Oligonucleotide probes labeled with pyrene pairs that form excimers have a number of applications in hybridization analysis of nucleic acids. A long excited state lifetime, large Stokes shift, and chemical stability make pyrene excimer an attractive fluorescent label. Here we report synthesis of chiral phosphoramidite building blocks based on (R)-4-amino-2,2-dimethylbutane-1,3-diol, easily available from an inexpensive d-(-)-pantolactone. 1-Pyreneacetamide, 1-pyrenecarboxamide, and DABCYL derivatives have been used in preparation of molecular beacon (MB) probes labeled with one or two pyrenes/quenchers. We observed significant difference in the excimer emission maxima (475-510 nm; Stokes shifts 125-160 nm or 7520-8960 cm) and excimer/monomer ratio (from 0.5 to 5.9) in fluorescence spectra depending on the structure and position of monomers in the pyrene pair. The pyrene excimer formed by two rigid 1-pyrenecarboxamide residues showed the brightest emission. This is consistent with molecular dynamics data on excimer stability. Increase of the excimer fluorescence for MBs after hybridization with DNA was up to 24-fold.

We developed a novel technique for the efficient conjugation of oligonucleotides with various alkyl azides such as fluorescent dyes, biotin, cholesterol, N-acetylgalactosamine (GalNAc), etc. using copper-catalysed alkyne-azide cycloaddition on the solid phase and CuI·P(OEt) as a catalyst. Conjugation is carried out in an oligonucleotide synthesizer in fully automated mode and is coupled to oligonucleotide synthesis and on-column deprotection. We also suggest a set of reagents for the construction of diverse conjugates. The sequential double-click procedure using a pentaerythritol-derived tetraazide followed by the addition of a GalNAc or Tris-GalNAc alkyne gives oligonucleotide-GalNAc dendrimer conjugates in good yields with minimal excess of sophisticated alkyne reagents. The approach is suitable for high-throughput synthesis of oligonucleotide conjugates ranging from fluorescent DNA probes to various multi-GalNAc derivatives of 2'-modified siRNA.

Dye-loaded polymeric nanoparticles emerge as powerful bioimaging tools, but their assembly is challenged by aggregation-caused quenching (ACQ) of flat fluorophores. Aggregationinduced emission (AIE) proposes an effective solution against ACQ by exploiting propellershaped neutral fluorophores without polymeric blending. Fighting ACQ of ionic dyes in polymeric nanoparticles can be achieved by bulky hydrophobic counterions. Here, we aim to generate an AIE phenomenon in poorly emissive ionic dyes using bulky counterions. Three cationic hemicyanine dyes of styryl pyridinium family have been synthesized: two planar fluorophores featuring ACQ and one propeller-shaped AIE fluorophore. We found that in water bulky fluorinated tetraphenylborates can light up all three dyes, including planar non-AIE fluorophores. The described "ionic" AIE (iAIE) with bulky counterions enables preparation of 50-nm dye-loaded polymeric NPs showing 40 % quantum yield at 500 mM dye loading, whereas small anions favor poorly emissive large aggregates. Single-particle microscopy reveals that these NPs emit without blinking 50-fold brighter than quantum dots-605 (at 470-nm excitation). iAIE opens the route to assembling charged intrinsically non-AIE fluorophores into bright fluorescent (nano)materials.

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