Combinatorial approaches are valuable in designing new biomaterials that exhibit specific interactions with biological moieties and in obtaining fundamental insights into biological processes by surveying the entire parameter space simultaneously. The research program in this area is divided into biomaterial-biomolecule and biomaterial-cell interactions.

The design and synthesis of novel materials for biological applications has, to date, been strongly empirical because of the complexities of biological systems. Serial approaches to understand interactions of biomaterials with biomolecules and cells and their effect on function reveal only a “small piece of the puzzle,” limiting the ability to predict optima in such a large search space. CombiSci approaches are therefore invaluable in gaining a more generalized mechanistic picture of structure and function by answering questions like:

1) what roles are played by materials chemistry and architecture in biomolecule/biomaterial interactions; and

2) how does biomaterial structure govern transport and recognition in biological systems? This research relies heavily on the intimate integration of library design and screening strategies with informatics.

CombiSci process applied to polyanhydride library for the development of vaccine delivery systems with tailored immune response.

Biomolecule/biomaterial interactions: High-throughput Investigation of Protein Function and Enzyme Activity (Pohl, Yeung): There are many proteins whose catalytic activity is valuable in the biosynthesis of saccharides, but chemical functions cannot be ascertained solely by primary sequence analysis or X-ray crystallography. Our objective is to develop libraries and new assays to identify the chemical function and synthetic and diagnostic utility of genes involved in carbohydrate biosynthesis and degradation. Preliminary data show that our electrospray ionization mass spectrometry (ESI-MS) assays on picomolar sample quantities can ameliorate the tedious synthesis of both carbohydrate substrates as well as active proteins. Based on these findings, we aim to: 1) develop high throughput assays and compounds for nucleotidyltransferases and glycosyltransferases, 2) discover the chemical function and synthetic utility of new proteins, and 3) develop compound libraries to discover the chemical functions of putative glycosidases using ESIMS- based assays. With these CombiSci studies, we have studied three major classes of carbohydrate-utilizing enzymes, thereby assessing virulence of cancer cells and microorganisms, screening for compounds targeting carbohydrate pathways, and harnessing biosynthesis for carbohydrate library construction. In parallel with this effort, we will identify the most efficient form(s) of any enzyme (e.g., carbonic anhydrase) by the massive alteration of reaction parameters such as genetic variants, conformers, ionic strength, pH, buffer additives, temperature, and co-substrates. We will design an appropriate non-fluorescent substrate for the enzyme that will form a fluorescent product via the catalyzed reaction of the target reactant, thus generating millions of fluorophors for detection and screening.

A) Visual map of a polyanhydride random copolymer library multiwell substrate with varying composition from 0 to 100 mol% SA and CPH. (B) IR map of SA (1815 cm^-2) in the multiwell substrates shown in (A). (C) Mol % CPH calculated from FTIR spectra extracted from six wells.

Cell/Biomaterial interactions: Aptamers (Nilsen-Hamilton, Cornick, Olsen, Porter): This area focuses on novel CombiSci methods for building aptamers as selective labels for microorganisms and cells.

 

1) Highly Selective Labels for Microorganisms: The well known limitations of antibodies in immunological recognition and labeling (e.g., batch-to-batch variability, temperature/sample matrix effects, and cross-reactivity) can potentially be overcome through the use of a new class of molecular recognition ligands called aptamers (nucleic acid oligomers). Aptamers are selectively isolated from pools of over 10^15 different random nucleic acid sequences through SELEX. Results from applying SELEX, starting with a pool of 1012 DNA molecules, to the development of tags for foodborne pathogens are shown in image to the right. Though qualitative, the images show that we can amplify the presence of aptamers that bind to microorganisms with high affinity because the fluorescence intensity is still detectable after extensive rinsing. We found that oligo-modified microorganisms can be concentrated after mixing the library with a solution of the target and then directly loaded into a thermal cycler for PCR amplification. This unexpected result eliminates the need to separate bound aptamers from the target organism, greatly simplifying the adaptation and allows automation of SELEX for identifying tags with enhance labeling capabilities.

Tagged with fluorescently labeled (hexachlorofluorescein phosphoramidite) aptamers : Light microscope image (left) fluorescence microscope image (right).

2) Highly Selective Tags for Cancer Therapy and Imaging: We have devised the concept of allosteric aptamers that bind to their primary target only when another part of the nucleic acid has bound to a second target. Localized therapy can be realized with these aptamers that then only bind to a prodrug and cell surface receptor. Thus, aptamers that bind to a cell surface will not bind to an imaging agent (e.g., fluorophor) or prodrug unless the allosteric aptamer is specifically coupled to cell surface receptor, circumventing difficulties in screening because of nonspecific adsorption.

FE-SEM images of organically functionalised mesoporous monoparticles of uniform shapes and sizes. The image magnification is same in all the images (scale bar = 3 um)

Combinatorial Strategies for Gene Therapy (Lin, Mallapragada, Nilsen-Hamilton): We have recently synthesized two types of libraries as vectors for gene therapy: 1) novel cationic water-soluble block and random copolymers with various block lengths and architectures and 2) mesoporous particles of various shapes and sizes functionalized with cationic polymers. Currently, biomaterial interactions with cells are poorly understood in the absence of any specific recognition tag. Based on our recent studies, the cellular uptake profiles of organically functionalized mesoporous nanoparticles with different sizes and shapes are very different and are strongly dependent on cell type. We are systematically exploring these variables with high throughput using microspotting techniques and standard well plate formats. DNA transfection studies use vectors mixed with reporter genes in multiple wells. Transfection efficiencies are obtained via fluorescence imaging. These studies will provide key clues about the role of vector shape, size, and chemistry on the mechanisms of cell uptake and expression of DNA.