Universal DNA Assembly via Methylation-Protected Type IIS Sites

The patent introduces an in vivo methylation system as an intermediary between the restriction enzyme and internal restriction sites. The methylase enzyme modifies internal type IIS restriction sites by methylation, preventing the restriction enzyme from cutting at these sites. This allows DNA parts to retain their internal restriction sites without interference, eliminating the need for their removal while maintaining controlled assembly at designated sites.

The patent changes the chemical state of restriction sites through methylation. By introducing methyl groups at specific positions in the DNA sequence, the recognition sites for type IIS restriction enzymes are modified. This parameter change (methylation status) selectively protects internal sites from cleavage while allowing assembly sites to remain accessible, thereby increasing sequence design freedom without compromising assembly precision.

The patent makes a single type IIS restriction enzyme system universal for all assembly stages by combining it with in vivo methylation. The same restriction enzyme can be used repeatedly for different assembly steps because the methylation status of restriction sites can be dynamically controlled. This eliminates the need to switch between multiple different restriction enzymes, simplifying the overall assembly process while enabling hierarchical assembly of large DNA constructs.

The patent introduces dynamic control over restriction site accessibility through regulated methylation. The methylation status of restriction sites can be changed on demand using inducible promoters or conditional expression systems. This dynamic switching allows the same restriction enzyme to be used multiple times with different specificity profiles, enabling complex hierarchical assembly without increasing procedural complexity.

The patent merges the methylation protection step with the restriction digestion and ligation steps into a single in vivo reaction. The DNA parts are transformed into host cells that express the methylase, allowing methylation to occur naturally within the cell. The restriction enzyme is then added to digest unprotected sites while methylated sites remain intact. This merging of steps eliminates separate in vitro methylation reactions and reduces overall system complexity.

The patent enables the DNA assembly system to perform its own methylation protection functionally within living cells. The host cell's metabolic machinery and protein expression systems are utilized to produce and activate the methylase enzyme automatically. This self-service approach eliminates the need for external methylation reagents or separate preparation steps, reducing system complexity while maintaining sequence design freedom.