Novel fluorescently labeled nucleotides: synthesis, spectral properties and application in polymerase chain reaction

Кузнецова, В. Е.; Спицын, М. А.; Шершов, В. Е.; Гусейнов, Т. О.; Фесенко, Е. Е.; Lapa, S. A.; Ikonnikova, A. Yu.; Avdonina, M. A.; Наседкина, Т. В.; Заседателев, А. С.; Чудинов, А. В.

doi:10.1016/j.mencom.2016.03.002

Cited by 14 publications

(3 citation statements)

References 27 publications

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“…Modified 2′-deoxyuridine triphosphate was synthesized by coupling 5-(3-aminoallyl)-2′-deoxyuridine 5′-triphosphate (Biosan, Russia) with activated Cy5 dye. Asymmetric Cy5 dye was prepared according to published procedures with modifications 50 – 54 . The details of the synthetic procedure are given in the Supplementary Information.…”

Section: Methodsmentioning

confidence: 99%

Mass-spectrometry analysis of modifications at DNA termini induced by DNA polymerases

Smirnov

Kolganova

Vasiliskov

et al. 2017

Sci Rep

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Non-natural nucleotide substrates are widely used in the enzymatic synthesis of modified DNA. The terminal activity of polymerases in the presence of modified nucleotides is an important, but poorly characterized, aspect of enzymatic DNA synthesis. Here, we studied different types of polymerase activity at sequence ends using extendable and non-extendable synthetic models in the presence of the Cy5-dUTP analog Y. In primer extension reactions with selected exonuclease-deficient polymerases, nucleotide Y appeared to be a preferential substrate for non-templated 3′-tailing, as determined by MALDI mass-spectrometry and gel-electrophoresis. This result was further confirmed by the 3′-tailing of a non-extendable hairpin oligonucleotide model. Additionally, DNA polymerases induce an exchange of the 3′ terminal thymidine for a non-natural nucleotide via pyrophosphorolysis in the presence of inorganic pyrophosphate. In primer extension reactions, the proofreading polymerases Vent, Pfu, and Phusion did not support the synthesis of Y-modified primer strand. Nevertheless, Pfu and Phusion polymerases were shown to initiate terminal nucleotide exchange at the template. Unlike non-proofreading polymerases, these two enzymes recruit 3′–5′ exonuclease functions to cleave the 3′ terminal thymidine in the absence of pyrophosphate.

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Section: Methodsmentioning

confidence: 99%

Mass-spectrometry analysis of modifications at DNA termini induced by DNA polymerases

Smirnov

Kolganova

Vasiliskov

et al. 2017

Sci Rep

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“…Developing novel biochemical and biophysical techniques has led to advances not only in elucidating the molecular basis of biological functions but also in discovering targeted drug molecules . Kinetic analysis of protein binding in real time is fundamental to developing new drugs and medical treatments. − In parallel, detection of a target molecule without prelabeling steps has been highly advantageous for many practical applications due to not only its experimental ease but also the fact that the labeling can perturb binding events and results. − …”

Section: Introductionmentioning

confidence: 99%

Label-Free Analysis of Multivalent Protein Binding Using Bioresponsive Nanogels and Surface Plasmon Resonance (SPR)

Yang

Teoh

Yim

et al. 2020

ACS Appl. Mater. Interfaces

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Precise identification of protein–protein interactions is required to improve our understanding of biochemical pathways for biology and medicine. In physiology, how proteins interact with other proteins or small molecules is crucial for maintaining biological functions. For instance, multivalent protein binding (MPB), in which a ligand concurrently interacts with two or more receptors, plays a key role in regulating complex but accurate biological functions, and its interference is related to many diseases. Therefore, determining MPB and its kinetics has long been sought, which currently requires complicated procedures and instruments to distinguish multivalent binding from monovalent binding. Here, we show a method for quickly evaluating the MPB over monovalent binding and its kinetic parameters in a label-free manner. Engaging pNIPAm-co-AAc nanogels with MPB-capable moieties (e.g., PD-1 antigen and biocytin) permits a surface plasmon resonance (SPR) instrument to evaluate the MPB events by amplifying signals from the specific target molecules. Using our MPB-based method, PD-1 antibody that forms a type of MPB by complexing with two PD-1 proteins, which are currently used for cancer immunotherapy, is detectable down to a level of 10 nM. In addition, small multivalent cations (e.g., Ca2+, Fe2+, and Fe3+) are distinguishably measurable over monovalent cations (e.g., Na+ and K+) with the pNIPAm-co-AAc nanogels.

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“…Fluorescent labeling of nucleotides and nucleic acids is an indispensable tool in chemical and molecular biology. − Environment-sensitive fluorophores are used as probes for changes of secondary structure or for sensing biomolecular interactions. − On the other hand, labeling of nucleic acids with bright and photostable fluorophores is needed for applications in imaging, microscopy, and metabolic studies − as well as for sequencing . Many diverse fluorophore-labeled 2′-deoxyribonucleoside triphosphates (dNTPs) were reported in literature. − Most of them bear cyanines, fluoresceins, and other bulky charged labels with limited photostability linked through a flexible tether at position 5 of dUTP, and some are even commercially available. They are used for enzymatic labeling of DNA through polymerase incorporation of modified nucleotides. ,,− Since some of these bulky modified dNTPs are rather poor substrates for DNA polymerases, and inherently they do not penetrate cell membranes, they cannot be directly used for in vivo or in cellulo incorporation to DNA, except for some studies involving highly invasive techniques temporarily disrupting the integrity of plasma membrane or fixed cells. − Very recently, Zawada et al reported the first synthetic nucleoside triphosphate transporter SNTT (see Figure , below), which enabled efficient and rapid intracellular delivery of modified dNTPs and their incorporation into genomic DNA for direct fluorescent staining and imaging using some commercially available fluorescent dNTPs.…”

Section: Introductionmentioning

confidence: 99%

Brightly Fluorescent 2′-Deoxyribonucleoside Triphosphates Bearing Methylated Bodipy Fluorophore for in Cellulo Incorporation to DNA, Imaging, and Flow Cytometry

et al. 2018

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Synthesis of cytosine, uracil, and 7-deazaadenine 2′-deoxyribonucleosides and triphosphates (dNTPs) bearing hexamethylated phenyl-bodipy fluorophore attached at position 5 of pyrimidines or at position 7 of 7-deazapurine was developed. All the title labeled nucleosides and dNTPs displayed bright green fluorescence with very high quantum yields. The modified dN mBdp TPs were good substrates to diverse DNA polymerases and were used for in vitro enzymatic synthesis of labeled DNA by primer extension or PCR. In combination with cationic cyclodextrin-peptide-based dNTP transporter, the dN mBdp TPs were successfully used for staining of genomic DNA in living cells for applications in confocal microscopy and in flow cytometry. The best performing cytosine nucleotide dC mBdp TP was used to monitor mitosis in live cells.

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Novel fluorescently labeled nucleotides: synthesis, spectral properties and application in polymerase chain reaction

Cited by 14 publications

References 27 publications

Mass-spectrometry analysis of modifications at DNA termini induced by DNA polymerases

Mass-spectrometry analysis of modifications at DNA termini induced by DNA polymerases

Label-Free Analysis of Multivalent Protein Binding Using Bioresponsive Nanogels and Surface Plasmon Resonance (SPR)

Brightly Fluorescent 2′-Deoxyribonucleoside Triphosphates Bearing Methylated Bodipy Fluorophore for in Cellulo Incorporation to DNA, Imaging, and Flow Cytometry

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