Transcription and reverse transcription of artificial nucleic acids involving backbone modification by template-directed DNA polymerase reactions.

Oligodeoxyribonucleotides (ODN) where the phosphodiester linkage had been replaced with an amide-type linker [-CH(2)C=ONH-] or an amine-type linker [-CH(2)CH(2)NH-] were synthesized to investigate the effect of these backbone modifications on polymerase reactions. In addition, a triphosphate analogue of thymidine dinucleotide with the amide-type linker was synthesized and enzymatic insertion of the amide linkage into ODN was attempted using this analogue for the polymerase reaction. Primer extension reactions using three types of thermostable DNA polymerases, KOD(exo-), Vent(exo-) and Taq were performed for the assays. Analysis of these data indicate that (i) the polymerase reaction tends to be affected much more by insertion of the cationic flexible amine-type linker than by insertion of the neutral rigid amide-type linker; (ii) the backbone modification has a greater effect on the polymerase reaction when it is adjacent to the 3'-end of a primer as the elongation terminus than when it is on the template, as well as in base or sugar modification; (iii) although the modified linker in the modified DNA template is passed beyond by the polymerase, it still affects the extension reaction several bases downstream from its location; (iv) the modified linker in the template, in some cases, also affects the extension reaction upstream from its location; (v) further improvement of the chemical structure is required for dinucleotide-mimic incorporation.

Smart conferring of nuclease resistance to DNA by 3'-end protection using 2',4'-bridged nucleoside-5'-triphosphates.

Incorporation of 2',4'-bridged nucleotides into the 3'-end of oligodeoxyribonucleotide (ODN) was examined using terminal deoxynucleotidyl transferase (TdT). The three types of 2',4'-bridged nucleoside-5'-triphospates with different bridging structures used were incorporated efficiently into the 3'-end of DNA by TdT, although only single nucleotide incorporation was observed. Nuclease resistance was conferred on DNA, depending on the types of bridging nucleotides added.

Effect of backbone-modification of oligodeoxyribonucleic acid on primer extension reactions.

We synthesized two types of modified DNA templates in which the phosphodiester linkage was replaced with an amide-type linker [-CH(2)C=ONH-] or an amine-type linker [-CH(2)CH(2)NH-]. Primer extension reactions were performed using these modified DNA templates to investigate the effect of backbone modification on polymerase reactions. Our results indicated that the polymerase reaction was affected much more by the insertion of the cationic flexible amine-type linker than by insertion of the neutral rigid amide-type linker.

Systematic analysis of enzymatic DNA polymerization using oligo-DNA templates and triphosphate analogs involving 2',4'-bridged nucleosides.

In order to systematically analyze the effects of nucleoside modification of sugar moieties in DNA polymerase reactions, we synthesized 16 modified templates containing 2',4'-bridged nucleotides and three types of 2',4'-bridged nucleoside-5'-triphospates with different bridging structures. Among the five types of thermostable DNA polymerases used, Taq, Phusion HF, Vent(exo-), KOD Dash and KOD(exo-), the KOD Dash and KOD(exo-) DNA polymerases could smoothly read through the modified templates containing 2'-O,4'-C-methylene-linked nucleotides at intervals of a few nucleotides, even at standard enzyme concentrations for 5 min. Although the Vent(exo-) DNA polymerase also read through these modified templates, kinetic study indicates that the KOD(exo-) DNA polymerase was found to be far superior to the Vent(exo-) DNA polymerase in accurate incorporation of nucleotides. When either of the DNA polymerase was used, the presence of 2',4'-bridged nucleotides on a template strand substantially decreased the reaction rates of nucleotide incorporations. The modified templates containing sequences of seven successive 2',4'-bridged nucleotides could not be completely transcribed by any of the DNA polymerases used; yields of longer elongated products decreased in the order of steric bulkiness of the modified sugars. Successive incorporation of 2',4'-bridged nucleotides into extending strands using 2',4'-bridged nucleoside-5'-triphospates was much more difficult. These data indicate that the sugar modification would have a greater effect on the polymerase reaction when it is adjacent to the elongation terminus than when it is on the template as well, as in base modification.

Arginine-modified DNA aptamers that show enantioselective recognition of the dicarboxylic acid moiety of glutamic acid.

We have screened glutamic acid-binding aptamers from a modified DNA pool containing arginine residues using the method of systematic evolution of ligands by exponential enrichment (SELEX). Thirty-one modified DNA molecules were obtained from the enriched pool after the 17th round of selection, and their binding affinities for the target were evaluated by binding assays using affinity gels. Three modified DNA molecules having higher affinity were sequenced and we determined their affinity and specificity for the target by surface plasmon resonance (SPR) measurements. The SPR studies indicated that two of these three aptamers distinguished the dicarboxylic acid moiety of the D-isomer from that of the L-isomer; however, the third aptamer did not show enantioselectivity.

Polymerisation of a DNA strand using oligo-DNA template with modified bases, sugars and phosphates.

We have attempted to synthesise a complimentary strand using oligo-DNA with modified bases, sugars and phosphates as a template strand by polymerase reaction. Analogues bearing C5-substituted uracil, those with amide linkage [-CH2C=ONH-] in place of phospho-diester linkage and those bearing 2'-O, 4'-C-bridged sugar were used. Primer extension reactions were carried out to synthesise complimentary DNA strands. The reactions depended on the thermostable DNA polymerase used, the type of modification or the number of the modified position on the template strand.

Differences in substrate specificity of C(5)-substituted or C(5)-unsubstituted pyrimidine nucleotides by DNA polymerases from thermophilic bacteria, archaea, and phages.

The pyrimidine bases of RNA are uracil (U) and cytosine (C), while thymine (T) and C are used for DNA. The C(5) position of C and U is unsubstituted, whereas the C(5) of T is substituted with a Me group. Miller et al. hypothesized that various C(5)-substituted uracil derivatives were formed during chemical evolution, and that C(5)-substituted U derivatives may have played important roles in the transition from an 'RNA world' to a 'DNA-RNA-protein world'. Hyperthermophilic bacteria and archaea are considered to be primitive organisms that are evolutionarily close to the universal ancestor of all life on earth. Thus, we examined the substrate specificity of several C(5)-substituted or C(5)-unsubstituted dUTP and dCTP analogs for several DNA polymerases from hyperthermophilic bacteria, hyperthermophilic archaea, and viruses during PCR or primer extension reaction. The substrate specificity of the C(5)-substituted or C(5)-unsubstituted pyrimidine nucleotides varied greatly depending on the type of DNA polymerase. The significance of this difference in substrate specificity in terms of the origin and evolution of the DNA replication system is discussed briefly.

Substrate property and incorporation accuracy of various dATP analogs during enzymatic polymerization using thermostable DNA polymerases.

DNA aptamers and DNAzymes with similar function to antibodies and enzymes can be produced by in vitro selection. They would be useful as research tools for molecular biology and as indicators of specific substances for the analysis of clinical and food samples. Furthermore, development of modified DNA molecules aimed to diversify function and improve activity has recently proceeded by introducing functionalities to these DNA molecules. Such functional modified DNA molecules with an aimed activity screened from a random sequence pool of modified DNA prepared by a polymerase reaction. To enhance potential of selection library and expand diversity of modified DNA that can be synthesized enzymatically, modified analogs of 2'-deoxyadenosine triphosphate were synthesized and their substrate properties for some thermostable DNA polymerases in polymerase chain reactions (PCR) were investigated. Modified DNAs were sequenced in order to analyze incorporation accuracy of modified dATP during PCR.

Systematic characterization of 2'-deoxynucleoside- 5'-triphosphate analogs as substrates for DNA polymerases by polymerase chain reaction and kinetic studies on enzymatic production of modified DNA.

We synthesized C5-modified analogs of 2'-deoxyuridine triphosphate and 2'-deoxycytidine triphosphate and investigated them as substrates for PCRs using Taq, Tth, Vent(exo-), KOD Dash and KOD(exo-) polymerases and pUC 18 plasmid DNA as a template. These assays were performed on two different amplifying regions of pUC18 with different T/C contents that are expected to have relatively high barriers for incorporation of either modified dU or dC. On the basis of 260 different assays (26 modified triphosphates x 5 DNA polymerases x 2 amplifying regions), it appears that generation of the full-length PCR product depends not only on the chemical structures of the substitution and the nature of the polymerase but also on whether the substitution is on dU or dC. Furthermore, the template sequence greatly affected generation of the PCR product, depending on the combination of the DNA polymerase and modified triphosphate. By examining primer extension reactions using primers and templates containing C5-modified dUs, we found that a modified dU at the 3' end of the elongation strand greatly affects the catalytic efficiency of DNA polymerases, whereas a modified dU opposite the elongation site on the template strand has less of an influence on the catalytic efficiency.

Cerebral blood flow index: dynamic perfusion MRI delivers a simple and good predictor for the outcome of acute-stage ischemic lesion.

OBJECTIVES: To evaluate the feasibility of utilizing cerebral blood flow (CBF) index images, calculated automatically and quickly from dynamic perfusion imaging (DPI), to identify acute cerebral ischemia. We attempted to investigate (1) whether the CBF index has a threshold for assessing tissue outcome, (2) whether CBF index images can predict the resulting infracted area, and if so, (3) whether the predictive capacity of the CBF index image is comparable to the regional CBF (rCBF) image delivered from singular value decomposition (SVD) deconvolution methods, which are regarded as most accurate in predicting the final infarct area. METHODS: Diffusion-weighted images (DWI) and DPI were obtained in 17 patients within 12 hours of stroke onset and follow-up magnetic resonance imaging (MRI). On 3 DPI-delivered images, namely relative regional cerebral blood volume (rrCBV), uncorrected mean transit time (MTTu) and CBF index images, univariate discriminant analysis was done to estimate cut-off values to discriminate between infarcted and noninfarcted areas. Subsequently, correlations between the initial lesion volume of 3 images together with rCBF images delivered with SVD methods and the final infarct volume on follow-up T2-weighted MRI taken at the 8th to 20th day were determined. RESULTS: Among the 3 images, only the CBF index image was able reveal the threshold of the ischemic region. Lesion volume of CBF index images against follow-up infarct volume had the highest correlation (r = 0.995) to a linear fit and the slope was closest to 1.0 (0.91) among the 3 and had identical accuracy to the regression coefficient of rCBF images. CONCLUSIONS: CBF index images can predict final infarct volume. Evaluating CBF index images together with DWI can guide the initial assessment in the acute stage of cerebral ischemia.