I. Background of DNA Nanotechnology and Functional Modification
DNA nanotechnology leverages base pairing principles (A-T, C-G) to construct self-assembled nanostructures (e.g., nanotubes, origami, hydrogels), while functional modification is key to endowing biological activity, catalytic capacity, or responsive properties. Fmoc-Arg(Pbf)-OH, as a protected arginine derivative, features:
Bifunctional amino and carboxyl groups: Enables chemical conjugation with DNA strand termini (5'-amino or 3'-carboxyl).
Charge property of arginine residue: Protonated to carry positive charges, forming electrostatic interactions with the negatively charged DNA phosphate backbone.
Modifiability of protecting groups: Fmoc (fluorenylmethoxycarbonyl) and Pbf (tert-butoxycarbonyl) can be removed under specific conditions to expose active groups for subsequent functionalization.
II. Modification Strategies of Fmoc-Arg(Pbf)-OH in DNA Self-Assembled Structures
1. DNA-Amino Acid Conjugation Methods
Solid-phase synthesis: Introduces amino or carboxyl groups at the end of DNA solid-phase synthesis, grafting Fmoc-Arg(Pbf)-OH onto DNA strands via carbodiimide (EDC/NHS) coupling reactions, e.g., linking arginine side chains to 5'-amino-modified DNA termini.
Click Chemistry: Utilizes azide-alkyne cycloaddition to conjugate alkyne-premodified DNA with azidated Fmoc-Arg(Pbf)-OH, ensuring high-efficiency and byproduct-free reactions.
2. Localization and Distribution in Self-Assembled Structures
Terminal modification: Connects Fmoc-Arg(Pbf)-OH to DNA duplex ends, forming "charge anchors" that affect self-assembly stability (e.g., neutralizing DNA backbone negative charges to reduce interchain repulsion).
Periodic insertion: Modifies multiple arginine residues at specific sites (e.g., edges or vertices) of DNA origami structures, forming positively charged functional clusters to enhance binding with negatively charged molecules (e.g., siRNA, nanoparticles).
III. Application Scenarios of Functionalized DNA Nanostructures
1. Biomolecular Recognition and Delivery
Protein Binding and Regulation
Arginine residues bind to negatively charged proteins (e.g., enzymes, antibodies) via electrostatic interactions. For example, Fmoc-Arg(Pbf)-OH-modified DNA nanocages encapsulate insulin, improving loading efficiency through interactions between arginine and insulin's surface negative charges.
After removing the Pbf protecting group, arginine guanidino groups synergize with aptamers to recognize target molecules, enhancing the targeting of DNA nanostructures.
Drug Delivery Systems
Arginine-modified DNA self-assemblies (e.g., tetrahedrons, nanospheres) electrostatically adsorb siRNA or oligonucleotide drugs, while arginine's positive charges facilitate membrane penetration (similar to cell-penetrating peptides). For instance, a study using Fmoc-Arg(Pbf)-OH-modified DNA origami to deliver anticancer siRNA showed a 3-fold increase in tumor drug accumulation compared to unmodified groups.
2. Nanointerface Engineering
Nanoparticle Assembly Regulation
Positively charged arginine-modified DNA acts as a "bridge molecule" to connect negatively charged nanoparticles (e.g., gold nanoparticles, quantum dots), controlling nanoparticle arrangement (e.g., linear arrays, 2D lattices) via DNA base pairing.
Deprotection of Fmoc groups exposes fluorenyl rings for π-π stacking with hydrophobic nanomaterials (e.g., carbon nanotubes), enabling DNA-nanomaterial composite assembly.
Surface Functional Coatings
Immobilizing Fmoc-Arg(Pbf)-OH-modified DNA strands on biomaterial surfaces (e.g., medical catheters, chips), arginine's positive charges inhibit bacterial adhesion (by disrupting membrane potential), while DNA structures can further conjugate antimicrobial peptides or antibodies for enhanced multifunctionality.
3. Responsive Intelligent Materials
pH/Temperature-Responsive Release
Fmoc groups are removed under alkaline conditions (pH>12), releasing free arginine to alter DNA nanostructure charge density and trigger structural dissociation (e.g., nanotube depolymerization into single strands) for controlled drug release.
Pbf protecting groups are removed under acidic conditions (e.g., tumor microenvironment pH≈6.5), exposing arginine guanidino groups to enhance DNA-target cell binding and local drug concentration.
Enzyme-Responsive Function Activation
Introducing enzyme cleavage sites (e.g., protease-recognizable peptides) at the Fmoc-Arg(Pbf)-OH-DNA junction releases arginine-modified groups upon exposure to specific enzymes (e.g., tumor-associated proteases), activating subsequent functions of DNA nanostructures (e.g., fluorescence signal activation).
IV. Technical Challenges and Optimization Directions
Modification efficiency and structural stability: Excessive Fmoc-Arg(Pbf)-OH modification may interfere with DNA base pairing. Modification density should be controlled (e.g., 1–2 arginine residues per 100 bases), monitored via circular dichroism (CD) and atomic force microscopy (AFM).
Selective deprotection of protecting groups: Fmoc/Pbf deprotection conditions must be compatible with DNA structures (e.g., avoiding DNA hydrolysis during alkaline Fmoc removal). Photo-responsive protecting groups (e.g., photocleavable Fmoc analogs) can enable spatiotemporally controlled modification.
Biocompatibility and immunogenicity: Arginine-modified DNA nanostructures may trigger immune responses in vivo, mitigated by PEGylation or natural amino acid analogs.
V. Future Development Trends
Integrating with gene editing technologies (e.g., CRISPR-Cas9), using Fmoc-Arg(Pbf)-OH-modified DNA nanostructures as delivery vectors to enhance cellular uptake of gene editing tools.
Developing "DNA-protein hybrid nanomachines" that utilize arginine charge regulation for dynamic nanostructure deformation under electrical or chemical signals.
Optimizing arginine modification sites via computational simulation to predict effects on DNA self-assembly thermodynamics, enabling rational design of functionalized structures.
Conclusion
The functionalized application of Fmoc-Arg(Pbf)-OH in DNA nanotechnology provides new approaches for constructing programmable and bioactive nanodevices, holding promise for breakthroughs in precision medicine, nanocatalysis, and intelligent materials.
