Self-assembled DNA crystals as scaffolds for macromolecules

PART 1: NON-TECHNICAL SUMMARY

Nature uses self-assembly in order to organize molecules into functional materials, with biological cells being a prime example of the great potential in this approach. DNA and RNA are two of the most promising molecules for constructing tailored self-assembling systems because they encode information in a programmable way, so many independent strands can be designed that come together in a predictable manner.

The molecular properties (e.g. the dimensions, geometry, and rigidity) of these nucleic acids are also well known, and they can form branched junctions that allow them to assemble in 2D and 3D space. One key goal of nucleic acid-based nanotechnology is to build 3D crystals with programmable void spaces to host various guest molecules. This project aims to show, for the first time, that molecules like RNA or proteins can be specifically positioned in these crystals, by tethering them to strands that make up the lattice.

The first goal is to incorporate RNA into the crystals, both to see how it changes the assembly of the lattice, but also to solve the structure of small, unknown RNA motifs like aptamers. In the second goal, proteins will be attached to the crystals, not to solve their structure, but rather to create a dense 3D array of these molecules (e.g. for catalytic reasons, by incorporating enzymes).

Finally, the third goal of this project is to create nano-crystals (which are roughly a billion-fold smaller than the crystals typically obtained), laden with either small interfering RNA (siRNA) or functional proteins. These crystals can be used to effectively deliver these cargoes into cells, given the extremely high density they can carry. Taken together, the goals in this proposal will create a new system of 3D scaffolds for solving RNA structures, attaching proteins to make catalytic materials, and more efficiently delivering important functional molecules into cells.

The project will also have significant societal and educational impact by developing an online curriculum and teacher training for K-12 education and a new self-assembly online game for exploring how these systems work. This program will engage undergraduate, graduate, and underrepresented minority students to gain knowledge and pursue research in the science, technology, engineering, and math (STEM) fields, and help develop a new course for teaching nanotechnology to a broad range of students.

PART 2: TECHNICAL SUMMARY

The goal of this project is to use self-assembled, 3D DNA crystals as functional macromolecular scaffolds that can immobilize guest molecules, such as RNA and proteins. These DNA crystals are programmable in both their lattice geometry and the size of the pores and channels that comprise them, and guest species can be site-specifically positioned by tethering them to the strands that assemble to form the crystal (either covalently or via supramolecular effects).

This project will create crystals that incorporate RNA as well as DNA, and determine the effects on crystal symmetry of this change. In addition, the cavities of the crystal will be used to host both RNA and DNA aptamers, and to solve their structure (including ones that are currently unknown) using X-ray crystallography. Small aptamers in particular are difficult to solve in any other way, and thus this method will facilitate the determination of more such structures.

Another goal of the project will be to create a high density of functions proteins (both model systems like GFP, and enzymes like horseradish peroxidase), by attaching them to molecules that site-specifically bind the minor groove of DNA. Although the proteins will not be tethered rigidly enough to solve their structure using crystallography, the lattices can serve as catalytic materials, as affinity scaffolds for protein purification, or ways to protect the proteins from degradation.

The third key goal of this project will be to scale down the size of the crystals to 100-200 nm in size (compared with traditional such crystals, which are hundreds of micrometers). These nano-crystals will be used to deliver functional cargoes (such as siRNA or functional proteins) to the interior of cells, and the regular lattice will provide an extremely high loading capacity, e.g. ~1,000 molecules for a crystal approximately 100 nm in diameter.

Taken together, the work will: (1) create a new set of scaffolds for determining RNA and DNA aptamer structure; (2) enable functional materials with controlled attachment of proteins in 3D space; and (3) design a new category of nanoparticles with extremely high loading capacity of proteins or siRNA for effective delivery to cells.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.