2-Allyl- and Propargylamino-dATPs for Site-Specific Enzymatic Introduction of a Single Modification in the Minor Groove of DNA
Abstract
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.