US 11,851,651 B2 · Automated methods for scalable, parallelized enzymatic biopolymer synthesis and modification using microfluidic devices

Abstract

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.

A method for synthesizing a biopolymer having a user-defined sequence of component building blocks on a microfluidic system, the method comprising:

(a) forming, on a microfluidic device, first, second, and third droplets, wherein the microfluidic device comprises:
(i) a multiplicity of spatially distinct addressed locations;
(ii) a multiplicity of distinct fluid reservoirs;
and
(iii) a microfluidic handling system that can actuate (1) splitting of droplets from each of the multiplicity of reservoirs through motive force induced by electric potential or optical excitation at the fluid reservoirs, and (2) movement of droplets from each of the addressed locations to another of the addressed locations through motive force induced by electric potential or optical excitation at the addressed locations,
wherein each of the multiplicity of fluid reservoirs is located at a spatially distinct addressed location on the microfluidic device,
wherein a first of the multiplicity of fluid reservoirs comprises a solution comprising a component initiation sequence,
wherein a second of the multiplicity of fluid reservoirs comprises a solution comprising a first component building block,
wherein a third of the multiplicity of fluid reservoirs comprises a solution comprising an attachment catalyst that can catalyze the attachment of the component building block to the component initiation sequence and/or a further component building block, and
optionally wherein a fourth or further of the multiplicity of fluid reservoirs comprises a solution comprising a further component building block;
wherein the first droplet comprises a component initiation sequence,
wherein the first droplet is formed by splitting from the first reservoir,
wherein the second droplet comprises a first component building block,
wherein the second droplet is formed by splitting from the second reservoir,
wherein the third droplet comprises an attachment catalyst,
wherein the third droplet is formed by splitting from the third fluid reservoir,
wherein the splitting comprises moving a droplet of fluid away from a fluid reservoir, and
wherein each of the droplets is formed at a distinct addressed location on the microfluidic device;
(b) combining the first, second, and third droplets to form a combined droplet,
wherein the combining comprises moving each of the droplets to the same addressed location on the microfluidic device,
wherein the combining is performed under conditions suitable for the attachment catalyst to attach the component initiation sequence to the first component building block within the combined droplet;
(c) forming, on the microfluidic device, a further droplet comprising a component building block,
wherein forming the further droplet comprises splitting a droplet from the second, or fourth or further reservoir, and
optionally forming an additional droplet comprising an attachment catalyst,
wherein forming the additional droplet comprises splitting a droplet from the third reservoir;
(d) combining the combined droplet with the further droplet and, optionally, with the additional droplet, to synthesize a biopolymer,
wherein the combining comprises moving the further droplet and/or the combined droplet and, optionally, the additional droplet, to the same addressed location, and
wherein the combining is performed under conditions suitable for the attachment catalyst to attach the component building block from the further droplet to the component building block within the combined droplet;
(e) optionally repeating steps (c) and (d) one or more times to perform step wise addition of component building blocks to the biopolymer;
and
(f) manipulating, purifying, or isolating the synthesized biopolymer on the microfluidic device,
wherein the sequence of the synthesized biopolymer is defined by the order in which droplets comprising component building blocks are combined with the combined droplet, and
wherein the order in which the droplets are combined is controlled by the user.

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

Life of this patent

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

Life of this patent

Abstract

Claims43