Shinya Y. Sawata

Application of peptide nucleic acid to the direct detection of deoxyribonucleic acid amplified by polymerase chain reaction

Double-stranded DNA amplified by polymerase chain reaction (PCR) was detected by peptide nucleic acid (PNA) using a BIAcore 2000 biosensor based on surface plasmon resonance (SPR). PNA is an artificial oligo amide that is capable of forming highly stable complexes with complementary oligonucleotides. We succeeded in the direct detection of double-stranded DNA, amplified by PCR with high-sequence specificity. It was shown that the target DNA was available for detection over the range of 40-160 nM. Therefore, the detection limit was 7.5 pmol of the target DNA (143 bases, applied volume 30 microliters). Our DNA detection system, the combination of BIAcore and the probe PNA, could detect the target DNA with good reproducibility. In this report, we show that our system is a powerful tool for the diagnosis of pathologically significant DNA.

Loss-of-function screening by randomized intracellular antibodies: Identification of hnRNP-K as a potential target for metastasis

We have developed a loss-of-function screening system on the basis of intracellular expression of single domain antibodies. We demonstrate its use in identification of potential targets of metastasis of human cancerous cells. Randomized intracellular antibodies were expressed in highly metastatic cells, and a derivative pool of cells with loss of migration phenotype in chemotaxis assay was isolated. Isolation of antibodies from cells with loss of migration phenotype and identification of their target proteins revealed the involvement of the heterogeneous nuclear ribonucleoprotein K (hnRNP-K), a multifunctional signaling protein, in metastasis. Furthermore, we found that the cytoplasmic accumulation of hnRNP-K is crucial for its role in metastasis. The results demonstrate (i) the advantages of our functional interference screening over the gene-knockouts and gene-silencing, (ii) hnRNP-K as a potential target of metastasis, and (iii) a potential anti-metastasis peptide validated in in vitro cell migration assays.

Molecular design and delivery of siRNA.

Double stranded short interfering RNAs (siRNAs) mediate gene silencing in a sequence specific manner. By virtue of their specific gene silencing activity and owing to the recent discoveries on their plasmid and virus driven expression, siRNAs are being widely adopted in research and therapeutics. Efforts were made to optimize the siRNA expression system for the application in therapy. One major obstacle in developing RNA interference (RNAi) therapy is the delivery of siRNAs to the target cells. Combination of novel molecular targeting technologies, such as recombinant protein technology and ribosome display technology, will enable to deliver gene silencing agents to target cells specifically and efficiently.

Specific N-Terminal Biotinylation of a Protein In Vitro by a Chemically Modified tRNAfmet can Support the Native Activity of the Translated Protein.

Biotinylation of a protein generally involves chemical modification of a translated protein. Using this methodology, however, biotinylation at a specific position remains difficult. We investigated whether it would be possible to use an Escherichia coli initiator tRNA(fmet) aminoacylated with methionine biotinylated at the alpha-amino group to introduce a biotin tag specifically at the N terminus. We report here that a biotin tag could be incorporated into the green fluorescent protein (GFP) at the N-terminal site, in the presence of an E. coli initiator tRNA(fmet) aminoacylated with methionine biotinylated at the alpha-amino group. The biotinylated GFP was purified by simple monomeric streptavidin-agarose affinity column chromatography. Based on the total amount of GFP molecules, the purification yield and the biotin labelling efficiency of this system were approximately 7% and 10-20%, respectively, according to the densitometric analysis of Western blots. Judging from the results of a fluorescence imaging experiment, almost all the purified GFP molecules retained the native fluorescence activity. Importantly, the present results support the hypothesis that the E. coli initiator tRNA(fmet) aminoacylated with a relatively large substituent can be recognized by an E. coli ribosome and adequately placed at the P site to initiate translation.