dA)20 non-coding sequence or survivin-encoding oligonucleotides. We reached excellent cell penetration and viability of electroporated cells in presence of DNA-decorated fluorescent NPs. Fluorescence microscopy revealed bright single spot fluorescence demonstrating random Brownian movement for nanoparticles covered with (dA)20 non-coding sequence and significantly different diffusion profile for surviving targeted NPs, suggesting their hybridization with the intracellular mRNA target of survivin.
Fluorescent methods of analysis in biology are fast spreading in different fields of applications related to molecular biology, drug development, advanced medicine and clinical diagnostics. Nowadays, the impact of modern nucleic acids diagnostics for a huge list of diseases could not be underestimated. Meanwhile, life sciences associated research requires new tools for precise control of RNA localization and quantification in living cells. The current research project made several advances towards development of such diagnostics tools, as follows: i) novel bright fluorescent nanomaterials of different color allowing enhanced signal amplification, and thus better limit of detection for an analyte; ii) novel biocompatible nanoparticles with improved optical properties and decreased toxicity allowing to perform in vitro and in vivo analysis; iii) new design for DNA-nanosensors based on light-up fluorophores for potential clinical diagnostics, e.g. analysis of microRNA in tumor biopsy; iv) robust methodology for delivery of DNA-decorated nanoparticles into the cytosol of live cells based on electroporation. To conclude, we believe that performed study laid the groundwork for future nanotechnology-based medical diagnostics and therapy, which can positively impact human health. Successful application of the nanoprobes in the field of biomedicine should also result in their commercialization, with a consecutive positive economic impact on society.
Graphical abstract for "DNANanoProbes" project.