Nucleoprotein assemblies for cellular biomarker detection.
In this report, we have used DNA Y-junctions as fluorescent scaffolds for EcoRII methyltransferase-thioredoxin (M.EcoRII-Trx) fusion proteins. Covalent links between the DNA scaffold and the methyltransferase were formed at preselected sites on the scaffold containing 5FdC. The resulting thioredoxin-targeted nanodevice was found to bind selectively to certain cell lines but not to others. The fusion protein was constructed so as to permit proteolytic cleavage of the thioredoxin peptide from the nanodevice. Proteolysis with thrombin or enterokinase effectively removed the thioredoxin peptide from the nanodevice and extinguished cell line specific binding measured by fluorescence. A number of potential applications for devices of this type can be envisioned. In particular, the ability of the fused protein to selectively target the nanodevice to certain tumor cell lines and not others suggests that this approach may serve as an adjunct to immunohistochemical methods in tumor classification as well as probe cell surface receptor architecture and function.
Singer
Elizabeth M
EM
City of Hope National Medical Center and Beckman Research Institute, 1500 East Duarte Road, Duarte, California 91010, USA. bsinger@coh.org
Smith
Steven S
SS
D015415:Biomarkers / Q000378:metabolism*; D002468:Cell Physiological Phenomena; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D015257:DNA-Cytosine Methylases / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D008856:Microscopy, Fluorescence / Q000379:methods*; D015336:Molecular Probe Techniques; D049329:Nanostructures / Q000737:chemistry* / Q000648:ultrastructure; D009698:Nucleoproteins / Q000737:chemistry* / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods
D015415:Biomarkers; D004269:DNA, Bacterial; D009698:Nucleoproteins; D011993:Recombinant Fusion Proteins; C029528:DNA modification methylase EcoRII; D015257:DNA-Cytosine Methylases