A set of modified 2'-deoxyribonucleoside triphosphates (dNTPs) bearing a linear or branched alkane, indole or phenyl group linked through ethynyl or alkyl spacer were synthesized and used as substrates for polymerase synthesis of hypermodified DNA by primer extension (PEX). Using the alkyl-linked dNTPs, the polymerase synthesized up to 22-mer fully modified oligonucleotide (ON), whereas using the ethynyl-linked dNTPs, the enzyme was able to synthesize even long sequences of >100 modified nucleotides in a row. In PCR, the combinations of all four modified dNTPs showed only linear amplification. Asymmetric PCR or PEX with separation or digestion of the template strand can be used for synthesis of hypermodified single-stranded ONs, which are monodispersed polymers displaying four different substituents on DNA backbone in sequence-specific manner. The fully modified ONs hybridized with complementary strands and modified DNA duplexes were found to exist in B-type conformation (B- or C-DNA) according to CD spectral analysis. The modified DNA can be replicated with high fidelity to natural DNA through PCR and sequenced. Therefore, this approach has a promising potential in generation and selection of hypermodified aptamers and other functional polymers.

• MeSH
• Adenine chemistry   metabolism   MeSH
• Aptamers, Nucleotide chemical synthesis   genetics   MeSH
• Cytosine chemistry   metabolism   MeSH
• Deoxyribonucleosides chemistry   genetics   metabolism   MeSH
• Dinucleoside Phosphates chemistry   genetics   metabolism   MeSH
• DNA-Directed DNA Polymerase genetics   metabolism   MeSH
• DNA chemistry   genetics   metabolism   MeSH
• Guanine chemistry   metabolism   MeSH
• Hydrophobic and Hydrophilic Interactions MeSH
• Base Pairing MeSH
• Polymerase Chain Reaction MeSH
• Polymers chemical synthesis   metabolism   MeSH
• DNA Replication * MeSH
• Base Sequence MeSH
• Uracil chemistry   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenine MeSH
• Aptamers, Nucleotide MeSH
• Cytosine MeSH
• Deoxyribonucleosides MeSH
• Dinucleoside Phosphates MeSH
• DNA-Directed DNA Polymerase MeSH
• DNA MeSH
• Guanine MeSH
• Polymers MeSH
• Uracil 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

2'-Deoxyadenosine triphosphate (dATP) derivatives bearing diverse substituents (Cl, NH2 , CH3 , vinyl, ethynyl, and phenyl) at position 2 were prepared and tested as substrates for DNA polymerases. The 2-phenyl-dATP was not a substrate for DNA polymerases, but the dATPs bearing smaller substituents were good substrates in primer-extension experiments, producing DNA substituted in the minor groove. The vinyl-modified DNA was applied in thiol-ene addition and the ethynyl-modified DNA was applied in a CuAAC click reaction to form DNA labelled with fluorescent dyes in the minor groove.

• Keywords
• DNA modification, DNA polymerase, bioconjugation, fluorescent labelling, nucleotides,
• MeSH
• Nucleic Acid Denaturation MeSH
• Deoxyadenine Nucleotides chemistry   metabolism   MeSH
• DNA-Directed DNA Polymerase metabolism   MeSH
• DNA chemistry   metabolism   MeSH
• Nucleic Acid Conformation MeSH
• Base Sequence MeSH
• Substrate Specificity MeSH
• Thermodynamics MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 2'-deoxyadenosine triphosphate MeSH Browser
• Deoxyadenine Nucleotides MeSH
• DNA-Directed DNA Polymerase MeSH
• DNA MeSH