ABSTRACT
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.
Subject(s)
DNA Replication , DNA-Directed DNA Polymerase/metabolism , DNA/genetics , Deoxyribonucleosides/chemistry , Dinucleoside Phosphates/chemistry , Polymers/chemical synthesis , Adenine/chemistry , Adenine/metabolism , Aptamers, Nucleotide/chemical synthesis , Aptamers, Nucleotide/genetics , Base Pairing , Base Sequence , Cytosine/chemistry , Cytosine/metabolism , DNA/chemistry , DNA/metabolism , DNA-Directed DNA Polymerase/genetics , Deoxyribonucleosides/genetics , Deoxyribonucleosides/metabolism , Dinucleoside Phosphates/genetics , Dinucleoside Phosphates/metabolism , Guanine/chemistry , Guanine/metabolism , Hydrophobic and Hydrophilic Interactions , Polymerase Chain Reaction , Polymers/metabolism , Uracil/chemistry , Uracil/metabolismABSTRACT
A new chemical labeling-based LC-MS/MS approach was developed for quantitative profiling of nine canonical bases and deoxynucleosides (dNs) in natural products. Using 2-bromo-1-(4-dimethylamino-phenyl)-ethaone (BrDPE) as the tagging reagent, a previously unexploited N-alkylpyrimidine derivative (Nad) was created for one-pot labeling of widescope nucleobases via a flexible bromophilic substitution under mild conditions. The derivatization notably improved the LC-MS detection sensitivity by 31-107 fold, enabling a fast dilute-and-shoot analysis of highly diluted samples. The optimized and validated method demonstrated satisfactory accuracy (87-107%), precision (RSDs < 7.5%), and recovery (89-105%) for matrix-matched standard addition. The method was applied to simultaneously determine all target analytes and four uncanonical analogues and base-modified species in seven traditional health foods, which differ in contents by up to 600-fold for discrimination among samples. Further, the base-labeled Nads exhibit a unique fragmentation signature that could be used for untargeted screening of nucleobase-containing metabolites by simplified LC-MS/MS workflow to improve quality evaluation of natural medicinal products.
Subject(s)
Biological Products/chemistry , Chromatography, High Pressure Liquid/methods , Nucleosides/chemistry , Plant Extracts/chemistry , Plants, Medicinal/chemistry , Tandem Mass Spectrometry/methods , Deoxyribonucleosides/chemistry , Limit of DetectionABSTRACT
Herein, the conventional and unconventional hydrogen bonding potential of adenine in APA for double zipper helical assembly of deoxyoligonucleotides is demonstrated under ambient conditions. The quantum mechanical calculations supported the formation of hybrid DNA ensembles.
Subject(s)
DNA/chemical synthesis , Deoxyribonucleosides/chemistry , Adenine/chemistry , DNA/chemistry , Hydrogen Bonding , Molecular Imprinting , Molecular Structure , Quantum Theory , Templates, GeneticABSTRACT
P-Stereodefined oligodeoxyribonucleoside boranophosphates (PB-ODNs) were successfully synthesized by the conversion of diastereopure H-phosphonate intermediates. Thermal denaturation studies clearly showed that the duplex stability of PB-ODNs with the complementary DNA and RNA depends on the stereochemistry of the boranophosphate linkages.
Subject(s)
Boranes/chemistry , Oligodeoxyribonucleotides/chemical synthesis , Organophosphonates/chemistry , Deoxyribonucleosides/chemistry , Nucleic Acid Denaturation , Oligodeoxyribonucleotides/chemistry , StereoisomerismABSTRACT
We present an ab initio study of the optical absorption and emission spectra of size-expanded nucleic acid base analogues (yA, yT, yT-m, yG, yG-t2, and yC) obtained by benzo homologation (see Krueger, A. T.; Lu, H.; Lee, A. H. F.; Kool, E. T. Acc. Chem. Res. 2007, 40, 141 and references therein). Also examined were the effects of linking to deoxyribose and hydrogen bonding to their natural complementary bases (T, A, C, and G, respectively). The calculated excitation and emission energies are in good agreement with the measured data where experimental results are available. The geometries corresponding to the first excited singlet state of yA and yT are found to be quasi-planar, while those for yG and yC are nonplanar. In general, binding to deoxyribose will red shift the absorbance and fluorescence emission maxima of the y-bases. The ground-state geometries of the Watson-Crick analog base pairs (yAT, yTA, yGC, and yCG) are found to be planar, and the calculated interaction energies are very close to those of natural base pairs, indicating that the y-bases can pair with their natural complementary partners to generate stable base pairs. The base pairing has no significant effects on the fluorescence emission of yA, yC, and yT, but blue shifts the fluorescence emission of yG by 22 nm.