This is a revision to our previous two-year R21 proposal submitted in February 2002. The National Institute of Neurological Disorders and Stroke (NINDS) invites applications for Exploratory/Developmental (R21) Grants relevant to its research mission. This proposal brings together two principal investigators, a neurobiologist and a bioanalytical chemist for a joint effort in developing and applying bio/nano technologies to answer important neurobiology questions related to learning, memory and neurodevelopment. Currently, we have a gene chip technology to study gene profiling of giant snail Aplysia cells (1 mm in size). But to proceed with single characterized neurons and synapses (submicrometer to about 30 mu m), we need bio-nanotechnologies to assay messenger RNA (mRNAs) contents of single neurons, neuronal processes and synapses. Our scientific goal comes from nanobiology, to measure the concentration of specific mRNAs in defined subcellular regions of single neurons, first from the snail Aplysia, and ultimately from mammals. This information will open new avenues for better understanding of how organisms respond to their environment by learning. Because few molecules must be assayed in subcellular structures to reach this goal, we will need nanofabricated eDNA arrays to capture the mRNAs for assay. We propose to develop a one-dimensional (1-D) eDNA nanoarray for gene profiling of single cells and processes. The 1-D linear eDNA nanoarray is a fluidic silica channel consisting of about 1,000 detection zones, each carrying different neuron or synapse-specific eDNA detection elements. The major advantages of the 1-D nanoarray in gene profiling are: ultra small sample volume (10-12 liter to 10-15 liter), extremely high sensitivity (down to a few mRNA molecules), no PCR amplification needed, rapid assay and analysis, and multiple gene analysis. The arrays will be made by nanofabrication and biomolecule functionalization. Within the arrays, molecular engineered DNA probes are needed to generate a fluorescence signal when they assemble with specific target mRNAs. These will focus on emerging technology in molecular beacons (MBs) and fluorescent nanoparticles. Nanofluidics and AFM will be used to deliver these MBs within the DNA array elements. Software specialized for analyzing biological molecules will be used to archive and interpret recovered mRNA sequences. In the process of meeting a challenging goal, we will be forced to improve and validate each component of the project, scientific and technological, in an environment where postdoctors and students at all levels will engage in multidisciplines in a collaborative setting for the training of the scientists of the future. By the end of this project, we will have generated pilot data to assess the feasibility of a novel avenue of research, involved high-risk experiments that could lead to breakthroughs in neuronal functions and neurogenomics, and demonstrated the potential of nanoscopic DNA arrays in answering basic questions in learning, memory and neurodevelopment. This grant will lay the foundation for a research program in using genomic tools and nanotechnology to answer critically important fundamental questions in neuroscience. How many and what genes are expressed in a specific neuron? How different are gene expression profiles among individual neurons or their subcellular regions? How do these molecular differences determine a unique neuronal phenotype (i.e. discrete neuronal morphology, synaptic patterning and path finding) and functions? The nanoarrays, developed and validated in this proposal, will provide revolutionary tools to understand how neurons and synapses operate, remember and learn, and how these behaviors are evolved.
