ArticleNucleic acids research2009
Highly efficient incorporation of the fluorescent nucleotide analogs tC and tCO by Klenow fragment.
Article in Nucleic acids research, 2009. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 19 papers.
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Who cites it
19 citing papers in PubMed, 53 citations in OpenAlex.
- Article
- RNase H-based analysis of synthetic mRNA 5' cap incorporation.RNA (New York, N.Y.) · 2022Article
- Structural and Molecular Kinetic Features of Activities of DNA Polymerases.International journal of molecular sciences · 2022Review
- Modified nucleoside triphosphates in bacterial research forRSC chemical biology · 2020Article
- Probing of Nucleic Acid Structures, Dynamics, and Interactions With Environment-Sensitive Fluorescent Labels.Frontiers in chemistry · 2020Review
- Fluorescent nucleobases as tools for studying DNA and RNA.Nature chemistry · 2017Review
- Synthesis and polymerase activity of a fluorescent cytidine TNA triphosphate analogue.Nucleic acids research · 2017Article
- Polymerase/DNA interactions and enzymatic activity: multi-parameter analysis with electro-switchable biosurfaces.Scientific reports · 2015Article
- Conformational Dynamics of DNA Repair by Escherichia coli Endonuclease III.The Journal of biological chemistry · 2015Article
- New environment-sensitive multichannel DNA fluorescent label for investigation of the protein-DNA interactions.PloS one · 2014Article
- Synthetic nucleotides as probes of DNA polymerase specificity.Journal of nucleic acids · 2012Article
- Fluorescent DNA-based enzyme sensors.Chemical Society reviews · 2011Review
- Characterization of photophysical and base-mimicking properties of a novel fluorescent adenine analogue in DNA.Nucleic acids research · 2011Article
- Fluorescent xDNA nucleotides as efficient substrates for a template-independent polymerase.Nucleic acids research · 2011Article
- Site-specific fluorescent probing of RNA molecules by unnatural base-pair transcription for local structural conformation analysis.Nature protocols · 2010Article
- Fluorescent analogs of biomolecular building blocks: design, properties, and applications.Chemical reviews · 2010Review
- Incorporation of the fluorescent ribonucleotide analogue tCTP by T7 RNA polymerase.Analytical chemistry · 2010Article
- A single-label phenylpyrrolocytidine provides a molecular beacon-like response reporting HIV-1 RT RNase H activity.Nucleic acids research · 2010Article
- High density labeling of polymerase chain reaction products with the fluorescent base analogue tCo.Analytical chemistry · 2009Article
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Authors and funding
6 authors at 3 institutions in 2 countries.
Funding
Abstract
Studies of the mechanisms by which DNA polymerases select the correct nucleotide frequently employ fluorescently labeled DNA to monitor conformational rearrangements of the polymerase-DNA complex in response to incoming nucleotides. For this purpose, fluorescent base analogs play an increasingly important role because they interfere less with the DNA-protein interaction than do tethered fluorophores. Here we report the incorporation of the 5'-triphosphates of two exceptionally bright cytosine analogs, 1,3-diaza-2-oxo-phenothiazine (tC) and its oxo-homolog, 1,3-diaza-2-oxo-phenoxazine (tC(O)), into DNA by the Klenow fragment. Both nucleotide analogs are polymerized with slightly higher efficiency opposite guanine than cytosine triphosphate and are shown to bind with nanomolar affinity to the DNA polymerase active site, according to fluorescence anisotropy measurements. Using this method, we perform competitive binding experiments and show that they can be used to determine the dissociation constant of any given natural or unnatural nucleotide. The results demonstrate that the active site of the Klenow fragment is flexible enough to tolerate base pairs that are size-expanded in the major groove. In addition, the possibility to enzymatically polymerize a fluorescent nucleotide with high efficiency complements the tool box of biophysical probes available to study DNA replication.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.