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.

Synthesis and properties of acridone-labeled base-discriminating fluorescent (BDF) nucleosides.

We have developed novel acridone-labelled BDF probe which showed its potential in recognizing opposite matched base from its target sequence via enhancement of fiuorescence intensity. This probe emit at a longer wavelength than previously reported pyrene-labelled BDF probe and thus can be used in DNA chip.

Synthesis of perylene-labeled base-discriminating fluorescent (BDF) nucleoside and its fluorescence properties.

We have synthesized perylene labeled BDF probe and its photophysical properties were explored. Perylene labeled oligonucleotide probe is capable of detecting cytosine from its target mismatch sequence by enhancement of fluorescence intensity.

Synthesis and properties of novel base-discriminating fluorescent (BDF) nucleosides.

We designed a new type of pyrene-labeled base-discrimination fluorescent (BDF) nucleosides (Py)U, (Py)C, (8Py)A and (MePy)dA, which emitted strong fluorescence only when the bases opposite the BDF base are A, G, T and C, respectively. The DNA probes containing four different BDF bases enable us to distinguish single base alterations by simply mixing with a sample solution of target DNA.

Intelligent fluorescent nucleoside in sensing cytosine base: importance of hydrophobic nature of perylene fluorophore.

Fluorescence response upon hybridization of perylene labeled oligonucleotide probes depends on the microenvironment experienced by the perylene fluorophore. In mismatched duplex ((Per)U-C), enhanced fluorescence was observed while in matched duplex ((Per)U-A) fluorescence intensity decreased considerably. This observation will be a promising research effort in giving rise to a new powerful tool in detection of SNP.

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.

Direct PCR amplification of various modified DNAs having amino acids: convenient preparation of DNA libraries with high-potential activities for in vitro selection.

We synthesized modified 2'-deoxyuridine triphosphates bearing amino acids at the C5 position and investigated their substrate properties for KOD Dash DNA polymerase during polymerase chain reaction (PCR). PCR using C5-modified dUTP having an amino acyl group (arginyl, histidyl, lysyl, phenylalanyl, tryptophanyl, leucyl, prolyl, glutaminyl, seryl, O-benzyl seryl or threonyl group) gave the corresponding full-length PCR products in good yield. Although dUTP analogues bearing aspartyl, glutamyl or cysteinyl were found to be poor substrates for PCR catalyzed by KOD Dash DNA polymerase, optimization of the reaction conditions resulted in substantial generation of full-length product.

Simultaneous incorporation of three different modified nucleotides during PCR.

Modified analogs of 2'-deoxycytidine triphosphates bearing (6-aminohexyl)carbamoylmethyl or 7-amino-2,5-dioxaheptyl linker at a C5 position were designed and synthesized. Both analogs were found to be good substrates for Vent(exo-) DNA polymerase during PCR, resulting in the corresponding full-length modified DNAs, respectively. Moreover, we have demonstrated simultaneous incorporation of three different modified nucleotides into a DNA strand by PCR using triphosphates of 5-(3-aminopropynyl)dUTP, 5-[(6-aminohexyl)carbamoylmethyl]dCTP and 2-amino-dATP (dDTP) or N6-methyl-dATP in place of the natural nucleoside triphosphates TTP, dCTP and dATP.

Enzymatic incorporation of chemically-modified nucleotides into DNAs.

Modified 2'-deoxyuridine triphosphates bearing proteinous amino acids at a C5 position were prepared, and their substrate properties were investigated for KOD Dash DNA polymerase during PCR. The modified dUTPs bearing histidyl, lysyl, glutaminyl or seryl group produced the aimed 108 nt PCR products in good yields. In contrast, the analog bearing glutamyl group did not work as a substrate for KOD Dash while the analog bearing aspartyl group gave the product in a low yield.