Cadnano Tutorial: From Design to Assembly of DNA Tiles

Cadnano Tutorial: From Design to Assembly of DNA Tiles

Overview

Cadnano is an open-source tool for designing DNA origami and DNA tile-based nanostructures. This tutorial covers a complete workflow: setting up a project, designing tiles, routing staples, exporting sequences, and preparing for assembly.

1. Setup

• Install cadnano (latest stable build for your OS).
• Prepare scaffold sequence (commonly M13mp18 or a custom scaffold).
• Create a working directory for design files and outputs.

2. Project structure

• Scaffold: long single-stranded DNA path.
• Helices: parallel double-helical domains where staples bind.
• Staples: short strands that fold the scaffold into the target shape.
• Tiles: repeating units built from helices and crossovers.

3. Designing a DNA tile (step-by-step)

1. Create new design: choose lattice (square or honeycomb) depending on target geometry.
2. Place helices/grid: set number of helices and scaffold start/end positions.
3. Route scaffold: use the scaffold tool to draw a continuous path through helices to form the tile shape.
4. Add crossovers: place crossovers between adjacent helices at integer helical turns (every ~21 bp for square, ~32 bp for honeycomb) to maintain correct twist.
5. Insert staple breaks: define staple strand boundaries so staples are typically 16–60 nt.
6. Check strand lengths: avoid very short (<8 nt) or very long (>70 nt) staples to reduce synthesis/assembly issues.
7. Adjust nicks and overhangs: add nicks for modular assembly and single-stranded overhangs for tile–tile binding if needed.

4. Routing and staple design tips

• Maintain consistent helical phasing: align crossovers to integer turns to prevent strain.
• Use symmetry: design one tile and replicate to reduce routing complexity.
• Minimize isolated scaffold loops: ensure scaffold path is mostly continuous.
• Split long staples: break >40 nt staples into shorter segments where necessary.

5. Exporting sequences and files

• Export staple sequences as CSV or TXT for ordering.
• Save design (.json/.cadnano) for future editing.
• Generate visualization snapshots for lab records.

6. Preparing for assembly

• Order staples with standard desalting or HPLC depending on purity needs.
• Prepare scaffold stock and staple mixes with accurate concentrations.
• Typical folding protocol: mix scaffold (5–10 nM) with 5–10× molar excess of each staple in folding buffer (e.g., 10 mM Tris, 1 mM EDTA, 12.5 mM MgCl2), heat to 80–95°C for 2 min, then slow-cool to 20°C over 12–48 hours or use programmed thermal ramp.
• Optimize Mg2+ concentration and annealing time empirically.

7. Validation and troubleshooting

• Agarose gel: check mobility shifts and monodispersity.
• AFM/TEM: visualize tile shape and assembly.
• Common issues: aggregation (reduce Mg2+), misfolding (check routing/phasing), missing bands (insufficient staple excess or degraded staples).

8. Assembly of tiles into larger arrays

• Design complementary sticky ends or connector staples on tile edges.
• Use controlled stoichiometry and annealing ramps to promote correct tile–tile binding.
• Consider hierarchical assembly: fold tiles separately, then mix and anneal at milder conditions.

9. Practical tips

• Start with simple rectangular tiles before complex shapes.
• Keep a lab notebook with design parameters, scaffold ID, staple concentrations, and thermal profile.
• Use simulation/visualization tools to inspect possible steric clashes.

If you want, I can:

• produce a sample cadnano tile design (assume a standard M13 scaffold),
• generate staple sequences for a simple rectangular tile, or
• provide a step-by-step thermal ramp script for a PCR machine. Which would you like?