Topologically Restricted Appearance in the Developing Chick Retinotectal System of Bravo, a Neural Surface Protein: Experimental Modulation by Environmental Cues

A novel neural surface protein, Bravo, shows a pattern of topological restriction in the embryonic chick retinotectal system. Bravo is present on the developing optic fibers in the retina; however, retinal axons in the tectum do not display Bravo. The appearance of Bravo in vitro is modulated by environmental cues. Axons growing out from retinal explants on retinal basal lamina, their natural substrate, express Bravo, whereas such axons growing on collagen do not. Retinal explants provide a valuable system to characterize the mechanism of Bravo restriction, as well as the cellular signals controlling it. Bravo was identified with monoclonal antibodies from a collection generated against exposed molecules isolated by using a selective cell surface biotinylation procedure. The NH2-terminal sequence of Bravo shows similarity with L1, a neural surface molecule which is a member of the immunoglobulin superfamily. This possible relationship to L1, together with its restricted appearance, suggests an involvement of Bravo in axonal growth and guidance

Uridine-Conjugated Ferrocene DNA Oligonucleotides: Unexpected Cyclization Reaction of the Uridine Base

The study of energy and electron-transfer processes through DNA duplexes and the development of DNA hybridization probes and electrochemical sensors have resulted in the incorporation of numerous transition-metal complexes into DNA oliognucleotides. These include ruthenium, osmium, iron, rhodium, and copper complexes. Ferrocene (Fc) and its derivatives are attractive electrochemical probes because of their stability and convenient synthetic chemistry. Fc-containing DNA oligonucleotides have been prepared by attaching ferrocenyl moieties to the 5‘ termini through either solid-phase synthesis using phosphoramidites or by reacting suitable ferrocenyl derivatives with end-functionalized oligonucleotides

Transfection of Folate-Polylysine DNA Complexes: Evidence for Lysosomal Delivery

We are utilizing the folate receptor for the intracellular delivery of DNA. In this study, a folate-poly-L-lysine (FPLL) conjugate was synthesized and equilibrated with plasmid DNA encoding the firefly luciferase gene. The FPLL-DN_A complexes were added to KB cells treated with chloroquine. Luciferase activity of cells incubated with FPLL-DNA was 6-fold higher than of cells exposed to poly-L-lysine. (PLL)-DNA. The addition of free folic acid competitively inhibited the enhancement of gene expression. Removal of chloroquine from the media significantly inhibited transfection efficiency of FPLL-DNA complexes. We conclude that FPLL-DNA complexes are delivered into KB cells via folate receptor-mediated endocytosis and likely follow a lysosomal pathway into the cytoplasm

Transfection of Folate-Polylysine DNA Complexes: Evidence for Lysosomal Delivery

We are utilizing the folate receptor for the intracellular delivery of DNA. In this study, a folate-poly-L-lysine (FPLL) conjugate was synthesized and equilibrated with plasmid DNA encoding the firefly luciferase gene. The FPLL-DN_A complexes were added to KB cells treated with chloroquine. Luciferase activity of cells incubated with FPLL-DNA was 6-fold higher than of cells exposed to poly-L-lysine. (PLL)-DNA. The addition of free folic acid competitively inhibited the enhancement of gene expression. Removal of chloroquine from the media significantly inhibited transfection efficiency of FPLL-DNA complexes. We conclude that FPLL-DNA complexes are delivered into KB cells via folate receptor-mediated endocytosis and likely follow a lysosomal pathway into the cytoplasm

Stretching and breaking duplex DNA by chemical force microscopy

AbstractBackground: Specific interactions between complementary strands of DNA and other molecules are central to the storage, retrieval and modification of information in biological systems. Although in many cases the basic structures of duplex DNA and the binding energetics have been well characterized, little information is available about the forces in these systems. These forces are of critical importance because they must be overcome, for example, by protein machines during transcription and repair. Recent developments in atomic force microscopy make possible direct measurements of such forces between the individual oligonucleotide strands that form DNA duplexes.Results: We used the chemical force microscopy technique, in which oligonucleotides are covalently linked to the force microscope probe tip and the sample surface, to measure the elongation and binding forces of individual DNA duplexes. The separation forces between complementary oligonucleotide strands were found to be significantly larger than the forces measured between noncomplementary strands, and to be consistent with the unbinding of a single DNA duplex. With increasing applied force, the separation of complementary strands proceeded in a stepwise manner: B-form DNA was stretched, then structurally transformed to a stable form of DNA approximately twice the length of the B form, and finally separated into single-stranded oligonucleotides. These data provide a direct measurement of the forces required to elastically deform and separate double-stranded DNA into single strands.Conclusions: Force microscopy provides a direct and quantitative measurement of the forces and energetics required to stretch and unbind DNA duplexes. Because the measurements can be carried out readily on synthetic oligonucleotides and in the presence of exogenous molecules, this method affords an opportunity for directly assessing the energetics of distorting and unbinding specific DNA sequences and DNA complexes. Such data could provide unique insights into the mechanistic steps following sequence-specific recognition by, for example, DNA repair and transcription factors