## Using the mfold Web Server With Constraints

- 1.
**Forcing**a string of consecutive bases to pair. - This is an interesting constraint. The partners for these bases are chosen by the program. To enter this type of constraint, type:
F i 0 k

on a line in the constraint box. The letter "F" means "Force"; the first number, "i", is the first base that must pair; the second number, "0" (zero), is not used, and the third number, "k", is the number of consecutive bases that must pair, beginning with the i

^{th}. For example,

F 23 0 5

would cause bases 23, 24, 25, 26 and 27 to pair. - 2.
**Forcing**a string of consecutive base pairs. - This is the same thing as forcing a helix to form. The helix is designated by its
*external closing base pair*. That is, by the base pair*i.j*where*i*is as small as possible and*j*is as large as possible. Thus the*k*consecutive base pairs beginning with*i*are:^{.}j*i*. To enter this type of constraint, type^{.}j, i+1^{.}j-1, i+2^{.}j-2, ..., i+k-1^{.}j-k+1F i j k

on a line in the constraint box. Note how 3 integers are necessary in this case. For example,

F 2 110 3

would force the following base pairs: 2^{.}110, 3^{.}109, 4^{.}108.**These base pairs must be able to form**Be aware also that mfold filters out isolated base pairs. In the example on the right, the G^{.}C base pair is isolated. - 3.
**Prohibiting**a string of consecutive bases from pairing. -
To enter this simple constraint, type:
P i 0 k

on a line in the constraint box. This command is similar to 1. above, except that the designated bases are prevented from pairing.

- 4.
**Prohibiting**a string of consecutive base pairs -
To enter this simple constraint, type:
P i j k

on a line in the constraint box. This command is similar to 2. above, except that the designated base pairs are prevented from forming.

- 5.
**Prohibiting**1 part of a sequence from pairing with another - If your molecule has
*N*bases, and*1 <= i <= j <= N*&*1 <= k <= l <= N*, are 2 segments, then these segments can be prevented from pairing with each other by enteringP i-j k-l

on 1 line in the constraint box. For example, in a molecule with 120 bases, the constraint

P 1-10 11-120

will prevent the first 10 bases from pairing in any way with the last 10 bases. The constraint

P i-j i-j

will prevent the segment*i-j*from pairing with itself. - 6.
**Annotated bases** - The software recognizes
**A**,**C**,**G**,**U**and**T**. In RNA folding, a 'T' will be converted to a 'U';*vice versa*for DNA folding. In addition**B**,**D**,**H**and**V**are recognized as**A**,**C**,**G**and**U/T**, respectively. Bases marked in this way are regarded as*susceptible to nuclease cleavage*. They are allowed to pair only if their 3' neighbor is unpaired. This is an old feature of mfold.The mfold web server also recognizes

**W**,**X**,**Y**and**Z**as**A**,**C**,**G**and**U/T**, respectively. These bases are regarded as "modified" and are allowed to pair only at the ends of helices.**Please note that the commonly used ambiguous codes shown in the table below**__ARE NOT SUPPORTED__by the mfold server.Unsupported ambiguous codes for DNA: A,G

RC,T

YA,T

WC,G

SA,C

MG,T

KC,G,T

BA,G,T

DA,C,T

HA,C,G

VA,C,G,T

N