A series of adenosine triphosphate (ATP) derivatives bearing chloro, fluoro, amino, methyl, vinyl, and ethynyl groups at position 2 are synthesized and tested as substrates for RNA and DNA polymerases. The modified nucleotides work well in in vitro transcription with T7 RNA polymerase and primer extension (PEX) using engineered DNA polymerases (TGK, 2M) except for the bulkier 2-vinyl- and 2-ethynyl-ATP derivatives that give truncated products. However, in single nucleotide incorporation followed by PEX, they still can be used for site-specific incorporation of reactive modifications into RNA that can be further used for postsynthetic labeling through thiol-ene or Cu-catalyzed alkyne-azide cycloadditions reactions. All modified ATPs work in polyadenylation catalyzed by poly(A) polymerase to form long 3'-polyA tails containing the modifications that also can be used for labeling.

• Keywords
• DNA polymerases, RNA polymerases, click reactions, nucleosides triphosphates, nucleotides, polyA polymerase, thiol‐ene addition,
• MeSH
• Adenosine Triphosphate * chemical synthesis   chemistry   analogs & derivatives   metabolism   MeSH
• Cycloaddition Reaction MeSH
• DNA-Directed RNA Polymerases * metabolism   chemistry   MeSH
• DNA-Directed DNA Polymerase * metabolism   chemistry   MeSH
• RNA * chemistry   metabolism   MeSH
• Viral Proteins metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adenosine Triphosphate * MeSH
• bacteriophage T7 RNA polymerase MeSH Browser
• DNA-Directed RNA Polymerases * MeSH
• DNA-Directed DNA Polymerase * MeSH
• RNA * MeSH
• Viral Proteins MeSH

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.

• Keywords
• DNA, fluorescent probes, nucleotides, oligonucleotides, polymerases,
• MeSH
• Allyl Compounds chemistry   MeSH
• Deoxyadenine Nucleotides chemistry   MeSH
• DNA chemistry   MeSH
• Fluorescent Dyes chemistry   MeSH
• Nucleic Acid Conformation MeSH
• Oligonucleotides analysis   MeSH
• Pargyline analogs & derivatives   chemistry   MeSH
• Propylamines chemistry   MeSH
• Fluorescence Resonance Energy Transfer methods   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 2'-deoxyadenosine triphosphate MeSH Browser
• Allyl Compounds MeSH
• Deoxyadenine Nucleotides MeSH
• DNA MeSH
• Fluorescent Dyes MeSH
• Oligonucleotides MeSH
• Pargyline MeSH
• propargylamine MeSH Browser
• Propylamines MeSH