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US-12624352-B2 - DNA-responsive hydrogels

US12624352B2US 12624352 B2US12624352 B2US 12624352B2US-12624352-B2

Abstract

Disclosed herein are hydrogels comprising a polynucleotide-based structural component. Methods of altering a property of a hydrogel based on user-defined nucleic acid input sequences are also disclosed. In addition, various applications are described that utilize these hydrogels and methods.

Inventors

  • James J. Collins
  • Helena de Puig Guixe
  • Luis Ruben Soenksen Martinez
  • Max English
  • Raphael Gayet
  • Nicolaas Angenent-Mari
  • Angelo S. Mao
  • Peter Q. NGUYEN

Assignees

  • MASSACHUSETTS INSTITUTE OF TECHNOLOGY
  • PRESIDENT AND FELLOWS OF HARVARD COLLEGE

Dates

Publication Date
20260512
Application Date
20200131

Claims (5)

  1. 1 . A composition comprising: (i) a hydrogel comprising a plurality of structural components, wherein the plurality of structural components comprise polyethylene glycol (PEG) covalently bound to one or more DNA molecules, wherein the one or more DNA molecules comprise a single-stranded region; and (ii) a Cas12a protein, a guide RNA corresponding to the Cas12a protein, and a double-stranded DNA trigger molecule, wherein the guide RNA comprises a spacer region that is at least 85% complementary to a target sequence of the double-stranded DNA trigger molecule, wherein the Cas12a protein is capable of cleaving the single-stranded region of the one or more DNA molecules in the hydrogel following cleavage of the double-stranded DNA trigger molecule.
  2. 2 . The composition of claim 1 , further comprising an additional guide RNA, wherein the additional guide RNA complements a sequence of the one or more DNA molecules in the hydrogel.
  3. 3 . The composition of claim 1 , wherein the spacer region of the guide RNA is at least 90% complementary to the target sequence of the double-stranded DNA trigger molecule.
  4. 4 . The composition of claim 1 , wherein the spacer region of the guide RNA is at least 95% complementary to the target sequence of the double-stranded DNA trigger molecule.
  5. 5 . The composition of claim 1 , wherein the spacer region of the guide RNA is 100% complementary to the target sequence of the double-stranded DNA trigger molecule.

Description

RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application No. 62/823,272, filed Mar. 25, 2019, and U.S. provisional application No. 62/889,234, filed Aug. 20, 2019, the entirety of each of which is incorporated herein by reference. GOVERNMENT SUPPORT This invention was made with Government support under Grant Nos. HDTRA1-15-1-0040 and HDTRA1-15-1-0051 awarded by the Defense Threat Reduction Agency (DTRA). The Government has certain rights in the invention. FIELD Disclosed herein are hydrogels comprising a polynucleotide-based structural component. Methods of altering a property of a hydrogel based on user-defined nucleic acid input sequences are also disclosed. In addition, various applications are described that utilize these hydrogels and methods. INCORPORATION BY REFERENCE The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 31, 2022, is named M065670465US02-SUBSEQ-CRP and is 9,874 bytes in size. BACKGROUND Smart materials, whose physicochemical properties can be modulated in response to external stimuli, are increasingly important for numerous biotechnology applications, including the fabrication of scaffolds for tissue engineering (6), the actuation of microfluidic valves (7), and the sensing of small molecule in diagnostic devices (8, 9). Recent advances in low-cost DNA synthesis have made DNA-based materials increasingly attractive. DNA-responsive hydrogels are well suited to interface with synthetic DNA constructs or naturally-occurring extracellular DNA (10). Current DNA-responsive hydrogels typically rely on toehold strand-displacement or hairpin formation (11, 12), which require high concentrations of DNA triggers for actuation. Adapting these DNA-hydrogels for activation with new trigger sequences usually involves extensive hydrogel redesign and optimization, limiting the programmability of these systems. Thus, the development of sensitive hydrogels capable of generating various types of outputs in response to specific, user-defined nucleic acid input sequences could significantly augment the applicability of these platforms. SUMMARY The use of CRISPR system components for effecting multiscale, programmable changes in the physical properties of macroscopic materials such as hydrogels remains unexplored. As disclosed herein, CRISPR system components were used to control the properties of DNA-based hydrogels. Different hydrogels were engineered to demonstrate a variety of responses for use in therapeutic, diagnostic and sensing applications, including the release of small molecules, nanoparticles (NPs), and live cells, as well as for the modulation of bulk electrical and permeability properties of DNA-hydrogels (FIG. 1). Accordingly, in some aspects, the disclosure relates to methods of altering a property of a hydrogel. In some embodiments, the method comprises contacting the hydrogel with a CRISPR component, wherein: (i) the hydrogel comprises a plurality of structural components, wherein one or more structural component is a nucleic acid molecule component comprising a polynucleotide sequence of at least 5 nucleotides; (ii) the CRISPR component comprises at least one guide RNA; and (iii) the polynucleotide sequence of at least one nucleic acid molecule component in (i) is cleaved when contacted with the CRISPR component of (ii), thereby altering a property of the hydrogel. In some embodiments, two or more structural components are nucleic acid molecule components comprising a polynucleotide sequence of at least 5 nucleotides. In some embodiments, at least two of the two or more nucleic acid molecule components comprise different polynucleotide sequences. In some embodiments, at least one nucleic acid molecule component comprises a double-stranded polynucleotide sequence. In some embodiments, at least one nucleic acid molecule component comprises a single-stranded polynucleotide sequence. In some embodiments, at least one nucleic acid molecule component comprises the polynucleotide sequence of a protospacer adjacent motif (PAM). In some embodiments, at least one nucleic acid molecule component lacks the polynucleotide sequence of a PAM. In some embodiments, a nucleic acid molecule component of the hydrogel further comprises a monomer species selected from the group consisting of an ethylene glycol monomer and an acrylamide monomer. In some embodiments, a nucleic acid molecule component of the hydrogel further comprises polyethylene glycol, polyacrylamide, gelatin, a fibrillar protein, or a combination thereof. In some embodiments, one or more structural component of the hydrogel is selected from the group consisting of a carbon black nanoparticle, a carbon nanocone, a carbon nanofiber, a carbon nanoscroll, a carbon nanothread, a diamondoid, a nanodiamond, a single-walled carbon nanohorn, a carbon nano