Programmable synthesis of DNA nanostructures for spatial and temporal control
Abstract
In the proposed effort, the Principal Investigator will continue the development of his newly invented molecular process for dynamic DNA circuitry, termed primer exchange reaction (PER). The basic primer exchange reaction has three steps. First, a primer (domain 1) binds reversibly to a hairpin, which facilitates specific elongation. Then, a strand displacement extends the primer to copy the stem sequence (domain 2) in the hairpin, until a stop sequence is reached. After elongation has terminated, the displaced stem region of the hairpin re-hybridizes with its opposing strand on the hairpin to displace the primer sequence plus extension to a point that it can spontaneously dissociate from the hairpin and is free to interact with another cognate hairpin in solution. All primer exchange reactions are powered by dNTPs in solution and operate isothermally. Conceptually, PER is capable of appending sequences onto growing strands in a specific, programmable manner, and the Principal Investigator has termed this reaction a ~molecular primitive~ because each modular reaction unit can be combined with other such reactions to create a molecular program with specific functions. PER has significant conceptual advantages over the present predominant technology in DNA circuitry, the toehold exchange reaction (TER). Rather than manipulating the sequestration states or pre-existing toehold sequences as in TER, PER synthesizes new sequences on demand. This critical difference renders PER much more programmable, less leaky, more robust, and the process has the potential to construct more sophisticated and complex functional dynamic systems. An additional advantage of PER systems is that they use inexpensive dNTPs as fuel versus the large, unstable, and expensive complexes of DNA used as fuel for TER systems. PER systems are also able to synthesize strands in situ, which enables many possibilities of growing large structures in situ. For example, if a strand has been conjugated to another biomolecule such as a protein in a fixed cell, structures could be grown directly in these locations. Finally, PER systems have single molecule resolution, automatically growing one transcript per primer molecule in solution, versus TER systems where states are generally inferred by the aggregates state of molecules in the system, which leads to limitations in the multiplexed readout strategies of such systems and difficulty in determining the progression of the computation. In PER systems, each transcript indicates the ordered set of states (history) it traversed in the state transition diagram graph over time, with course length information that can be read out on gels and more precise information that can be obtained with sequencing. These transcripts can be engineered to record transcripts of when environmental signals were present over time. - The Principal Investigator~s specific aims in the proposed research involve combining PER with his previous success in programming DNA nanostructures to engineer: (1) the triggered assembly of complex DNA nanostructures in situ; (2) molecular clocks for measuring elapsed time and timers for controlling signals after a time delay; and (3) environmentally responsive nanomachines that differentiate in response to environmental signals and record those signals over time. The research will provide the basis for applications such as the in situ growth of markers for Cryo-Electron Microscopy imaging, long term environmental surveillances of pollutants, conditional gene regulation, and the triggered encapsulation of toxins in situ.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Aug 12, 2016
- Source ID
- N000141612410
Entities
People
- Peng Yin
Organizations
- Office of Naval Research
- President and Fellows of Harvard College
- United States Navy