Sequence File Format

An RNAstructure sequence file has the following format:

1. Comment lines must be at the beginning of the file and must start with a semicolon. There needs to be at least one comment line in the file.
2. Any number of comments are allowed, but each line before the title must start with a semicolon.
3. After the comments, a title must be given and must be contained on one line. (The title cannot be empty.)
4. The line after the title must start the sequence, entered from 5’ to 3’. The sequence can be on any number of lines and can contain U or T (T's are translated as U's for RNA structure prediction). Spaces are allowed in the sequence. Normal bases must be in capital letters. Lowercase letters indicate a base that will be single-stranded in the predicted structure. "XXXX" can be used in the sequence to indicate that some bases have been left out of the prediction.
5. Finally, the sequence must end in 1.

An example of the format is given here:

; (first line of file) Comments must start with a semicolon.
; There can be any number of comments.
A title must immediately follow on the next line and be on one line.
AAA GCGG UUTGTT UTCUTaaTCTXXXXUCAGG1

FASTA Sequence File Format

Sequences can also be provided to RNAstructure in FASTA format. For FASTA, the first line, a title line, needs to start with >. Subsequent lines should only contain sequence and whitespace, which is ignored. Lowercase nucleotides will be forced single stranded in structure prediction.

An example of the format is:

>Title of Sequence
AAA GCGG UUTGTT UTCUTaaTCTXXXXUCAGG
UUA GCCG UUTGTT UTCUTaaTCTGGG

CT File Format

A CT (Connectivity Table) file contains secondary structure information for a sequence. When entering a structure to calculate the free energy, the following format must be followed:

1. Start of first line: number of bases in the sequence
2. End of first line: title of the structure
3. Each of the following lines provides information about a given base in the sequence. Each base has its own line, with these elements in order:
1. Base number: index n
2. Base (A, C, G, T, U, X)
3. Index n-1
4. Index n+1
5. Number of the base to which n is paired. No pairing is indicated by 0 (zero).
6. Natural numbering. RNAstructure ignores the actual value given in natural numbering, so it is easiest to repeat n here.

The CT file may hold multiple structures for a single sequence. This is done by repeating the format for each structure without any blank lines between structures.

The CT file format is such that files generated by RNAstructure are compatible with mfold/Unafold, available from Michael Zuker.

Constraint File Format

Folding constraints can be specified using plain text files. For multiple entries of a specific type of constraint, entries are each listed on a separate line. When there is no constraint of a type, there are no lines required. Note that all specifiers, followed by "-1" or "-1 -1", are expected by RNAstructure. The file format is as follows:

DS:
XA
-1
SS:
XB
-1
Mod:
XC
-1
Pairs:
XD1 XD2
-1 -1
FMN:
XE
-1
Forbids:
XF1 XF2
-1 -1

• XA: Nucleotides that will be double-stranded
• XB: Nucleotides that will be single-stranded (unpaired)
• XC: Nucleotides accessible to chemical modification
• XD1, XD2: Forced base pairs
• XE: Nucleotides accessible to FMN cleavage
• XF1, XF2: Prohibited base pairs

A sample file with constraints is:

DS:
15
25
76
-1
SS:
17
18
20
35
-1
Mod:
2
15
-1
Pairs:
16 26
-1 -1
FMN:
-1
Forbids:
15 27
-1 -1

SHAPE Reactivity File Format

The file format for SHAPE reactivity comprises two columns. The first column is the nucleotide number, and the second is the reactivity.

Nucleotides for which there is no SHAPE data can either be left out of the file, or the reactivity can be entered as less than -500. Columns are separated by any white space.

Note that there is no header information. Nucleotides 1 through 10 have no reactivity information. Nucleotide 11 has a normalized SHAPE reactivity of 0.042816. Nucleotide 12 has a normalized SHAPE reactivity of 0, which is NOT the same as having no reactivity when using the pseudo-energy constraints.

By default, RNAstructure looks for SHAPE data files to have the file extension SHAPE, but any plain text file can be read. An example of such a file is given below.

9 -999
10 -999
11 0.042816
12 0
13 0.15027
14 0.16201

List File Format

This file contains any number of sequences of any length or nucleic acid, each on its own line. This is used by the program oligoscreen. For example:

CUGAGCCAAG
GGGCUCAACG
GGCGUGAGAAAC