Jon Faiz Kayyem

Stretching and breaking duplex DNA by chemical force microscopy

BACKGROUNDnSpecific 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.nnnRESULTSnWe 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.nnnCONCLUSIONSnForce 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.

Receptor-targeted co-transport of DNA and magnetic resonance contrast agents

BACKGROUNDnLigand molecules conjugated to polylysine can be electrostatically bound to DNA and can bind receptors or antigens on the surface of cells, delivering the DNA into specific cells and tissues. Several researchers have used this approach to generate non-viral vehicles for the efficient delivery of DNA to specific cells. We have attempted to adopt this general approach to the cell-specific delivery of magnetic contrast agents for use in magnetic resonance imaging (MRI).nnnRESULTSnWe have synthesized a new class of agents capable of both transfecting genes into cells and enhancing the contrast of the targeted cells for MRI. DNA is used both to encode a marker gene and as a molecular scaffold, which electrostatically binds polylysine conjugated to transferrin, an iron uptake protein, and polylysine modified with gadolinium chelated to diethylenetriaminepetaacetic acid. When cells displaying the transferrin receptor are treated with these particles, high levels of gene expression are observed, higher than with control particles composed only of transferrin, polylysine and DNA. The treated cells show specific MRI contrast enhancement, which did not require expression of the marker gene.nnnCONCLUSIONSnThe development of this class of particles permits the use of novel protocols by which genes for genetic therapy and agents for MRI contrast are co-transported. These protocols may allow non-invasive MRI monitoring of DNA delivery for gene therapy in real time.

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.