Synthesis of adenoviral targeting molecules by intein-mediated protein ligation.

Adenoviral vectors infect cells through the binding of capsid proteins to cell-surface receptors. The ubiquitous expression of adenoviral receptors in human tissues represents an obstacle toward the development of systemically deliverable vectors for cancer therapy, since effective therapy may require delivery to specific sites. For these reasons, major efforts are directed toward the elimination of the native tropism combined with identification of ligands that bind to tumor-specific cell-surface proteins. Highthroughput technologies have identified potential targeting ligands, which need to be evaluated for their ability to retarget adenovirus to alternative receptors. Here, we present a strategy that permits the routine analysis of adenoviral targeting ligands. We use intein-mediated protein ligation as a means to produce functional biological molecules, that is, adenoviral targeting molecules that function as adapters between cellular receptors and the adenovirus fiber protein. We demonstrate the versatility of the present system by conjugating targeting ligands that differ in size and nature including an apolipoprotein E synthetic peptide, the basic fibroblast growth factor and folic acid. The resulting adenoviral targeting molecules mediate adenoviral gene delivery in cells that express the corresponding receptor.

Template-directed interference footprinting of protein-phosphate contacts in DNA.

[figure: see text] We have developed a method for interference footprinting of contacted phosphates in protein-DNA complexes. Template-directed enzymatic polymerization using a synthetic triphosphate analogue (alpha Me-dTTP) generates a product having a modified Internucleotide linkage, which perturbs protein-phosphate contacts. We found that treatment of the methylphosphonodiester-substituted extension product under nonaqueous conditions (MeO-/MeOH) led to the formation of a single cleavage product at each T residue but to two cleavage products when treated under the standard aqueous piperidine cleavage protocol.

A versatile framework for the design of ligand-dependent, transgene-specific transcription factors.

The ability to regulate transgene expression will be essential for the safety and efficacy of many gene therapies. Various ligand-dependent transcription factors, including steroid hormone receptors, have been modified to enable transgene-specific regulation. To minimize effects on cellular gene expression, chimeric steroid receptors have been constructed by replacing their native DNA binding domain (DBD) with a heterologous DBD, like that from the yeast transcription factor GAL4. This approach has limitations for human gene therapy, including the potential immunogenicity of the GAL4 domain and the inability to discriminate between different GAL4-linked transgenes in the same cell. To address this, we have constructed chimeric regulators containing the human estrogen receptor (ER) ligand binding domain (LBD) and a Cys(2)-His(2)-type zinc finger DBD. Cys(2)-His(2) zinc finger domains are common among human DNA binding proteins and can be engineered to selectively bind different DNA sequences. We demonstrate over 500-fold drug-dependent transgene induction with these chimeric regulators in vitro and the ability to regulate an adenovirus-delivered transgene in mice. Two chimeras containing different Cys(2)-His(2) domains displayed highly sequence-specific binding and regulation. Incorporating a point mutation in the ER LBD that ablates estrogen binding enables selective in vivo regulation with the clinically useful anti-estrogen tamoxifen. These Cys(2)-His(2)-ER LBD chimeras represent a versatile framework for creating transgene-specific regulators potentially useful for human gene therapy applications.

Induced alpha helix in the VP16 activation domain upon binding to a human TAF.

Activation domains are functional modules that enable sequence-specific DNA binding proteins to stimulate transcription. The structural basis for the function of activation domains is poorly understood. A combination of nuclear magnetic resonance (NMR) and biochemical experiments revealed that the minimal acidic activation domain of the herpes simplex virus VP16 protein undergoes an induced transition from random coil to alpha helix upon binding to its target protein, hTAFII31 (a human TFIID TATA box-binding protein-associated factor). Identification of the two hydrophobic residues that make nonpolar contacts suggests a general recognition motif of acidic activation domains for hTAFII31.

A nonnatural transcriptional coactivator.

In eukaryotes, sequence-specific DNA-binding proteins activate gene expression by recruiting the transcriptional apparatus and chromatin remodeling proteins to the promoter through protein-protein contacts. In many instances, the connection between DNA-binding proteins and the transcriptional apparatus is established through the intermediacy of adapter proteins known as coactivators. Here we describe synthetic molecules with low molecular weight that act as transcriptional coactivators. We demonstrate that a completely nonnatural activation domain in one such molecule is capable of stimulating transcription in vitro and in vivo. The present strategy provides a means of gaining external control over gene activation through intervention using small molecules.