Expansion of Combinatorial DNA Nanoparticle Libraries for Materials Research & Structural Biology
Abstract
Structured DNA nanoparticles programmed using scaffolded DNA origami offer the unique ability to fabricate nanoscale materials with molecular-level precision. This revolutionary nanoscale materials fabrication platform has opened numerous transformative research avenues in structural biology, quantum sensing and computing, light harvesting, therapeutics, and molecular data storage. To facilitate the design and synthesis of structured DNA and RNA assemblies for these application areas, our lab has developed several top down sequence design algorithms that offer the ability to program nearly arbitrary 2D (PERDIX and METIS) and 3D (DAEDALUS and TALOS) nanoscale objects with precise nanoscale dimensions. We have also designed and implemented the biological production of custom circular single-stranded DNA scaffolds on the 14kb scale that produce raw scaffold material with cost and scale that are on par with solid-state oligonucleotide synthesis, also performed in-house in our lab. In order to further enable the production of large-scale libraries of structured DNA assemblies with diverse geometries and chemical compositions, here we seek to acquire high-throughput assembly and characterization tools that we can leverage for synthesis scale-up, purification, and quantitative downstream characterization of structured DNA assemblies with unique optical, biochemical, and structural properties. This workflow requires multistep liquid handling in which large numbers of distinct oligonucleotide staple strands are first combined, and subsequently folding buffer is transferred together with unique small molecule dyes and other templated materials, thermal annealing, washing, purification, and high-throughput quantitative characterization using optical methods offered by multi-well plate readers. These numerous steps are prohibitive to perform manually and suffer from reproducibility issues that lead to waste of both raw material and time. Thus, to mitigate these issues, we propose to integrate automated pipetting equipment with microliter to milliliter precision that will complement our in-house DNA synthesis and LabCyte Echo liquid handling instruments, as well as a plate washer, thermal cyclers, incubators, plate reader, and nanosizer. The proposed system will complement our existing Echo liquid handling instrument that offers the generation of distinct oligo pools by offering access to larger volumes of materials and, most critically, larger scale library preparation and purification of functional DNA origami-based materials for diverse applications being pursued in our lab. Toward this end, we propose a high- throughput system for assembly of nanoscale DNA devices which includes a FLO I8 intelligent liquid handler to automate pipetting, a Biotek EL406 with a BioSpa accessory to handle our plate incubation and washing workflows, and a Tecan M200 Infinite and Malvern NanoSight NS300 for quality control for high-throughput downstream assays using the synthesized DNA assemblies. These instruments would enable our goals towards implementing large-scale functional DNA- based devices and meet our objectives that are currently funded by the ONR, DoE, NIH, and NSF. We would make available our automated liquid handling capabilities to the MIT Biological Engineering Department Teaching Laboratory, facilitating practical learning by undergraduate and graduate students interested in nucleic acid nanotechnology and combinatorial library generation for synthetic biology.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Apr 29, 2020
- Source ID
- N000142012202
Entities
People
- Mark Bathe
Organizations
- Massachusetts Institute of Technology
- Office of Naval Research
- United States Navy