Cadnano vs. Alternatives: Choosing the Best DNA Origami Design Tool

Beginner’s Guide to Cadnano: Designing DNA Origami Step by StepDNA origami is a technique that uses the predictable base-pairing rules of DNA to fold a long single-stranded scaffold into precise two- and three-dimensional shapes with the help of many short “staple” strands. Cadnano is a free, open-source design tool created specifically to make the process of designing DNA origami structures faster and more intuitive. This guide walks you step by step through the basics of cadnano, from installation and interface overview to creating your first design, exporting staple lists, and preparing for experimental assembly.

What you’ll learn

What cadnano is and when to use it
How cadnano represents DNA structures (helix arrays, routing, and staples)
Step-by-step workflow: project setup → scaffold routing → staple design → export
Tips for common design pitfalls and how to check your design
How to prepare output for ordering oligos and experimental folding

1. Why use cadnano?

Cadnano is built for DNA origami designers and provides a visual, grid-based interface specifically tailored for scaffolded DNA origami. Compared with general-purpose molecular modeling tools, cadnano simplifies the common tasks of:

Laying out parallel bundles of helices on honeycomb or square lattices
Routing a long scaffold strand through the helix lattice
Designing staple strands automatically based on scaffold routing
Exporting staple sequences and visualization files for downstream use

Cadnano is ideal for beginners because it abstracts much of the low-level sequence management while keeping the critical details visible.

2. Installing cadnano

Cadnano has historically been available as a desktop application. As of 2025, several variants exist (cadnano 2.x, cadnano 3.x, and web-based implementations). To install:

Visit the cadnano project page or GitHub repository for the latest release.
Download the package for your OS (Windows, macOS, Linux) or use the web app if available.
Follow installation instructions in the README; some versions require Python or Qt dependencies.

If you encounter dependency issues, consider using the web-based version or a pre-built binary release.

3. Cadnano interface overview

Cadnano’s main workspace represents helices laid out on a lattice (square or honeycomb). Key UI elements:

Toolbar: tools for routing, selecting, deleting, and editing strands
Helix view: grid of parallel helices where you draw scaffold and staples
3D/2D preview: many versions offer a 3D viewer or export options for 3D rendering
Strand inspector: shows sequence, length, and crossover positions
Export menu: outputs staple CSVs, JSON design files, and images

Understanding the helix coordinate system (row, column, base index) helps when making precise edits.

4. Basic concepts: scaffold, staples, crossovers, and nicks

Scaffold: the long single-stranded DNA (often M13mp18, ~7249 nt) that is routed through the design.
Staples: short synthetic oligos that bind segments of the scaffold and fold it into the desired shape. Cadnano auto-generates staples from scaffold routing.
Crossover: points where a strand switches between adjacent helices—critical for mechanical stability and correct folding. Cadnano supports both inter-helix crossovers and intra-helix nicks.
Nick: the end of a staple where it doesn’t continue; staples typically have nicks at their ends.

Staple lengths are usually between ~16–60 nt; typical DNA origami uses staples around 20–40 nt.

5. Choosing a lattice: honeycomb vs square

Honeycomb lattice: helices arranged like hexagons; gives 120° angular geometry and is often used for compact, rounded cross-sections. It has 21-base pair (bp) repeat units per crossover spacing commonly used.
Square lattice: helices arranged on a square grid; simpler to visualize for rectangular beams and sheets, with 10.5 bp per turn considerations.

Choose the lattice based on the desired cross-sectional geometry and the types of crossovers you intend to use.

6. Step-by-step design workflow

6.1 Start a new design

Create a new file and select lattice type and number of helices (rows/columns).
Set scaffold length to match your scaffold (e.g., M13 7249 nt) or a custom scaffold length.

6.2 Lay out helices and perimeter

Use the helix tools to add or remove helices and define the perimeter of your shape.
For 2D shapes, sketch a contour in the helix grid; for 3D bundles, arrange multiple helices into bundles.

6.3 Route the scaffold

Switch to scaffold routing mode. Click to place the scaffold path along contiguous base positions and add crossovers where necessary.
Ensure the scaffold forms one continuous path entering and exiting helices at proper helical turns (respect lattice repeat rules).

Practical tips:

Follow lattice-specific crossover spacing (e.g., every 7 bases on honeycomb for certain offsets, or multiples of full/half-turns on square).
Avoid isolated small loops of scaffold; the scaffold should be a single continuous strand.

6.4 Generate staples

Use the automatic staple-generation tool. Cadnano segments the scaffold into staple lengths and inserts nicks and crossovers to form staples that hybridize to scaffold segments.
Review staple lengths: adjust cut points if staples are too short/long.

6.5 Edit staples manually

Use the strand inspector to merge/split staples, move nicks, and add or remove crossovers.
Consider symmetry: design repeating staple sequences to reduce unique oligo count if desired.
Rename or annotate key staples (e.g., handle/barcode positions).

6.6 Validate your design

Check for unpaired scaffold bases, unexpected breaks, or non-standard crossovers.
Use sequence viewer to ensure staples do not form long unintended complementary regions with each other (reduce unwanted self-assembly).
Run any built-in design checks or plugins (some cadnano forks include verification tools).

7. Exporting sequences and files

After finalizing:

Export staple sequences as CSV/TSV for ordering oligos. Cadnano typically outputs columns: staple ID, sequence, length, position.
Export the scaffold sequence and a mapping file if needed.
Save the cadnano JSON design file (.json) to preserve the project.
Export images (SVG/PNG) or 3D files (if supported) for documentation and visualization.

Order staples with appropriate modifications (e.g., 5’ phosphorylation, fluorescent labels) noted in the order file.

8. Preparing for folding experiments

Assemble staple pool: resuspend oligos, mix equimolar or use a pre-mixed plate; typical final staple:scaffold ratio is 10:1–20:1 for each staple.
Folding buffer: common buffer is 1× TAE-Mg2+ (e.g., 40 mM Tris-acetate, 1 mM EDTA, 12–20 mM MgCl2), but exact Mg2+ depends on design.
Thermal annealing: typical protocol—heat to 80–95°C then slow cool to room temperature over several hours (ramps vary). Many groups use programmable thermocyclers with stepwise cooling (e.g., 80°C → 65°C → 25°C over 12–24 hours).
Purification: remove excess staples via agarose gel extraction, spin columns, or PEG precipitation depending on downstream needs.

Follow institutional lab safety rules and standard molecular biology practices.

9. Common pitfalls and troubleshooting

Short unexpected staples: adjust crossover positions to lengthen staples to practical synthesis limits (~16–60 nt).
Broken scaffold path: ensure the scaffold is continuous; missing bases or gaps cause design failure.
Excessive blunt ends or dangling strands: trim or redesign perimeter to eliminate frayed ends.
Misfolding or aggregates: reduce staple concentration, adjust Mg2+ concentration, optimize annealing ramp.

Testing small design changes incrementally helps isolate problems.

10. Advanced tips

Use symmetry to reduce distinct staples and cost.
Implement sequence optimization: avoid long runs of a single base and minimize complementarity between staples. Some pipelines post-process cadnano output to adjust sequences while preserving binding locations.
Integrate with tools like oxDNA for coarse-grained simulations to predict mechanical properties and folding pathways.
For multi-layer or 3D objects, consider scaffold routing strategies that minimize strain and optimize crossover placements.

11. Resources and next steps

Cadnano documentation and GitHub repo for version-specific instructions and plugins.
Tutorials and example designs from the DNA nanotechnology community (papers, lab websites).
Simulation tools (oxDNA, CanDo) and experimental protocols from established labs.

If you want, I can:

Convert this into a printable PDF or slide deck.
Walk through creating a simple cadnano design (I can provide step-by-step clicks and screenshots).
Generate a sample staple CSV from a simple rectangular design.