Duplexr documentation – how to fold and read a DNA or RNA duplex

Quick start

Five minutes is enough. Paste two sequences, press Fold, read four numbers off the top of the canvas.

Everything below the quick start is reference material — look it up when you need it. Duplexr needs no account and stores nothing: your sequences are sent to the folding engine, folded, and returned.

Paste both strands

Two fields, Strand A and Strand B. Letters A C G T U, up to 300 nt each. The counter under each field shows the length.

Check the temperature

The slider starts at 60 °C, an annealing temperature typical for PCR. Move it to the temperature your experiment actually runs at — it changes the answer.

Fold and read

With Auto-refold on change ticked the fold runs by itself; otherwise press Fold. The energies appear above the canvas, the structure inside it.

Try it with this pair. Strand A GGGAAAUCC, Strand B GGAUUUCCC, 60 °C, default parameters. You should get a fully paired 9 bp duplex, ΔG (MFE) −3.10, ensemble −3.44, binding −3.12 kcal/mol. If your numbers match, everything is working.

Reference 01

The sequence fields

Duplexr folds exactly two strands. Both live in the Setup panel on the left, and both accept only the five nucleotide letters — anything else is rejected before the fold runs.

Alphabet — A C G T U. Lower case and whitespace are cleaned up for you; digits and FASTA headers are not.
Length — 1 to 300 nt per strand. The counter below each field is the check.
T and U are interchangeable — the letter you get back depends on the parameter set, not on what you typed. A DNA set labels everything T, an RNA set labels it U.
Colour — the swatch row picks one of nine palettes per strand. It only changes the drawing, never the prediction. Strong pairs (G·C) take the saturated tone, weak pairs (A·T) the pale one.
The A C G T U buttons insert a base at the caret — handy for testing a point mutation without retyping the sequence.

The Setup panel. Highlighted bases in Strand A are the ones currently selected on the canvas — selection is shared between the two.

Reference 02

Temperature

The slider covers 20 to 90 °C and starts at 60. The folding engine interpolates its thermodynamic tables to whatever you set, so temperature is not a cosmetic setting — it is usually the single most consequential number on the page.

Same duplex, two temperatures	ΔG (MFE)	Ensemble ΔG	ΔG binding
37 °C — body temperature	−7.00	−7.07	−6.47
60 °C — typical annealing	−3.10	−3.44	−3.12

GGGAAAUCC against GGAUUUCCC, DNA · Mathews 1999, all other parameters at their defaults. The structure is identical at both temperatures; only its stability changes — by more than a factor of two. Values in kcal/mol.

Set it before you read anything else. A duplex that looks convincing at 37 °C may barely hold at your annealing temperature, and that is exactly the question most people come here to answer.

Reference 03

Fold parameters

Hidden behind Fold parameters in the Setup panel. The defaults reproduce the invocation OligoPAD uses, so you can leave every one of them alone unless you have a reason not to.

Parameter	Default	What it does
Energy parameters	DNA · Mathews 1999	Which thermodynamic table to fold with. Also decides the alphabet: the two DNA sets fold DNA and label bases `T`; Turner 2004, Turner 1999 and Andronescu 2007 fold RNA and label them `U`.
Dangles	`0 — off`	How unpaired bases next to a helix end contribute. `0` ignores them, `1` lets each dangle on at most one stem, `2` always counts them. `2` is the folding engine’s own default; `0` matches OligoPAD.
No lonely pairs	on	Forbids isolated single base pairs. Real helices stack cooperatively, so a lone pair is usually a numerical artifact. Leave this on.
Forbid G·U pairs	off	Removes the G·U wobble everywhere. Switch on for a strict Watson–Crick DNA·DNA model.
Forbid G·U closure	off	Allows G·U inside helices but not as the closing pair of a loop — a weak position where it often over-predicts.
Min loop size	`3`	Fewest unpaired bases a hairpin may enclose. Three is the physical floor; lower values are diagnostic only.
Layout	Simple radial	Only affects where nucleotides are drawn, never the structure or the energies. NAView untangles heavily branched folds; Simple radial is faster and deterministic.

Reset to defaults at the bottom of the panel puts all seven back.

Reference 04

Reading the results

Three numbers sit above the canvas. They answer different questions, and the difference between them is where the interesting information lives. All are in kcal/mol, and more negative means more stable.

ΔG (MFE)	The minimum free energy: the single most stable structure the two strands can form. This is the structure drawn on the canvas.
Ensemble ΔG	The free energy of all structures the pair can adopt, weighted by how likely each is. When it sits close to the MFE, the molecule has essentially one shape. When it is much lower, the MFE picture is one of many competing folds — trust the drawing less.
ΔG binding	F_AB − F_A − F_B — what the two strands gain by pairing with each other rather than folding alone. This is the number to quote when asking whether two strands will hybridize.
Dot-bracket	The structure as text. A dot is an unpaired base, matching brackets are a pair, and `&` marks the boundary between the two strands. The example above folds to `(((((((((&)))))))))`: nine bases, nine partners, nothing left over.

A large gap between MFE and ensemble is a warning, not an error. It means the prediction is not confident. Narrow it by lowering the temperature, shortening the strands, or checking whether one strand has a strong self-structure competing with the duplex.

Reference 05

The canvas

The structure is drawn as a real graph, not a bitmap — every base stays crisp however far you zoom in.

Action	Mouse and trackpad	Touch
Pan	Drag anywhere on the canvas	Drag with one finger
Zoom	Scroll wheel	Pinch with two fingers
Rotate	Ctrl + scroll wheel	Twist with two fingers
Jump to the caret	Zoom to cursor in the toolbar	Same button

Panning, zooming and rotating change nothing about the prediction — and exports always use the structure’s own bounding box, so how you have the view arranged never affects the saved file.

Reference 06

Display styles

The View menu in the toolbar switches how nucleotides are drawn. Same fold, same energies — only the ink changes. Shown here on the 9 bp demo duplex.

Duplexr

Filled bases with letters. The default, and the most readable at a glance.

Outline

Hollow bases. Lighter on the page — good for print and for long structures.

Letters only

No circles at all. The most compact way to read a sequence off the structure.

Dots

Position without identity. Use it when the shape matters more than the bases.

Backbone only

Topology alone. The clearest view of a large, heavily branched fold.

The dashed lines and the 5′/3′ labels mark where each strand begins and ends — they stay in every style.

Reference 07

Selecting and editing bases

The canvas is not a picture you can only look at. Clicking into it moves the caret in the sequence field, so you can go from „this loop looks wrong“ to a corrected sequence without counting positions by hand.

Click a base to place the caret there. A red arrow marks the insertion point.
Shift-click a second base to select everything between the two. The same bases highlight in the sequence field.
Type, or use the A C G T U buttons, to insert at the caret.
With Auto-refold on change on, the structure re-folds as you edit — you see the consequence of a mutation immediately.

Four bases selected with Shift-click. The halo marks the selection, the red arrow the caret.

Reference 08

Exporting

Four buttons, two formats, two destinations. Copy puts the image on the clipboard for pasting straight into a document; Download writes a file.

SVG	Vector. Scales to any size without losing an edge, and stays editable in Illustrator or Inkscape. The right choice for a paper or a poster.
PNG	Pixels. Use it where SVG is awkward — slides, a lab notebook, a chat message.
PNG settings	The gear button opens two options: a transparent background for placing the structure on a coloured slide, and a resolution — Low 800 px, Mid 1600 px, High 3200 px wide. High is worth it for print.
What gets exported	The complex’ own bounding box, not what happens to be on screen. Selection halos and the caret are removed first, so an export never carries your editing state into the figure.

The watermark. Duplexr is free without an account, and guests get a GNWI mark on the canvas and in every export. Signing in removes it from both. Nothing else changes — same engine, same numbers, same limits.

Reference 09

Limits and messages

If you see	What it means
The Fold button stays disabled	One field is empty, contains a character outside `A C G T U`, or exceeds 300 nt. Check the counters under the fields.
An error above the canvas	The fold request was rejected or the connection dropped. The previous structure stays on screen so you do not lose your place — fix the input and fold again.
A structure with almost no pairs	Physically meaningful: at this temperature these two strands do not hybridize. Lower the temperature to see whether they ever would.
Ensemble far below the MFE	Many competing structures. See Reading the results.

Duplexr folds exactly two strands. Three or more, sequence design, and structure storage are deliberately out of scope — for assay-scale work, see OligoPAD.

Still stuck?

If something here does not match what you see, or Duplexr is missing a feature you need for your work, tell us. It is a prototype, and the roadmap follows what people actually ask for.

Contact GNWI

Credits. Folding is powered by the ViennaRNA Package (Hofacker lab, University of Vienna). Lorenz, R., Bernhart, S. H., Höner zu Siederdissen, C., Tafer, H., Flamm, C., Stadler, P. F., & Hofacker, I. L. (2011). ViennaRNA Package 2.0. Algorithms for Molecular Biology, 6:26.

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