DNA Origami Scaffolds for Single-particle Cryo-Electron Microscopy of Viral RNA
Abstract
The proposed research will develop a novel technical platform based on DNA nanotechnology for the high-throughput and high-resolution determination of the 3-dimensional (3D) structure of RNAs, and apply the method to determine the structure of the entire 9,100 nucleotide human immunodeficiency virus type 1 (HIV-1) RNA genome for proof of concept. First, computational resources and experimental automation will be used to generate large libraries of rigid DNA origami nanostructures of arbitrary size that have complementary single-stranded DNA bait sequences that will bind selectively to loops of a target RNA in order to ~capture~ it. A chemical footprinting method that specifically modifies single-stranded regions of RNA (i.e. selective 2~hydroxyl acylation analyzed by primer extension (SHAPE)), and next generation sequencing will then be conducted with the bound RNAs to obtain the secondary structure and distance constraints of tightly bound RNA-DNA nanostructure pairs. The tightest bound complexes will then be used to solve in atomic resolution the tertiary structure of the RNA genome in 3D. Results of this work will offer basic insight into the 3D structure of the viral HIV-1 genome structure, as well as offer a technological platform for broader classes of viral and other RNAs. Furthermore, the technology developed here will apply to the development of RNA viral detection devices for the military and civilian populations. The technology developed here will also impact high-throughput DNA nanostructure library generation in general, which will affect biosensing, the delivery of biologics, nanoreactors/nanomolds, and DNA-base memory storage. To develop the methodology, the proposed research is structured into three aims. In Aim 1, a large scale library of approximately 3,000 DNA objects with designed single-stranded DNA overhangs complimentary to the RNA bait sequences of interest will be developed in order to probe across a broad diversity of 3D structural space. In Aim 2, the DNA objects will be used capture the single-stranded loops of the 5~ untranslated region of the HIV-1 RNA genome and solve the bound structure to atomic detail using cryo-electron microscopy. In Aim 3, the high-throughput library will be applied to the entire HIV-1 genome to solve its 3D structure, as well as map distances between single stranded RNA loops present on its surface and interior cavities.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Sep 23, 2016
- Source ID
- N000141612953
Entities
People
- Mark Bathe
Organizations
- Massachusetts Institute of Technology
- Office of Naval Research
- United States Navy