Cryoprotective composition comprising DNA nanostructures and method of use thereof

Cryoprotectant using DNA origami conjugated with antifreeze peptides to inhibit ice crystal growth

This technology is a cryoprotective composition created by attaching linkers to the single-stranded ends of a DNA nanostructure—formed by binding staple nucleic acids to a scaffold nucleic acid folded at specific positions—and conjugating antifreeze peptides to those linkers. This composition can be used for the cryopreservation of cells and tissues, as well as for the freezing of food products.

Existing antifreeze materials have limitations: small-molecule substances require very high concentrations (over 60%), raising concerns about tissue toxicity, while naturally derived antifreeze proteins are difficult to purify and mass-produce, making them prohibitively expensive. DMSO, widely used as a cell cryoprotectant, also requires high concentrations (around 10% of the culture medium) and is cytotoxic, necessitating its rapid removal immediately after thawing.

This technology is designed so that some staple nucleic acids leave non-complementary ends, creating single-stranded protrusions. Using 5–20 nucleotide-long DNA, RNA, or PNA linkers, short peptides consisting of 1–10 alanine or threonine amino acids are precisely positioned. By creating surfaces that interface with ice crystals using two or more helical bundles, or by utilizing twisted or curved helical bundles, the peptide arrangement can be locked onto the ice crystal surface. Applicable to stem cell and germ cell cryopreservation, organ and tissue banking, and the processing of frozen meat and seafood, this technology reduces the required amount of toxic cryoprotectants, improves post-thaw cell viability, and prevents texture degradation in frozen foods.

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Key Features:

• A scaffold nucleic acid folded at predetermined positions to form a plurality of strands, and a plurality of first staple nucleic acids that bind to it to form double strands
• A plurality of second staple nucleic acids where one end is not complementary to the scaffold nucleic acid, forming double strands with single-stranded ends
• A 5 to 20 nucleotide-long linker made of DNA, RNA, or PNA that binds to at least one of the single strands of the nucleic acid structure
• An antifreeze peptide 1 to 10 amino acids in length, containing alanine or threonine, that binds to at least one of the linkers

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This invention was developed with support from the Ministry of Science and ICT for the development and evaluation of DNA-based ice control materials.

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