The LutR protein represses the transcription of genes encoding enzymes for the utilization of l-lactate in Bacillus subtilis through binding to a specific DNA region. In this study, we employed oligonucleotide probes modified by viscosity-sensitive tetramethylated thiophene-BODIPY fluorophores to investigate the impact of selected metabolites on the LutR-DNA complex. Our goal was to identify the effector molecule whose binding alters the protein-DNA affinity, thereby enabling gene transcription. The designed DNA probes exhibited distinctive responses to the binding and release of the protein, characterized by significant alterations in fluorescence lifetime. Through this method, we have identified l-lactate as the sole metabolite exerting a substantial modulating effect on the protein-DNA interaction and thus confirmed its role as an effector molecule. Moreover, we showed that our approach was able to follow conformation changes affecting affinity, which were not captured by other methods commonly used to study the protein-DNA interaction, such as electro-mobility shift assays and florescence anisotropy binding studies. This work underlines the potential of environment-sensitive fluorophore-linked nucleotide modifications, i.e. dCTBdp, for studying the dynamics and subtle changes of protein-DNA interactions.

• Publication type
• Journal Article MeSH

While bioorthogonal reactions are routinely employed in living cells and organisms, their application within individual organelles remains limited. In this review, we highlight diverse examples of bioorthogonal reactions used to investigate the roles of biomolecules and biological processes as well as advanced imaging techniques within cellular organelles. These innovations hold great promise for therapeutic interventions in personalized medicine and precision therapies. We also address existing challenges related to the selectivity and trafficking of subcellular dynamics. Organelle-targeted bioorthogonal reactions have the potential to significantly advance our understanding of cellular organization and function, provide new pathways for basic research and clinical applications, and shape the direction of cell biology and medical research.

• Keywords
• Bioconjugations, Bioorthogonal reactions, Cellular organelles, Click chemistry, Targeting,
• MeSH
• Cell Biology MeSH
• Organelles * chemistry   MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

Surgery is an efficient way to treat localized prostate cancer (PCa), however, it is challenging to demarcate rapidly and accurately the tumor boundary intraoperatively, as existing tumor detection methods are seldom performed in real-time. To overcome those limitations, we develop a fluorescent molecular rotor that specifically targets the prostate-specific membrane antigen (PSMA), an established marker for PCa. The probes have picomolar affinity (IC50 = 63-118 pM) for PSMA and generate virtually instantaneous onset of robust fluorescent signal proportional to the concentration of the PSMA-probe complex. In vitro and ex vivo experiments using PCa cell lines and clinical samples, respectively, indicate the utility of the probe for biomedical applications, including real-time monitoring of endocytosis and tumor staging. Experiments performed in a PCa xenograft model reveal suitability of the probe for imaging applications in vivo.

• MeSH
• Antigens, Surface chemistry   metabolism   MeSH
• PC-3 Cells MeSH
• Endocytosis MeSH
• Spectrometry, Fluorescence methods   MeSH
• Glutamate Carboxypeptidase II chemistry   metabolism   MeSH
• Humans MeSH
• Models, Molecular MeSH
• Molecular Probes chemistry   metabolism   MeSH
• Mice, Inbred BALB C MeSH
• Mice, Nude MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Prostatic Neoplasms diagnosis   metabolism   MeSH
• Optical Imaging methods   MeSH
• Protein Domains MeSH
• Transplantation, Heterologous MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Antigens, Surface MeSH
• FOLH1 protein, human MeSH Browser
• Glutamate Carboxypeptidase II MeSH
• Molecular Probes MeSH

Thymidine triphosphate bearing benzylidene-tetrahydroxanthylium near-IR fluorophore linked to the 5-methyl group via triazole was synthesized through the CuAAC reaction and was used for polymerase synthesis of labelled DNA probes. The fluorophore lights up upon incorporation to DNA (up to 348-times) presumably due to interactions in major groove and the fluorescence further increases in the single-stranded oligonucleotide. The labelled dsDNA senses binding of small molecules and proteins by a strong decrease of fluorescence. The nucleotide was used as a light-up building block in real-time PCR for detection of SARS-CoV-2 virus.

• Keywords
• DNA, fluorescence, nucleotides, real-time PCR,
• MeSH
• COVID-19 * MeSH
• DNA Probes MeSH
• Humans MeSH
• Nucleotides MeSH
• DNA Replication * MeSH
• SARS-CoV-2 MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA Probes MeSH
• Nucleotides MeSH

A series of 2-alkylamino-2'-deoxyadenosine triphosphates (dATP) was prepared and found to be substrates for the Therminator DNA polymerase, which incorporated only one modified nucleotide into the primer. Using a template encoding for two consecutive adenines, conditions were found for incorporation of either one or two modified nucleotides. In all cases, addition of a mixture of natural dNTPs led to primer extension resulting in site-specific single modification of DNA in the minor groove. The allylamino-substituted DNA was used for the thiol-ene addition, whereas the propargylamino-DNA for the CuAAC click reaction was used to label the DNA with a fluorescent dye in the minor groove. The approach was used to construct FRET probes for detection of oligonucleotides.

• Keywords
• DNA, fluorescent probes, nucleotides, oligonucleotides, polymerases,
• MeSH
• Allyl Compounds chemistry   MeSH
• Deoxyadenine Nucleotides chemistry   MeSH
• DNA chemistry   MeSH
• Fluorescent Dyes chemistry   MeSH
• Nucleic Acid Conformation MeSH
• Oligonucleotides analysis   MeSH
• Pargyline analogs & derivatives   chemistry   MeSH
• Propylamines chemistry   MeSH
• Fluorescence Resonance Energy Transfer methods   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 2'-deoxyadenosine triphosphate MeSH Browser
• Allyl Compounds MeSH
• Deoxyadenine Nucleotides MeSH
• DNA MeSH
• Fluorescent Dyes MeSH
• Oligonucleotides MeSH
• Pargyline MeSH
• propargylamine MeSH Browser
• Propylamines MeSH

2'-Deoxyadenosine triphosphate (dATP) derivatives bearing diverse substituents (Cl, NH2 , CH3 , vinyl, ethynyl, and phenyl) at position 2 were prepared and tested as substrates for DNA polymerases. The 2-phenyl-dATP was not a substrate for DNA polymerases, but the dATPs bearing smaller substituents were good substrates in primer-extension experiments, producing DNA substituted in the minor groove. The vinyl-modified DNA was applied in thiol-ene addition and the ethynyl-modified DNA was applied in a CuAAC click reaction to form DNA labelled with fluorescent dyes in the minor groove.

• Keywords
• DNA modification, DNA polymerase, bioconjugation, fluorescent labelling, nucleotides,
• MeSH
• Nucleic Acid Denaturation MeSH
• Deoxyadenine Nucleotides chemistry   metabolism   MeSH
• DNA-Directed DNA Polymerase metabolism   MeSH
• DNA chemistry   metabolism   MeSH
• Nucleic Acid Conformation MeSH
• Base Sequence MeSH
• Substrate Specificity MeSH
• Thermodynamics MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 2'-deoxyadenosine triphosphate MeSH Browser
• Deoxyadenine Nucleotides MeSH
• DNA-Directed DNA Polymerase MeSH
• DNA MeSH

A nucleoside bearing a solvatochromic push-pull fluorene fluorophore (dCFL ) was designed and synthesized by the Sonogashira coupling of alkyne-linked fluorene 8 with 5-iodo-2'-deoxycytidine. The fluorene building block 8 and labeled nucleoside dCFL exerted bright fluorescence with significant solvatochromic effect providing emission maxima ranging from 421 to 544 nm and high quantum yields even in highly polar solvents, including water. The solvatochromism of 8 was studied by DFT and ADC(2) calculations to show that, depending on the polarity of the solvent, emission either from the planar or the twisted conformation of the excited state can occur. The nucleoside was converted to its triphosphate variant dCFLTP which was found to be a good substrate for DNA polymerases suitable for the enzymatic synthesis of oligonucleotide or DNA probes by primer extension or PCR. The fluorene-linked DNA can be used as fluorescent probes for DNA-protein (p53) or DNA-lipid interactions, exerting significant color changes visible even to the naked eye. They also appear to be suitable for time-dependent fluorescence shift studies on DNA, yielding information on DNA hydration and dynamics.

• Publication type
• Journal Article MeSH