Cadnano Workflows: From Concept to Laboratory-Ready FilesCadnano is a widely used open-source software for designing DNA origami and other DNA-based nanostructures. It provides a user-friendly graphical interface for routing scaffold and staple strands on 2D and 3D lattice geometries, making it a cornerstone of many DNA nanotechnology workflows. This article walks through an end-to-end Cadnano workflow: conceptualization, design, validation, export, sequence optimization, and preparing files for synthesis and laboratory assembly. It includes practical tips, common pitfalls, and examples to help move a design from idea to experiment-ready materials.
Overview: What Cadnano Does and Why It Matters
Cadnano lets designers convert geometric concepts into sequence-level DNA designs. It handles scaffold routing, staple placement, crossovers, and produces the staple sequences required to fold a long scaffold strand (commonly M13mp18) into the intended shape. Because DNA nanotechnology relies on precise base-pairing, Cadnano’s role in producing accurate designs is crucial for reproducible, foldable structures.
1) Concept and Design Goals
Before opening Cadnano, define clear design goals:
- Purpose: structural (e.g., tile, box), functional (e.g., dynamic device, aptamer display), or conductive (e.g., wire, scaffold for proteins).
- Size and resolution: determine approximate dimensions and whether a honeycomb (HC) or square lattice (SL) geometry is better. Square lattice tends to produce flatter sheets with right angles; honeycomb lattice produces more compact, triangular packing with better curvature control.
- Scaffold length constraint: the standard M13mp18 scaffold is ~7249 nt; designs must either fit this length or plan for scaffold splitting or using custom scaffolds.
- Mechanical and thermal stability targets: these will influence staple density and crossover placement.
2) Choosing Lattice and Geometry
Cadnano supports two primary lattice types:
- Honeycomb lattice (3-helix per node): good for curved/compact 3D shapes and multi-layer constructs.
- Square lattice (4-helix per node): simpler for planar sheets and right-angled features.
Choose based on shape complexity:
- For flat sheets, arrays, and rectangular prisms, use square lattice.
- For curved surfaces, complicated 3D folding, or compact volumes, use honeycomb lattice.
Tip: sketch the target shape on paper or in a vector program, marking expected cross-sections and layering, before mapping to the lattice.
3) Setting Up Cadnano: Basic Workflow
- New Design: launch Cadnano and create a new document choosing SL or HC and the number of layers (for 3D).
- Scaffold Path: draw the scaffold route. Cadnano’s GUI allows click-and-drag to place scaffold segments and crossovers between adjacent helices. Maintain continuous routing whenever possible to avoid scaffold breaks.
- Staples: Cadnano auto-generates staples based on scaffold routing and specified crossover rules. You can edit staples manually to change lengths, add nicks, or split staples.
- Crossover Management: place crossovers to control rigidity. Standard crossover spacing is often 16 bp (approx. 1.5 turns) on square lattice designs and 21 bp (approx. 2 turns) for honeycomb geometries, but empirical testing may vary.
- Nicks and Staple Ends: ensure staple ends are accessible for purification/labeling; avoid placing many nicks at structurally critical points.
- Labels and Markers: use cadnano’s annotation features to label staple groups, positions for modifications (biotin, dyes), and reference coordinates for assembly instructions.
4) Practical Design Considerations
- Scaffold Length Matching: track cumulative scaffold length as you route. Cadnano reports scaffold mapping so you can stop before exceeding scaffold length.
- Staple Length Distribution: typical staples are 16–48 nt. Avoid very short staples (<8–10 nt) which may not bind stably, and very long staples (>60 nt) that can self-fold or mispair.
- GC Content and Melting Temperatures: while Cadnano does not automatically optimize GC content, aim for even GC distribution across staples. This yields more uniform melting behavior during thermal annealing.
- Repetitive Sequences Avoidance: repetitive or symmetric arrays can promote misfolding. Introduce design asymmetry if needed to reduce kinetic traps.
- Strand Break Placement: position staple nicks away from high-strain crossover hubs.
- Incorporating Functional Sites: for aptamers, protein binding, or chemical modifications, leave single-stranded overhangs or designated staple extensions. Mark these clearly for later sequence modification or ordering.
5) Validation and In-Silico Testing
- Visual Inspection: rotate and inspect your design in Cadnano for unexpected crossings, unconnected scaffold segments, or floating staples.
- Simulations: export to tools like CanDo (for mechanical modeling) or oxDNA (coarse-grained dynamics) to predict folding behavior, flexibility, and possible misfolding states. oxDNA can simulate thermal annealing pathways when run with appropriate parameters.
- Use automated checks: some Cadnano forks or plugins provide automated design rule checking for staple length bounds, crossover spacing, and isolated helices.
6) Exporting Files and Generating Sequences
- Export Formats: Cadnano can export JSON design files (primary editable format) and staple sequence lists. Export to CSV or FASTA for ordering staples from oligo suppliers.
- Sequence Assignment: map scaffold sequence (e.g., M13) to the routed scaffold in Cadnano. The program will compute the complementary staples. Confirm scaffold version matches the sequence you’ll use (M13mp18 vs. other variants).
- Custom Scaffolds: if using a custom scaffold, import its sequence into Cadnano before generating staples.
Example staple export workflow:
- Set the scaffold sequence in Cadnano’s sequence panel.
- Use “Export -> Sequences” to produce a CSV with staple IDs, sequences, and lengths.
- Optionally, run a script to split long staples into synthesizable oligos or to append purification/labeling tails.
7) Sequence Optimization and Ordering
- Oligo Synthesis Constraints: most commercial oligo providers reliably synthesize up to ~60–80 nt oligos; longer strands are possible but more expensive and lower-yield. If staples exceed vendor limits, split them logically in non-critical regions.
- Purification: for critical strands (labelled or long staples), order PAGE purification. For routine staples, standard desalting is usually sufficient.
- Modifications: add 5’ or 3’ modifications (fluorophores, biotin, amine) in the sequence export stage or via the vendor’s order form. Keep modification positions consistent with Cadnano annotations.
- Pooling Strategy: order staples individually or as pooled libraries. For high-throughput or cost-saving, pooled stoichiometric mixes can be used, but be aware of potential unequal concentrations.
8) Preparing Lab-Ready Files and Protocols
- Create an assembly spreadsheet containing:
- Staple IDs, sequences, modification notes, concentrations, and plate positions (if pre-plating).
- Master mix recipes and per-sample staple mix recipes (e.g., equimolar pools).
- Annealing Protocol: provide precise thermal ramp profiles. Typical thermal annealing:
- Heat to 80–95°C for 2–5 minutes (to denature),
- Rapidly cool to 65°C,
- Slow cooling from 65°C to 25°C over 12–48 hours (ramp rates depend on design and buffer).
- Buffer and Ion Conditions: DNA origami often requires Mg2+ (5–20 mM). Optimize MgCl2 concentration empirically: insufficient Mg2+ causes unfolding; excess causes aggregation.
- Concentrations: scaffold typically 5–20 nM in folding reactions; staples in 5–10× molar excess over scaffold (commonly 100–500 nM each staple, depending on protocol).
- Quality Controls: plan gel electrophoresis (AGE), TEM/AFM, and possibly dynamic light scattering or native PAGE to verify folding and monodispersity.
9) Common Problems and Fixes
- Misfolding/Smearing on Gel:
- Check Mg2+ concentration and annealing ramp speed.
- Increase staple excess or separately fold problematic regions using helper strands.
- Aggregation:
- Reduce Mg2+, add mild detergents or crowding agents carefully, or alter staple design to reduce blunt-end stacking.
- Missing Features in TEM/AFM:
- Verify staple presence (mass spec for modified staples), check purification, and confirm folding conditions.
- Incomplete Scaffold Routing:
- Re-open the Cadnano JSON and inspect for breaks; re-route scaffold to be continuous or provide a scaffold staple to bridge gaps.
10) Automation and High-Throughput Considerations
- Scripting Exports: use Cadnano JSON parsers (Python scripts exist) to automatically generate plate maps and vendor-ready CSVs.
- Robotic Liquid Handling: prepare normalized staple plates and use pipetting robots for mixing to reduce human pipetting error and increase reproducibility.
- Version Control: store Cadnano JSON and sequence export files in a version-controlled system (git) to track design iterations and link to experimental results.
11) Case Study Example (Simple 2D Rectangle)
- Goal: 50 nm × 100 nm rectangular tile on square lattice using M13 scaffold.
- Steps:
- Sketch rectangle and map to SL grid; estimate required helices and base pairs per helix.
- Open Cadnano SL template, route scaffold in a raster pattern across helices with crossovers every 16 bp.
- Inspect auto-generated staples; split any >60 nt staples at low-strain locations.
- Assign M13 sequence, export staple CSV, and order staples (standard desalting).
- Folding: scaffold 10 nM, staples 100 nM each, buffer 1× TAE, 12.5 mM MgCl2, anneal 80°C → 65°C → 25°C over 24 h.
- Validate by agarose gel and AFM imaging.
12) Best Practices Checklist
- Choose lattice and scaffold compatible with design goals.
- Keep scaffold continuous and track cumulative length.
- Maintain staple lengths within vendor limits; aim for uniform Tm.
- Annotate modifications and experimental notes in Cadnano file.
- Simulate mechanically (when possible) and run design checks.
- Export clean sequence files and prepare plate maps for ordering.
- Standardize annealing and buffer conditions; document every parameter.
- Use robotics and version control for scale-up and reproducibility.
Final Thoughts
A systematic Cadnano workflow bridges creative design and practical laboratory execution. Careful planning — from lattice choice and scaffold routing to sequence export and annealing protocols — reduces trial-and-error cycles. Combine Cadnano’s intuitive design environment with simulation tools, rigorous checks, and organized lab preparation to move reliably from concept to laboratory-ready DNA origami constructs.
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