Sequence-directed covalent protein-RNA linkages in a single step using engineered HUH-tags.

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Replication-initiating HUH-endonucleases (reps) are enzymes crucial for DNA replication.
• These enzymes, termed 'HUH-tags', are utilized in biotechnology for sequence-specific protein-ssDNA bioconjugation.
• Existing HUH-tags primarily interact with single-stranded DNA (ssDNA).

Purpose of the Study:

• To engineer novel HUH-tag derivatives with enhanced reactivity towards RNA substrates.
• To characterize the in vitro performance of these engineered enzymes.
• To establish a new class of RNA-reactive HUH-tags for biotechnological applications.

Main Methods:

• Laboratory evolution of HUH-tag derivatives (E1 and E2) from PCV2.
• In vitro characterization of enzyme activity on ssDNA and RNA substrates.
• Benchmarking against existing engineered variants (e.g., rHUH) and sequence specificity analysis.

Main Results:

• Engineered HUH-tags (E1 and E2) demonstrate robust covalent bond formation with RNA.
• Directed evolution significantly enhanced promiscuous rep-RNA interaction.
• Engineered enzymes exhibit increased activity on both ssDNA and ssRNA.
• Sequence specificity was analyzed across various metal ion concentrations.

Conclusions:

• A new class of RNA-reactive HUH-tags has been established.
• These engineered enzymes expand the biochemical capabilities of the HUH-tag family.
• The technology offers a promising platform for site-specific protein-RNA covalent bioconjugation.
• Potential applications in biotechnology and molecular engineering are significantly broadened.