Nucleoprotein Assemblies for Cellular Biomarker Detection

Abstract

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.

A directed evolution design of a GCG-specific DNA hemimethylase

“…Targeted heritable gene silencing can be achieved by MTases fused with sequence-specific Zn-finger domains (3,4). Formation of irreversible inhibitory complexes between DNA cytosine-5 methyltransferases (C5-MTases) and nucleotide analogs such as 5-fluorocytosine in DNA can be used for the design of covalently functionalized DNA-based nanostructures (5). Recently, novel approaches for sequence-specific functionalization and labeling of DNA using synthetic analogs of the cofactor AdoMet have been proposed (6).…”

Biomarker Identification with Ligand-Targeted Nucleoprotein Assemblies

“…Molecular modeling methods have been described previously [15]. The ND-Trx 3 model was refined in Insight II (Acellerys Inc, CA, USA) to demonstrate that the peptide linkers that join the methyltransferases to the thioredoxins have the flexibility needed to allow two of the three ligands to bind a putative homodimeric target protein.…”

“…The DNA itself can be labeled with a variety of chromophores or functional groups during synthesis, adding increased versatility to the approach. A schematic of the process used for the construction of the trivalent nanodevice (ND-Trx 3 ) displaying bacterial thioredoxin (Trx) [15] as a targeting ligand and a molecular model of ND-Trx 3 is depicted in Figure 1. Previous work, in which prostate cancer cell lines were targeted with this system [15], suggested it might selectively bind to human prostate cancer tissues specimens.…”

“…A schematic of the process used for the construction of the trivalent nanodevice (ND-Trx 3 ) displaying bacterial thioredoxin (Trx) [15] as a targeting ligand and a molecular model of ND-Trx 3 is depicted in Figure 1. Previous work, in which prostate cancer cell lines were targeted with this system [15], suggested it might selectively bind to human prostate cancer tissues specimens. Since human and bacterial thioredoxin are structural isomers [15], each of the four well-characterized thioredoxin interacting proteins in human tissue: thioredoxin reductase 1 (TxnRD1), thioredoxin reductase 2 (TxnRD2), thioredoxin-glutathione reductase (TxnRD3/TGR) and thioredoxin binding protein 2 (Txnip/VDUP-1), is a candidate for ND-Trx 3 target.…”

“…Previous work, in which prostate cancer cell lines were targeted with this system [15], suggested it might selectively bind to human prostate cancer tissues specimens. Since human and bacterial thioredoxin are structural isomers [15], each of the four well-characterized thioredoxin interacting proteins in human tissue: thioredoxin reductase 1 (TxnRD1), thioredoxin reductase 2 (TxnRD2), thioredoxin-glutathione reductase (TxnRD3/TGR) and thioredoxin binding protein 2 (Txnip/VDUP-1), is a candidate for ND-Trx 3 target. In this report, we compare the binding of ND-Trx 3 with a fluorescently labeled thioredoxin bioconjugate (Trx-F) for their ability to bind specific cell surface receptor targets on cell lines and tumor specimens.…”