Methods of serial assembly of DNA bricks into larger structures

• US 9,073,962 B2
• Filed: 07/12/2013
• Issued: 07/07/2015
• Est. Priority Date: 07/12/2012
• Status: Active Grant

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Fracchia, Charles, Gershenfeld, Neil, Cheung, Kenneth
• Primary Examiner(s): Kelly, Robert M
• Application Number:
  US13/941,405
• Publication Number:
  US 20140018441A1
• Time in Patent Office: 725 Days
• US Class Current: 1/1
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical

Abstract

In exemplary implementations of this invention, hierarchical, nanometer-precise assembly is performed: A first structural unit is attached to a solid substrate in a first fluidic flow. A second structural unit is attached to the first structural unit in a second fluidic flow, a third structural unit is attached to the second structural unit in a third fluidic flow, and so on, until a target structure comprising the structural units is assembled. The first, second, third and so on fluidic flows are separate and occur in order in a temporal sequence. During the temporal sequence, a specific permutation of nucleobases is used repeatedly, in separate fluidic flows which occur at different times, to form multiple attachments between structural units in an assembly. The assembled target structure is removed from the solid substrate. Attachments between the structural units may be formed by nucleobase pairing.

Claims

1. A method comprising the following steps, in combination:

(a) attaching a first deoxyribonucleic acid (DNA) brick to a solid substrate in a first fluidic flow;

(b) attaching a second DNA brick to the first DNA brick in a second fluidic flow;

(c) then attaching, to a previous DNA brick, an additional DNA brick in an additional fluidic flow,

(d) then repeating step (c) at least one more time; and

(e) removing a structure from the solid substrate, which structure comprises the DNA bricks attached to each other in steps (b), (c) and (d);

wherein

(i) the previous DNA brick mentioned in step (c) is (I) in the first iteration of step (c), the second DNA brick, and (II) in each subsequent iteration of step (c) pursuant to step (d), the additional DNA brick attached in the previous iteration of step (c),

(ii) the fluidic flows are separate from each other and occur in order in a temporal sequence,

(iii) attachments between structural units comprise nucleobase pairing, and

(iv) during the temporal sequence, a specific attachment permutation is used repeatedly, in separate fluidic flows which occur at different times, to form multiple attachments between structural units in an assembly sequence.

2. The method of claim 1, wherein:

(a) the target structure, when assembled, includes a physical sequence of DNA bricks; and

(b) overall direction of the physical sequence varies as a function of position; and

(c) a majority of the changes of the overall direction consist of 90 degree turns, each 90 degree turn being a 90 degree angle between long axes of adjacent structural units.

3. The method of claim 1, wherein each attachment site on a DNA brick comprises three elongated structures that comprise nucleobases for selective docking with other structural units.

4. The method of claim 3, further comprising the step of attaching or confining a payload to or in the target structure, which payload does not consist of the structural units.

5. The method of claim 1, wherein:

(a) attachment between DNA bricks occurs at attachment sites on the structural units; and

(b) a DNA brick includes exactly four attachment sites.

6. The method of claim 1, wherein DNA strand displacement reconfigures one or more attachments between structural units.

7. The method of claim 1, wherein toehold-mediated activation activates attachment sites.

8. The method of claim 1, wherein one or more nucleobases used in the nucleobase pairing are unnatural.

9. The method of claim 1, wherein sensor measurements assess yield or other metrics of an assembly step.

10. The method of claim 1, further comprising using microscopy imaging of fluorescence from FRET fluorophores to determine, based on light or lack thereof from three distinct fluorophore pairs, whether an assembly step yielded a right turn, left turn, go straight, or no connection.

11. The method of claim 1, wherein:

(a) a DNA brick has a main body; and

(b) overall shape of an external planar face of the main body, not including any elongated protrusions from the main body, is a parallelogram.

12. The method of claim 1, wherein, when the target structure is assembled, a plurality of DNA bricks are connected in-plane.

13. The method of claim 1, wherein the target structure, when assembled, is configured to attach to, or confine, a payload.