Four-stranded G-quadruplex nucleic acid structures are of great interest as their high thermodynamic stability under near-physiological conditions suggests that they could form in cells. Here we report the generation and application of an engineered, structure-specific antibody employed to quantitatively visualize DNA G-quadruplex structures in human cells. We show explicitly that G-quadruplex formation in DNA is modulated during cell-cycle progression and that endogenous G-quadruplex DNA structures can be stabilized by a small-molecule ligand. Together these findings provide substantive evidence for the formation of G-quadruplex structures in the genome of mammalian cells and corroborate the application of stabilizing ligands in a cellular context to target G-quadruplexes and intervene with their function.

New ways of making antibodies have recently been demonstrated using gene technology. Immunoglobulin variable (V) genes are amplified from hybridomas or B cells using the polymerase chain reaction, and cloned into expression vectors. Soluble antibody fragments secreted from bacteria are then screened for binding activities. Screening of V genes would, however, be revolutionized if they could be expressed on the surface of bacteriophage. Phage carrying V genes that encode binding activities could then be selected directly with antigen. Here we show that complete antibody V domains can be displayed on the surface of fd bacteriophage, that the phage bind specifically to antigen and that rare phage (one in a million) can be isolated after affinity chromatography.

To generate a stable resource from which high affinity human antibodies to any given antigen can be rapidly isolated, functional V-gene segments from 43 non-immunized human donors were used to construct a repertoire of 1.4 x 10(10) single-chain Fv (scFv) fragments displayed on the surface of phage. Fragments were cloned in a phagemid vector, enabling both phage displayed and soluble scFv to be produced without subcloning. A hexahistidine tag has been incorporated to allow rapid purification of scFv by nickel chelate chromatography. This library format reduces the time needed to isolate monoclonal antibody fragments to under two weeks. All of the measured binding affinities show a Kd < 10 nM and off-rates of 10(-3) to 10(-4) s-1, properties usually associated with antibodies from a secondary immune response. The best of these scFvs, an anti-fluorescein antibody (0.3 nM) and an antibody directed against the hapten DTPA (0.8 nM), are the first antibodies with subnanomolar binding affinities to be isolated from a naive library. Antibodies to doxorubicin, which is both immunosuppressive and toxic, as well as a high affinity and high specificity antibody to the steroid hormone oestradiol have been isolated. This work shows that conventional hybridoma technology may be superseded by large phage libraries that are proving to be a stable and reliable source of specific, high affinity human monoclonal antibodies.

In vitro display technologies, best exemplified by phage and yeast display, were first described for the selection of antibodies some twenty years ago. Since that time a large number of antibodies, some with remarkable properties, have been selected and improved upon using these methods. The first antibodies derived using in vitro display methods are now in the clinic, with many more waiting in the wings. Here we discuss the scope of the technology, some of the powerful antibodies selected, and the future potential in a post-genomic world. Unique advantages offered by in vitro display technologies include the ability to carefully define selection conditions, allowing the derivation of antibodies recognizing predefined epitopes or conformations, the further improvement of selected antibodies, the potential for high throughput applications and the immediate availability of genes encoding the selected antibody. We anticipate that the high throughput potential of these technologies will soon lead to their use to select antibodies against all human proteins.