Biomedical subjects
Publications and source records attributed to Kevin J Luebke.
We describe a novel photolithographic approach to the synthesis of peptoids (oligo-N-substituted glycines). This strategy enables the construction of a spatially addressable peptoid microarray, thus providing a potentially powerful tool for the discovery of protein ligands.
Patterns of cellular adhesion were created on a surface using novel photochemistry that is stimulated with visible light. A glass surface coated with polyethylene glycol is nonadhesive to a variety of adherent mammalian cell types. Treatment of that surface with a mixture of tris(bipyridyl)ruthenium(II) chloride, ammonium persulfate, and a tryptophan derivative or tryptophan-bearing peptide in conjunction with irradiation with visible light (447 nm) made the surface adhesive to several cell types including mouse fibroblasts, human myoblasts, and human lung tumor cells. Immunostaining data suggest that tryptophan-containing peptides are crosslinked intact to the surface by this chemistry, which enables patterning of peptides containing only naturally occurring amino acids. Microscopic patterns of cellular adhesion were created with this chemistry by projecting microscopic patterns of visible light with a digital micromirror array. Using this method, regions of cellular adhesion were patterned with single-cell resolution.
Only a small fraction of short oligonucleotide probes bind efficiently to complementary segments in long RNA transcripts. Technologies such as array-based transcript profiling and antisense control of gene expression would benefit greatly from a method for predicting probes that bind well to a given target RNA. To develop an algorithm for prioritizing selection of probes, we have analyzed predicted thermodynamic parameters for the binding of several large sets of probes to complementary RNA transcripts. The binding of five of these sets of probes to their RNA targets has been reported by others. In addition, we have used a method for light-directed synthesis of oligonucleotide arrays that we developed to generate two new arrays of surface-bound probes and measured the binding of these probes to their RNA targets. We considered predicted free energies for intramolecular base pairing of the oligonucleotide and its RNA target as well as the predicted free energy of intermolecular hybridization of probe and target. We find that a reliable predictor of probes that will hybridize significantly with their targeted transcripts is the predicted free energy of hybridization minus the predicted free energy for intramolecular folding of the probe.
We have developed a method for the parallel analysis of multiple CpG sites in genomic DNA for their state of methylation. Hypermethylation of CpG islands within the promoters and 5' exons of genes has been found to be a mechanism of transcriptional inactivation associated with a variety of tumors. The method that we developed relies on the differential reactivity of methylated and unmethylated cytosines with sodium bisulfite, which exclusively converts unmethylated cytosines to deoxyuracils. The resulting sequence changes are determined with single-nucleotide resolution by hybridization to an oligonucleotide array. Cohybridization with a reference sample containing a different label provides an internal standard for assessment of methylation state. This method provides advantages in parallelism over existing methods of methylation analysis. We have demonstrated this technique with a region from the promoter of the tumor suppressor gene p16, which is hypermethylated in many cancers.
A machine that employs a novel reagent delivery technique for biomolecular synthesis has been developed. This machine separates the addressing of individual synthesis sites from the actual process of reagent delivery by using masks placed over the sites. Because of this separation, this machine is both cost-effective and scalable, and thus the time required to synthesize 384 or 1536 unique biomolecules is very nearly the same. Importantly, the mask design allows scaling of the number of synthesis sites without the addition of new valving. Physical and biological comparisons between DNA made on a commercially available synthesizer and this unit show that it produces DNA of similar quality.
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