The successful sequencing of the human genome has provided us with the blueprints of life.... but now the greater challenge of understanding those blueprints lies squarely before us. New tools are needed to unravel the complexities of biological systems, and the development of such tools is the focus of research efforts in our group. In this talk I will present three short stories describing technology projects currently under development. These are:
1) GENECAPP (Global ExoNuclease-based Enrichment of Chromatin-Associated Proteins for Proteomics), a strategy for Sequence-Specific Capture of Protein-DNA Complexes for Mass Spectrometric Protein Identification.
In this approach, formaldehyde cross-linking is employed to covalently link DNA to its associated proteins; subsequent fragmentation of the DNA, followed by exonuclease digestion, produces a single-stranded region of the DNA that enables sequence-specific hybridization capture of the protein-DNA complex on a solid support. Mass spectrometric (MS) analysis of the captured proteins is then used for their identification and/or quantification. We have recently obtained excellent results with this strategy in E.coli, and efforts are underway to extend it to more complex organisms such as yeast and human.
2) A Mechanical Nanomembrane Detector for Time-of-Flight (TOF) Mass Spectrometry
A mass spectrometer is a system comprised of three major parts: an ionization source, which converts molecules to ions; a mass analyzer, which separates ions by their mass to charge ratio; and an ion detector. Current ion detectors perform poorly at detecting large ions in low charge states, such as those produced in the matrix-assisted laser desorption/ionization (MALDI) process, or by electrospray ionization (ESI) with charge reduction. We describe here a new principle for ion detection in such TOF mass spectrometry, in which an impinging ion packet excites mechanical vibrations in a 46 nm thick silicon nitride nanomembrane. The nanomembrane oscillations are detected by means of a time-varying field emission of electrons from the oscillating nanomembrane. Ion detection is demonstrated in the MALDI-TOF analysis of proteins varying in mass from 5,729 (insulin) to 150,000 (Immunoglobulin G) daltons.
3) RNA-Mediated Gene Assembly from DNA Arrays
A strategy is presented for RNA-mediated gene assembly from oligonucleotide sequences on a DNA array. In contrast to previously reported approaches in which the oligonucleotides themselves serve as the building blocks for hybridization-based gene assembly, we append a T7 promoter to each surface-bound oligonucleotide and produce many RNA copies of each with T7 RNA polymerase. These RNA molecules self-assemble into the desired full-length transcript by hybridization and ligation, which is then converted into dsDNA by reverse transcription PCR. This single-day gene assembly technology was demonstrated in synthesis of the gene encoding a 231-amino-acid green fluorescent protein. Sequence fidelity and functionality of the synthesized gene were evaluated via Sanger sequencing and in vitro protein expression. A 40% yield of the correct gene sequence was obtained.