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Seminars
Nucleic Acid Technology: New Methods and Applications
Dr. Richard C. Willson
Department of Chemical and Biomolecular Engineering
University of Houston, Houston, Texas
When: Thursday, August 30, 2007
Time: 3:30PM - 4:30PM
Where: 100 Keck Hall
Abstract:
Immobilized metal chelate affinity chromatography (IMAC) is widely used for purification of proteins, especially "hexa-histidine tagged" recombinant proteins. We have recently demonstrated the use of metal chelate affinity to selective capture of nucleic acids, including RNA and selectively-denatured genomic DNA, through interactions with purine base exposed in single-stranded regions. We also found that the binding affinity of nucleic acids for IMAC adsorbents can be increased several-fold by addition of neutral solutes, and that enhancement varies with reduction of water activity. Bound nucleic acids can be effectively eluted with water instead of the usual imidazole-containing competitive eluants when the surface density of negative charges is enhanced by operation at alkaline pH, or by deliberate metal-underloading of the anionic chelating ligands.
Aptamers are synthetic nucleic acid ligands selected *in vitro* from large combinatorial libraries. Aptamers can recognize a variety of targets with high affinity and specificity; one is now an FDA-approved pharmaceutical for treatment of wet macular degeneration by sequestration of VEGF. We have characterized the biophysical chemistry of the binding of DNA aptamers to VEGF using fluorescence anisotropy, SPR, and isothermal titration calorimetry. We have also recently inserted known aptamer-coding sequences into an artificial 5S ribosomal RNA deletion cassette that is strongly expressed in *E. coli* bacteria, and shown that aptamers' binding properties are not lost when they are expressed in the context of the stable 5S rRNA carrier. Stabilization of aptamers in rRNA carriers may facilitate production of large quantities of RNA aptamers, and may open the possibility of screening aptamer libraries *in vivo.*
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