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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 genesilencing, (ii) hnRNP-K as a potential target of metastasis, and (iii) a potential anti-metastasis peptide validated in in vitro cell migration assays.heterogeneous nuclear ribonucleoprotein K ͉ randomized antibodies ͉ functional screening ͉ cytoplasmic accumulation ͉ antimetastasis M etastasis is a complex phenomenon that involves an array of genetic changes and coordination of many positive and negative factors leading to motile cells that can penetrate into and travel through the bloodstream forming secondary tumors elsewhere in the body. Despite the advances in cancer therapies and the understanding that metastasis is a selective, nonrandom and inefficient process, it significantly contributes to morbidity and mortality of cancer patients and is a major challenge yet to be met in cancer therapeutics (1-4). Increased cell motility is one of the early and essential phenotype of metastatic cancers. Although it has been demonstrated to involve concerted reorganization of the actin cytoskeleton that regulates cell shape, and motility through spatiotemporal activation of Rho-family GTPases (5-9), the molecular mechanisms and its upstream regulators, the potential targets of metastasis, have not been sufficiently defined (10). Amongst a large variety of approaches that can be adopted to identify ''metastasis genes,'' we chose to employ a functional interference of proteins by expression of intracellular antibodies, also called as ''specific functional ablators'' and ''functional therapeutical molecules'' (11-13). We anticipated that the functional interference may serve as a milder and finer approach to reveal biological complexities and pathways that cannot be resolved by gene-knockouts and genesilencing strategies that challenge structural integrity of cells.We constructed a randomized intracellular antibody library (iAb-lib) on the basis of the single heavy-chain small antigenbinding fragments (VHH) of camel antibody. By employing iAb-lib against cell migration of highly malignant human fibrosarcoma, we obtained a derivative cell population in which cell migration was compromised. Cloning and characterization of antibodies from the derivative nonmigrating...

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Polymerase chain reaction (PCR) products were detected using a flow injection-type sensor based on surface plasmon resonance. Asymmetric PCR was used to amplify the target DNA sequence, and two products with different length were produced. The novelty of our DNA detection system was that our target DNA was double stranded but the probe binding site, located in the 3'-terminus, was single stranded. This avoids the formation of intra- and intermolecular complexes. This novel design permitted us not only to detect PCR product but also to develop a rapid detection system for the detection of the verotoxin 2 gene of Escherichia coli O157:H7.