NUPACK: help

Utilities

Evaluate, display, and annotate the equilibrium properties of an ordered complex. The Utilities page accepts as input either sequence information, structure information, or both, performing diverse functions based on the information provided.

Nucleic acid type:

Select RNA (default) or DNA for strand type. DNA/RNA hybrids are not allowed.

Update:

Update all calculations and graphics.

Temperature:

Enter the temperature (default 37 °C).

Structure:

Specify a secondary structure in dot-parens-plus notation (each unpaired base is represented by a dot, each base pair by matching parentheses, and each nick between strands by a plus). Structures must be connected and free of pseudoknots. For example:

..(((...((((((..+.)))))).((((....)))))))

yields:

MFE:

Use the MFE structure as the specified structure.
To Design:

Export structure information to the Design page, carrying available sequence information as sequence constraints for the redesign. This is useful when re-dimensioning duplex and loop lengths in a target secondary structure while retaining sequence information for a portion of the design.

Sequence:

Enter strand sequences with nicks betweem strands denoted “+”. T's or U's are acceptable for both RNA and DNA and will be appropriately converted. Each base can be any of the standard nucleic acid codes (depicted below for RNA; T replaces U for DNA).

N	A,C,G,U
R	A,G
Y	C,U
M	A,C
K	G,U
S	C,G
W	A,U
V	A,C,G
H	A,C,U
B	C,G,U
D	A,G,U

Cursor:

Position of the cursor within the ordered complex.
To Analysis:

Export sequence information to the Analysis page (e.g., to check for the formation of unintended ordered complexes in the context of a dilute solution).

Specified secondary structure:

Depiction of the specified secondary structure in any of a variety of formats.

Format:

Depict helicity:

Depict with ideal helical geometry (A-form helices for RNA, B-form helices for DNA).
Base Style:

Depict bases as either circles or tick marks.
Base Shading:

Shade each base according to: probability (the probability that it adopts the depicted paired or unpaired state at equilibrium), identity (A = green, U/T = red, G = black, C = blue, all others = gray), or none (all bases are black).
Annotation:

Annotate the drawing with any of: base letters, base numbers, top text, bottom text.

Download SVG or PNG:

Download an editable SVG file (without helicity) or a PNG file (with helicity).
Layout:

Edit the secondary structure layout (e.g., to eliminate overlaps).

Pair probability plot:

Click the thumbnail for a larger version. Depicts equilibrium base-pairing probabilities for the ordered complex, treating all strands as distinct. By definition, these data are independent of concentration and of all other ordered complexes in solution. The area and color of each dot scale with the equilibrium probability of the corresponding base pair (probabilities below 0.001 are not depicted). With this convention, the plot is symmetric, with the upper and lower triangles separated by a diagonal line. The area and color of each dot in the column at right scale with the equilibrium probability that the corresponding base is unpaired.

Download SVG:

Download a scalable vector graphics version of the plot that can be edited with standard vector graphics software.

Details:

Sequence properties:

Properties of the specified sequence information.

Free Energy:

Free energy of the ordered complex (-kT log Q).
NUPACK calculates free energies and equilibrium concentrations of ordered complexes as described in Dirks, et al. 2007 (if you plan to use NUPACK free energies to calculate equilibrium concentrations by hand, be sure to see endnote 13 regarding strand association penalties). For example, the following holds at equilibrium for a dilute solution containing strands A and B that can interact to form ordered complex AB:

$Mole fraction equation$

where for each ordered complex i, x_i is the mole fraction, [i] is the concentration (e.g. in units of molar), Δ G_i is the free energy as reported by NUPACK, and ρ_H₂O (≈ 55.14 mol/L at 37.0 °C) is the concentration of water.
Nucleotides:

The number and percentage of nucleotides that are A,C,G,U/T or “?” (corresponding to all other nucleic acid codes).

Structure/sequence properties:

Properties of the specified structure and sequence information.

Free energy:

Free energy of the specified secondary structure.
Ensemble defect:

The average number of nucleotides that are incorrectly paired at equilibrium relative to the specified secondary structure, evaluated over the Boltzmann-weighted ensemble of (unpseudoknotted) secondary structures (0 is best, N is worst, for a strand with N bases).
Normalized ensemble defect:

The average percentage of nucleotides that are incorrectly paired at equilibrium relative to the specified secondary structure, evaluated over the Boltzmann-weighted ensemble of (unpseudoknotted) secondary structures (0% is best, 100% is worst).

Advanced options:

Select among available energy models, specify dangle treatments, and specify salt concentrations.

Energy parameters:

For RNA, there are two parameter sets: (Serra and Turner, 1995; default) and (Mathews et al., 1999; valid only at 37 °C). For DNA, there is one parameter set: (SantaLucia 1998).
Dangle treatment:

  None: no dangle energies are considered.
  Some (default): a dangle energy is incorporated for each unpaired base flanking a duplex (a base flanking two duplexes contributes only the minimum of the two possible dangle energies).
  All: a dangle energy is incorporated for each base flanking a duplex regardless of whether it is paired.
Salt concentrations:

For RNA, the salt concentrations are 1.0 M Na⁺ and 0.0 M Mg⁺⁺. For DNA, the user-defined salt concentrations can be set in the ranges 0.05 – 1.1 M Na⁺ (SantaLucia and Hicks, 2004; default 1.0 M) and 0.0 – 0.2 M Mg⁺⁺ (Koehler and Peyret, 2005; default 0.0 M).

Download job:

Download all data and plots for the utilities job as a single compressed file.