Massachusetts Institute of Technology

Mark Bathe

Tyson Shepherd

Remi Veneziano

Sakul Ratanalert

James Banal

Do-Nyun Kim

Darrell J Irvine

Tyson Moyer

Joseph Don Berleant

Hyungmin Jun

Hendrik Dietz

Jun He

Syuan-Ming Guo

Nilah Monnier

Keyao Pan

Eike-Christian Wamhoff

Rebecca Du

Benjamin Joseph Read

James Earl Banal

Methods for controlled segregation of blocks of information encoded in the sequence of a biopolymer, such as nucleic acids and polypeptides, with rapid retrieval based on multiply addressing nanostructured data have been developed. In some embodiments, sequence controlled polymer memory objects include data-encoded biopolymers of any length or form encapsulated by natural or synthetic polymers and including one or more address tags. The sequence address labels are used to associate or select memory objects for sequencing read-out, enabling organization and access of distinct memory objects or subsets of memory objects using Boolean logic. In some embodiments, a memory object is a single-stranded nucleic acid scaffold strand encoding bit stream information that is folded into a nucleic acid nanostructure of arbitrary geometry, including one or more sequence address labels. Methods for controlled degradation of biopolymer-encoded blocks of information in the memory objects are also developed.

Sequence-controlled polymer random access memory storage

Methods for the automated template-free synthesis of user-defined sequence controlled biopolymers using microfluidic devices are described. The methods facilitate simultaneous synthesis of up to thousands of uniquely addressed biopolymers from the controlled movement and combination of regents as fluid droplets using microfluidic and EWOD-based systems. In some forms, biopolymers including nucleic acids, peptides, carbohydrates, and lipids are synthesized from step-wise assembly of building blocks based on a user-defined sequence of droplet movements. In some forms, the methods synthesize uniquely addressed nucleic acids of up to 1,000 nucleotides in length. Methods for adding, removing and changing barcodes on biopolymers are also provided. Biopolymers synthesized according to the methods, and libraries and databases thereof are also described. Modified biopolymers, including chemically modified nucleotides and biopolymers conjugated to other molecules are described.

Automated methods for scalable, parallelized enzymatic biopolymer synthesis and modification using microfluidic devices

Compositions containing a nucleic acid nanostructure having a desired geometric shape and antigens bound to its surface are provided. The nanostructures can be, for example, in the form of a 6-helix bundle or icosahedron. The nanostructure design allows for control of the relative position and/or stoichiometry of the antigen bound to its surface. The antigens displayed on the nanostructure surface are arranged with the preferred number, spacing, and 3D organization to elicit a robust immune response. The displayed antigen can be an HIV immunogen such as eOD-GT6, eOD-GT8, or variants thereof. The compositions may thus be useful as immunogens, vaccines, immunostimulators, adjuvants, and the like. Methods of inducing immune responses, inducing protective immunity, inducing the production of neutralizing antibodies or inhibitory antibodies, inducing tolerance, and treating cancer, infectious or autoimmune diseases are also provided.

Nucleic acid nanostructure platform for antigen presentation and vaccine formulations formed therefrom

Methods for the top-down design of nucleic acid nanostructures of arbitrary geometry based on target shape of spherical or non-spherical topology are described. The methods facilitate 3D molecular programming of lipids, proteins, sugars, and RNAs based on a DNA scaffold of arbitrary 2D or 3D shape. Geometric objects are rendered as node-edge networks of parallel nucleic acid duplexes, and a nucleic acid scaffold routed throughout the network using a spanning tree formula. Nucleic acid nanostructures produced according to top-down design methods are also described. In some embodiments, the nanostructures include single-stranded nucleic acid scaffold, DX crossovers, and staple strands. In other embodiments, the nanostructures include single-stranded nucleic acid scaffold, PX crossovers and no staples. Modified nanostructures include chemically modified nucleotides and conjugated to other molecules are described.

Stable nanoscale nucleic acid assemblies and methods thereof

Methods and compositions for bacterial production of pure single-stranded DNA (ssDNA) composed of custom sequence and size have been developed. The methods enable scalability and bio-orthogonality in applications of scaffolded DNA origami, offering one-step purification of large quantities of pure ssDNA amendable for immediate folding of DNA nanoparticles. The methods produce pure ssDNA directly from bacteria. In some embodiments thehelper strain M13cp combined with a phagemid carrying only an f1-origin allows for, without the need for additional purification from contaminating dsDNA. This system is useful for generalized circular ssDNA synthesis, and here is applied to the assembly of DNA nanoparticles folded both in vitro and direct from phage.

Microbial production of pure single stranded nucleic acids

Techniques for controlling nucleic acid structures include determining, for each junction type, values for parameters indicating ground-state geometry and both translational and rotational stiffness coefficients. Topological design data indicates a number of bases in each helix connected to corresponding junctions. Initial positions of each base are determined by connecting helices to junctions using the ground-state geometry and arbitrary coordinates not confined to lattice coordinates. Misalignment vectors each indicate a difference in coordinates and orientations between initial positions of a pair of bases that are not adjacent in the initial positions but are adjacent or coincident in the design data. Forces and moments at the junctions to reduce misalignment magnitudes are determined based on the translational and rotational stiffness coefficients at each junction. Position and orientation in 3D coordinates of each base are determined by reducing or eliminating the misalignment magnitudes and balancing forces and moments across the nanostructure.

Techniques for controlling spatial structure of nucleic acid structures based on lattice-free, three dimensional junction coordinates

Techniques for controlling properties of nucleic acid nanostructures include receiving data that indicates a sequence of nucleotides on at least a first strand of a nucleic acid. Values are determined for at least one physical property for each portion of the at least first strand. Based at least in part on a numerical model and the physical properties for each portion, a value is determined of at least one derived property of a nanostructure that comprises the at least first strand of nucleic acid. In some embodiments, information gained from the numerical model is used iteratively in order to optimize or improve one or more of the properties of the target DNA origami structure.

Method and apparatus for controlling properties of nucleic acid nanostructures

Techniques for inferring particle dynamics from certain data include determining multiple models for motion of particles in a biological sample. Each model includes a corresponding set of one or more parameters. Measured data is obtained based on measurements at one or more voxels of an imaging system sensitive to motion of particles in the biological sample; and, determining noise correlation of the measured data. Based at least in part on the noise correlation, a marginal likelihood is determined of the measured data given each model of the multiple models. A relative probability for each model is determined based on the marginal likelihood. Based at least in part on the relative probability for each model, a value is determined for at least one parameter of the set of one or more parameters corresponding to a selected model of the multiple models.

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Cambridge, MA, United States of America

Mark Bathe