Abstract:
:We report a rapid method for the covalent modification of commercially available amino-modified DNA oligonucleotides with a cysteine moiety. The resulting DNA-cysteine conjugates are versatile reagents for the efficient preparation of covalent DNA-protein conjugates by means of expressed protein ligation (EPL). The EPL method allows for the site-specific coupling of cysteine-modified DNA oligomers with recombinant intein-fusion proteins, the latter of which contain a C-terminal thioester enabling the mild and highly specific reaction with N-terminal cysteine compounds. We prepared a cysteine-modifier reagent in a single-step reaction which allows for the rapid and near quantitative synthesis of cysteine-DNA conjugates. The latter were ligated with the green fluorescent protein mutant EYFP, recombinantly expressed as an intein-fusion protein, allowing for the mild and selective formation of EYFP-DNA conjugates in high yields of about 60%. We anticipate many applications of our approach, ranging from protein microarrays to the arising field of nanobiotechnology.
journal_name
Biochem Biophys Res Communjournal_title
Biochemical and biophysical research communicationsauthors
Lovrinovic M,Niemeyer CMdoi
10.1016/j.bbrc.2005.08.001subject
Has Abstractpub_date
2005-09-30 00:00:00pages
943-8issue
3eissn
0006-291Xissn
1090-2104pii
S0006-291X(05)01675-Xjournal_volume
335pub_type
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journal_title:Biochemical and biophysical research communications
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