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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We introduce and analyse brightness of fluorescent organic nanomaterials as an essential characteristic for biosensing and bioimaging applications.

Super-resolution fluorescence imaging based on single-molecule localization microscopy (SMLM) enables visualizing cellular structures with nanometric precision. However, its spatial and temporal resolution largely relies on the brightness of ON/OFF switchable fluorescent dyes. Moreover, in cell plasma membranes, the single-molecule localization is hampered by the fast lateral diffusion of membrane probes. Here, to address these two fundamental problems, we propose a concept of ON/OFF switchable probes for SMLM (points accumulation for imaging in nanoscale topography, PAINT) based on fluorogenic dimers of bright cyanine dyes. In these probes, the two cyanine units connected with a linker were modified at their extremities with low-affinity membrane anchors. Being self-quenched in water due to intramolecular dye H-aggregation, they displayed light up on reversible binding to lipid membranes. The charged group in the linker further decreased the probe affinity to the lipid membranes, thus accelerating its dynamic reversible ON/OFF switching. The concept was validated on cyanines 3 and 5. SMLM of live cells revealed that the new probes provided higher brightness and ∼10-fold slower diffusion at the cell surface, compared to reference probes Nile Red and DiD, which boosted axial localization precision >3-fold down to 31 nm. The new probe allowed unprecedented observation of nanoscale fibrous protrusions on plasma membranes of live cells with 40 s time resolution, revealing their fast dynamics. Thus, going beyond the brightness limit of single switchable dyes by cooperative dequenching in fluorogenic dimers and slowing down probe diffusion in biomembranes open the route to significant enhancement of super-resolution fluorescence microscopy of live cells.

Remote control of cells and single molecules by magnetic nanoparticles in nonheating external magnetic fields is a perspective approach for many applications such as cancer treatment and enzyme activity regulation. However, the possibility and mechanisms of direct effects of small individual magnetic nanoparticles on such processes in magnetomechanical experiments still remain unclear. In this work, we have shown remote-controlled mechanical dissociation of short DNA duplexes (18−60 bp) under the influence of nonheating low-frequency alternating magnetic fields using individual 11 nm magnetic nanoparticles. The developed technique allows (1) simultaneous manipulation of millions of individual DNA molecules and (2) evaluation of energies of intermolecular interactions in short DNA duplexes or in other molecules. Finally, we have shown that DNA duplexes dissociation is mediated by mechanical stress and produced by the movement of magnetic nanoparticles in magnetic fields, but not by local overheating. The presented technique opens a new avenue for high-precision manipulation of DNA and generation of biosensors for quantification of energies of intermolecular interaction.

4-Chloro-L-kynurenine (3-(4-chloroanthraniloyl)-L-alanine, L-4-ClKyn), an amino acid known as a prospective antidepressant, was recently for the first time found in nature in the lipopeptide antibiotic taromycin. Here, we report another instance of its identification in a natural product: 4-chloro-L-kynurenine was isolated from acidic hydrolysis of a new complex peptide antibiotic INA-5812. L-4-ClKyn is a fluorescent compound responsible for the fluorescence of the above antibiotic. Whereas fluorescence of 4-chlorokynurenine was not reported before, we synthesized the racemic compound and studied its emission in various solvents. Next, we prepared conjugates of DL-4-ClKyn with two suitable energy acceptors, BODIPY FL and 3-(phenylethynyl)perylene (PEPe), and studied fluorescence of the derivatives. 4-Chloro-DL-kynurenine emission is not detected in both conjugates, thus evidencing effective energy transfer. However, BODIPY FL emission in the conjugate is substantially reduced, probably due to collisional or photoinduced charge-transfer-mediated quenching. The intrinsic fluorescence of L-4-ClKyn amino acid in antibiotics paves the way for spectral studies of their mode of action.