Site-Specific One-Pot Dual Labeling of DNA by Orthogonal Cycloaddition Chemistry.
Summary of "Site-Specific One-Pot Dual Labeling of DNA by Orthogonal Cycloaddition Chemistry."
Bioorthogonal reactions are of high interest in biosciences as they allow the introduction of fluorescent dyes, affinity tags or other unnatural moieties into biomolecules. The site-specific attachment of two or more different labels is particularly demanding and typically requires laborious multi-step syntheses. Here we report that the most popular cycloaddition in bioconjugation, the copper-catalyzed azide-alkyne click reaction (CuAAC), is fully orthogonal to the inverse electron-demand Diels-Alder reaction (DAinv). We demonstrate that both bioorthogonal reactions can be conducted concurrently in a one-pot reaction by just mixing all components. Orthogonality has been established even for highly reactive trans-cyclooctene-based dienophiles (with rate constants up to 380.000 M(-1)s(-1)). These properties allow for the convenient site-specific one-step preparation of oligonucleotide FRET probes and related reporters needed in cellular biology and biophysical chemistry.
Affiliation
Journal Details
This article was published in the following journal.
Name: Bioconjugate chemistry
ISSN: 1520-4812
Pages:
Links
- PubMed Source: http://www.ncbi.nlm.nih.gov/pubmed/22709568
- DOI: http://dx.doi.org/10.1021/bc300181n
Medical and Biotech [MESH] Definitions
Chromosome Fragile Sites
Specific loci that show up during KARYOTYPING as a gap (an uncondensed stretch in closer views) on a CHROMATID arm after culturing cells under specific conditions. These sites are associated with an increase in CHROMOSOME FRAGILITY. They are classified as common or rare, and by the specific culture conditions under which they develop. Fragile site loci are named by the letters "FRA" followed by a designation for the specific chromosome, and a letter which refers to which fragile site of that chromosome (e.g. FRAXA refers to fragile site A on the X chromosome. It is a rare, folic acid-sensitive fragile site associated with FRAGILE X SYNDROME.)
Isotope Labeling
Techniques for labeling a substance with a stable or radioactive isotope. It is not used for articles involving labeled substances unless the methods of labeling are substantively discussed. Tracers that may be labeled include chemical substances, cells, or microorganisms.
Rna, Small Nucleolar
Small nuclear RNAs that are involved in the processing of pre-ribosomal RNA in the nucleolus. Box C/D containing snoRNAs (U14, U15, U16, U20, U21 and U24-U63) direct site-specific methylation of various ribose moieties. Box H/ACA containing snoRNAs (E2, E3, U19, U23, and U64-U72) direct the conversion of specific uridines to pseudouridine. Site-specific cleavages resulting in the mature ribosomal RNAs are directed by snoRNAs U3, U8, U14, U22 and the snoRNA components of RNase MRP and RNase P.
Integrons
DNA elements that include the component genes and insertion site for a site-specific recombination system that enables them to capture mobile gene cassettes.
Click Chemistry
Organic chemistry methodology that mimics the modular nature of various biosynthetic processes. It uses highly reliable and selective reactions designed to "click" i.e., rapidly join small modular units together in high yield, without offensive byproducts. In combination with COMBINATORIAL CHEMISTRY TECHNIQUES, it is used for the synthesis of new compounds and combinatorial libraries.
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