We will develop an AFM-based, single-molecule, gene expression analysis technology. This technology meets a pressing need for order of magnitude improvements in detection sensitivity, and three-to-four orders of magnitude lower cost. This will allow for the first time, routine, high-resolution expression profiling of clinical biopsy samples, and single cells. Our technology is ultra sensitive and extremely low cost. This approach utilizes nanoscale, 'coded'cDNA molecular 'tracks'that are interrogated, molecule by molecule, using AFM. Each cDNA molecule is 'coded'by in situ digestion with sequence-specific restriction enzymes, that produce narrow, detectible, breaks in individual polynucleotide backbones whose locations correspond to particular 4bp or 6bp enzyme recognition sequences. These slight depressions or dimples in the molecule backbone serve as digital 1's and 0's, uniquely identifying each cDNA. In the future these molecular 'tracks'can be arrayed on inexpensive 'cDNA disks', analogous to today's DVDs, for high-speed readout. This technology requires no signal amplification (PCR, etc), and can uniquely identify up to 107 distinct cDNA species in an individual sample, with single molecule resolution. We estimate that throughput of 1,000+ cDNAs per second is technically achievable, at a cost 104 times less than exiting methods. Our integrated team incorporates expertise in nanotechnology, biochemistry, and bioinformatics, in collaboration between the UCLA Department of Chemistry and Biochemistry (Gimzewski group) and the New York University Departments of Computer Science and Mathematics (Mishra group). Our team has worked together on this concept for two years prior to this RFA. We have explored the concept with a mathematical model, AFM imaging and tests of the basic surface/biochemistry. Our Preliminary Studies clearly specify quantitative, experimental benchmarks that will validate the concept, and which are necessary for engineering scale-up. The Specific Aims of this Proposal are to achieve those benchmarks. R21 Proposal: Single Molecule cDNA profiling with AFM Achieving the Specific Aims of this proposal will establish complete proof-of-concept, and a technology development path for Single Molecule cDNA profiling with AFM. After engineering scale-up, this technology will allow precise and cost efficient gene expression profiling of thousands of individual cells. Routinely conducting such analysis would significantly advance disease prediction, diagnostics and treatment, as well as drug discovery.