Nucleic acids symposium series (2004)最新文献

Sequence-specific DNA alkylating agents have great interest for novel approach to cancer chemotherapy. We designed the conjugates between pyrrole (Py)-imidazole (Im) polyamides and DNA alkylating chlorambucil moiety possessing at different positions. The sequence-specific DNA alkylation by conjugates was investigated by using high-resolution denaturing polyacrylamide gel electrophoresis (PAGE). The results showed that polyamide chlorambucil conjugates alkylate DNA at flanking adenines in recognition sequences of Py-Im polyamides, however, the reactivities and alkylation sites were influenced by the positions of conjugation. In addition, we synthesized conjugate between Py-Im polyamide and another alkylating agent, 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI). DNA alkylation reactivies by both alkylating polyamides were almost comparable. In contrast, cytotoxicities against cell lines differed greatly. These comparative studies would promote development of appropriate sequence-specific DNA alkylating polyamides against specific cancer cells.

In this paper, the nonenzymatic detection of 5-methylcytosine with high selectivity is described. We present a new methylation detection method by using template-directed photoligation through 5-cyanovinyl-2'-deoxyuridine ((C)U). Significantly, the photoligation yield of the 5-methylcytosine case was approximately 9.1-fold higher than that in the case of cytosine.

We investigated the efficiency of osmium complex formation at 5-methylcytosine in mismatched DNA duplexes. Osmium complexation was not observed in fully matched duplexes, whereas the complexation site and efficiency in mismatched duplexes depended on the 5'-neighboring base of the 5-methylcytosine. In particular, when the base adjacent to the 5' side of the mismatched base pair was thymine, a unique side reaction was observed. However, the mismatched base pairs did not influence the selectivity of osmium complexation with methylated DNA.

Development of methods for in vitro selection of peptide aptamers with target binding affinities have been expected to construct biocatalysts, biosensors, and molecular targeted drugs in leading-edge fields of biotechnology and medical therapies. Therefore, a highly stabilized ribosome display method has been devised for efficient selection of various peptide aptamers from an artificial peptide library (APL). This ribosome display selection is performed by using a ribosomal conjugate consisted of APL, Cv RNA-associating protein (Cvap), and mRNA having Cv RNA motif at 5' terminus. The conjugate generated by in vitro translation can automatically link a peptide as phenotype and its mRNA as genotype, which can be stabilized by a high affinity association between Cv motif and Cvap. Here, in order to demonstrate the utility of this ribosome display method, we performed in vitro selection of peptide aptamers against a metal complex, and succeeded in identification and characterization of peptide aptamers with specific metal binding affinity. These results validate the concept to design APL in DNA level and indicate the versatility of the highly stabilized ribosome display selection.

Mouse telomeric DNA sequence, Tel3.5: 5'-AGGG(T TAGGG)(3)-3', has the ability to form antiparallel tetraplex structure in the presence of Na(+). We examined the interaction between the antiparallel tetraplex structure of Tel3.5 and each of two single-stranded telomeric DNA-binding domains of mouse telomere binding protein Pot1, mPot1OB1 and mPot1OB2. The antiparallel tetraplex of Tel3.5 became unfolded upon the interaction with mPot1OB1. On the other hand, no significant structural change of the antiparallel tetraplex of Tel3.5 was observed upon the interaction with mPot1OB2. Considering that the antiparallel tetraplex inhibits telomerase-mediated telomere elongation, we conclude that the ability of mPot1OB1 to unfold the antiparallel tetraplex of the mouse telomeric DNA is required for telomerase-mediated telomere elongation.

The (6-4) photoproduct, which is one of the major UV-induced DNA lesions formed at bipyrimidine sites, causes carcinogenesis at high frequency. The (6-4) photolyases restore the (6-4) photoproducts to their intact bases in a light-dependent manner, but its overall repair mechanism remains obscure. To investigate the light-dependent conversion of the (6-4) photoproduct, we prepared a (6-4) photoproduct analog with modification at 3' pyrimidone ring, in which the carbonyl group was replaced with an imine to apply to the (6-4) photolyase assay. The (6-4) photolyase shows affinity to an oligonucleotide carrying this imine analog of the (6-4) photoproduct, though the imine analog is not repaired by the (6-4) photolyase.

Small silencing RNAs, including small interfering RNAs (siRNAs) and microRNAs (miRNAs), regulate expression of their target genes via RNA-induced silencing complex (RISC). RISC assembly follows complex, ordered pathways, and RISC function is as diverse as cleavage, translational repression, and deadenylation of the target. We have recently shown how siRNAs and miRNAs are assembled into distinct types of RISC, and how differently they function, using Drosophila as a model organism.

Deoxyuridine triphosphate derivatives modified with pyrene was synthesized to functionalize DNA with fluorescent molecules based on the template DNA sequence. Incorporation of pyrene-labeled deoxyuridine triphosphates into DNA by DNA polymerase was investigated by using reverse-phase HPLC and polyacrylamide gel electrophoresis. The fluorescent properties of functionalized DNA were characterized by the steady-state fluorescence measurements.

L/F-transferase is known to catalyze transfer of hydrophobic amino acids from aminoacyl tRNA to the N-terminus of a protein possessing lysine or arginine as the N-terminus. Combining L/F-transferase with E. coli phenylalanyl-tRNA synthetase (ARS), we achieved non-ribosomal N-terminal-specific introduction of various kinds of nonnatural amino acids to a protein. A nonnatural amino acid is once charged onto an E. coli tRNA(Phe) by a mutant ARS in situ, and successively transferred from the tRNA to a target protein, namely the NEXT-A reaction. Besides alphaA294G mutation on the ARS, alphaT251A, betaG318W, or betaA356W double-mutation were effective to increase the introduction efficiency through the NEXT-A reaction. Protein specific fluorescence labelling via the NEXT-A reaction followed by Huisgen cycloaddition was also demonstrated.

We developed a new nucleic acid-based fluorescence probe for protein detection. The method is based on the scission of an aptamer into two probes, which are then attached with a chemically reactive fluorogenic compound. The protein-dependent association of the two probes accelerates a chemical reaction and indicates the presence of the target protein, which is detected using a fluorescence readout. The arginine-rich motif peptide was targeted by this type of probe. In presence of the peptide, the fluorescence signal at 450 nm increased, and no significant increase in fluorescence was observed in the absence of the peptide. An oligonucleotide-based fluorescence probe was successfully applied to the detection of the ARM peptide in solution.