An Evolutionary Approach to the Discovery of New Materials for the Production and Utilization of Alternative Fuels
The challenge of providing cost-effective sources of non-fossil fuel energy demands new materials for converting sunlight to electricity or stored chemical energy such as hydrogen. Unfortunately, the landscape of possible inorganic solid-state materials is infinitely vast, and predicting the right combination of elements for a desired application is nearly impossible. How can we possibly hope to find the perfect combination of elements for the materials needed advanced energy technology?
Our lab has been applying an approach to materials discovery that seeks to exploit biological evolution and selection. Indeed, nature has provided some of the best examples of materials synthesis. Nearly every living organism on Earth synthesizes a solid-state material, some for structural integrity or protection, others for biosphere function such as light focusing or magnetotaxis. We have asked the question: Can biomolecules evolve in vitro in response to certain selection criteria until they are capable of forming a solid-state material with a desired property? The answer is yes, and the particles shown below are some examples. These particles were all grown in the presence of specific RNA sequences that were selected from large random RNA sequence libraries (~1014 unique sequences) using a process known as in RNA vitro selection. These results are important because they suggest that biomolecules such as RNA can assemble metal precursors into nanoparticles with sizes, shapes, and compositions that depend upon RNA sequence. This in turn suggests that new materials with advanced catalytic or photonic properties could emerge from a large library of RNA sequences. We are currently attempting to exploit RNA in vitro selection to discover new catalyst and photonic materials for photovoltaics and fuel cells.
Methods for synthesizing Pd and Pt nanoparticles using RNA can be challenging and another lab has shown how improper protocols can lead to anomalous results. Proper protocols have been published in The Journals of Materials Chemistry (2010, 20, 8394 - 8398).
Pt spheres (left) and porous Pt "crystals of nanocrystals" synthesized with RNA.
Cobalt-doped iron oxide nanoparticles (left), and Pd hexagons and cubes synthesized with different RNA sequences isolated from large random sequence RNA libraries using RNA in vitro selection.
Nanomaterials in the Diagnosis and Treatment of Infectious Disease
One-third of the global population (3 billion people) is estimated to be infected with M. tuberculosis (TB). A majority of those infected will never contract active TB and will remain asymptomatic. However, for immunocompromised patients TB is a major cause of death worldwide. For example, TB is the leading cause of death in HIV-positive patients. The World Health Organization estimates that 9 million people contracted active TB in 2006 (~6 million from Southeast Asia and Africa alone) and over 1 million died from the disease. Yet over 1.5 million people in India alone are believed to undergo useless TB tests each year, and both HIV and TB have become resistant to current treatments and can hide in "sanctuary sites" such as the brain, where small molecule drugs often have difficulty penetrating.
Our lab is addressing challenging problems in the diagnosis and treatment of infectious diseases such as HIV and TB. In developing new diagnostic methods and therapeutics, we seek to exploit many of the novel physical and chemical properties of nanoscale materials. A few examples are highlighted below.
Nanoscale HIV and TB therapeutics. Synthetic nanometer-scale systems have the potential to overcome many limitations of conventional small molecule therapeutic agents. For instance, small molecules typically have short blood circulation times (hrs), rely on a single high-affinity contact to a disease target, and are incapable of disrupting protein-protein interactions that often drive disease pathogenisis. In contrast, nanoscale systems can provide long circulation times (days to weeks), have tunable valency, and are adept at preventing protein-protein interactions. We have hypothesized that gold nanocrystals may possess a number of attributes that make them useful drug candidates. Gold nanocrystals are now accessible in a range of well defined sizes from ca. 1.0 nm to 10 nm. Gold nanocrystals also enable one to rapidly synthesize combinatorial libraries of nanoscale compounds; using organothiol exchange reactions, combinations of two or more chemically distinct ligands can be attached to a single particle to create multi-ligand and multi-functional systems. The ability to rapidly assemble mixed thiol monolayers on a nanoscale platform provides a powerful tool that can be used to tune particle binding affinity to a disease target, and control cellular internalization and sub-cellular localization.
The potential benefits of gold nanocrystal therapeutics were demonstrated in our lab recently with the synthesis of a multivalent gold conjugate that effectively inhibited HIV entry in T-cells. We have also shown that lactam antibiotics, rendered ineffective at bacterial growth inhibition due to bacterial mutation, can be converted back into potent therapeutics via appropriate design and conjugation to gold nanocrystals. We are currently adapting these formulations to cross the blood-brain barrier and enter other sanctuary sites.
Model of a 2.0 nm diameter gold nanocrystal therapeutic that effectively inhibits HIV entry into human T cells.
Materials DNAzymes in Disease Diagnostics. Current diagnostic methods for active TB include culture, nucleic acid amplification, sputum stain, and the Quantiferon antigen test. When applied to resource-limited settings, these methods suffer from significant limitations. Collectively, they are too expensive, too time consuming (24 hrs or more), and require specialized equipment, expertise, and power. Furthermore, they have low sensitivities (50% in the sputum smear test), or are unreliable when used on HIV-positive patients or children (antigen tests). It has been estimated that more rapid and accurate TB diagnostic methods that could be deployed in resource-limited settings such as those that exist in many parts of Southeast Asia and Africa would save 400,000 lives per year.
We are combining the stability and specificity of DNA aptamers with the catalytic activity of materials DNAzymes to create new diagnostic assays for the detection of TB biomarkers in urine. DNAzymes are DNA sequences capable of converting metal or organic precursors into metal or polymeric nanoparticles that are detected either optically or electronically (similar to the RNA sequences described above). But why use these DNA reagents rather than adapting an existing diagnostic platform that uses capture antibodies and enzymatic detection? The answer is that modified DNA aptamers and DNAzymes are more suited for use in resource-limited settings. DNA aptamers are isolated rapidly using straightforward chemical methods. They are less costly to discover and produce than antibody reagents. Their synthesis is reproducible, with no batch-to-batch variation in binding affinity as is frequently encountered with antibodies. Finally, they are more thermally stable than protein reagents, enabling shipping and long-term storage without refrigeration.
Mapping the Visual Interactome
In addition to developing nanocrystals as new therapeutics for treating disease, our lab is developing new cellular imaging tools for understanding disease at the molecular level. Our long-term ambition is to generate a complete 3D map of the spatial arrangement of RNA and proteins in eukaryotic cells: the "visual interactome". This goal will be accomplished through the implementation of a new concept in biomolecule tagging, which will be used in conjunction with cryoelectron tomography (cryoET). CryoET combines multiple images of a biological sample acquired at different tilt angles to obtain a 3D volume representation of the sample. With achievable 5 nm resolution and perfect preservation of cellular structure, cryoET now represents the highest resolution technique for examining cells in their native state. As advanced as cryoET is, one problem still prevents the localization and identification of all but the most dense of structures in a cell: it is exceedingly difficult to know which biomolecule one is visualizing when looking at a 3D whole cell reconstruction. The problem is analogous to the one in fluorescence microscopy. Although many proteins autofluoresce, one cannot tell them apart in the absence of a specific fluorescent marker, such as the green fluorescent protein (GFP). Our lab is thus developing a new tagging strategy for use in cryoET experiments. The molecular tags we are developing consist of RNA or peptide sequences that are selected in vitro to mediate the formation of inorganic nanoparticles. Once genetically encoded in cells as RNA concatemers and protein chimeras, these sequences will be able to mediate the formation of inorganic nanoparticles inside living cells exclusively at the site of interest. These particles will be electron dense for visualization in a cryoET reconstruction; when a biomolecule is observed in a tomogram, the presence of a nanoparticle will allow the identity of the biomolecule to be known.