Dr. Richard Willson's laboratory works on biomolecular recognition, and its applications in separations and molecular diagnostics.

Biomolecular Recognition

Willson’s research group is interested in the structural determinants of molecular recognition in complexes of proteins with recognition agents such as monoclonal antibodies and aptamers. Their primary techniques are expression, mutagenesis, fluorescence anisotropy (kinetics) and titration calorimetry. Topics of current interest include the recognition of hen egg lysozyme by a “homologous series” of antibodies differing in combining site rigidity and cross-reactivity (with S. Smith-Gill of NIH), and the biophysical chemistry of aptamer/protein recognition (with A. Ellington of UT-Austin).

Biomolecular Separations

New chemistries for DNA/RNA purification

One example is metal-chelate affinity, also known as Immobilized Metal Affinity Chromatography, IMAC. IMAC is famous for its use in the purification of hexa-histidine tagged recombinant proteins, as well as large-scale purification of pharmaceutical proteins. Willson’s laboratory have found that the same chemistry binds purines in single-stranded RNA and DNA (which is quite useful for capturing RNA, polyA mRNA, primers, etc). They have combined the ideas of condensation conformational control and DNA metal affinity into a selective renaturation scheme in which genomic DNA is endowed with kinetically-trapped single-stranded "purification handles" that allow it to be captured by metal chelates while plasmid DNA renatures completely and does not bind. This method allows remarkable (million-fold) selectivity between plasmid (e.g., a DNA vaccine) and contaminating genomic DNA despite their chemical similarity.

Nanostructured adsorbents

Conventional bioseparations adsorbents (e.g., ion exchangers) are derivitized with functional groups (e.g., charges), randomly distributed over their surface area. This produces a functional polyclonality or heterogeneity, in that there are some places where several charges are close together, and many where a smaller number are clustered. So the binding properties are heterogeneous, and the selectivity for purification (e.g., of protein pharmaceuticals) or analysis (e.g., proteomics) is inherently limited. Willson’s research group is controlling the distribution of charges on a nanometer scale by immobilizing groups of charges all at once. This reduces the heterogeneity of adsorption, and confers interesting new specificity for proteins displaying clusters of charges on their surfaces. They are now also exploring nanoclustered metal-chelates for DNA/RNA separations.

Molecular Diagnostics and Sensors

Genome-based Identification of Microorganisms

As hundreds of microbial, viral, and metazoan genome sequences become available, it is increasingly possible to use this base of knowledge to design DNA probe/primer sets for organisms of interest known not to interact with background sequences (e.g., environmental background, human sequences, etc.). This HSARPA-funded project, with George Fox and Yuriy Fofanov of UH Computer Science, relies on the remarkable computational prowess of the Fofanov group in identifying sequences of interest by exhaustive calculation. Wilson’s research group use these results in several ways: 1. Custom DNA arrays for identification of pathogens; 2. Developing custom cocktails of RT-PCR primers rigorously known not to interact with each other, or with any human sequence or known SNP, with at least two mismatches and designed to amplify e.g., viral sequences against an overwhelming background of human DNA; 3. Engineering of hybridization-responsive fluorescent probes for these sequences. These resemble molecular beacons, but we directly incorporate environment-sensitive fluorescent nucleotide analogs such as 2-aminopurine during synthesis, etc.

Nano- and Micro-scale Molecular Labels

Many methods in diagnostics, genomic technology, and proteomics rely upon the sensitive detection of labels added to target or reporter molecules, to facilitate the detection of an analyte or a binding event. Together with Paul Ruchhoeft of UH, they are making 1-micron cubic retroreflectors (small-scale analogs of the lunar retroreflectors which are possibly the most detectable objects ever produced by mankind) for use as labels and in one-step assays based on self-assembly. With Dmitri Litvinov of UH (formerly Seagate) they are seeking to adapt the GMR technology which has radically improved data-storage hard disk drive performance, to produce a biosensor array of extremely high feature density and number (millions), capable of single-molecule detection (using 50 nm magnetic particle labels) and magnetic pull-off “melting curves” for each spot to ensure high data quality.