Freiburg RNA Tools
SECISDesign - Help
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Introduction

SECISDesign is a server for the design of SECIS-elements within the coding sequence of an mRNA with both structure and sequence constraints. This will trigger the insertion of a selenocystein at a preceding STOP codon. Furthermore, a certain similarity to the original protein sequence is kept. It can be used e.g. for recombinant expression of selenoproteins in E. coli.

A SECIS-element (SEC Insertion Sequence) is an mRNA motif with both structural and sequential constraints, that is required for the insertion of selenocysteine into a protein. Selenocysteine (Sec) is the rare 21st amino acid and is incorporated in a particular class of proteins, called selenoproteins. Selenocysteine is encoded by the UGA-codon, which is usually a STOP-codon. It has been shown that, in the case of selenocysteine, termination of translation is inhibited in the presence of a specific mRNA sequence in the 3'-region after the UGA-codon that forms a hairpin-like structure (the SECIS-element).



Selenoproteins have gained much interest, since they are of fundamental importance to human health and an essential component of several major metabolic pathways, such as antioxidant defence systems, the thyroid hormone metabolism, and the immune function. For this reason, there is an enormous interest in the catalytic properties of selenoproteins, especially since a selenoprotein has greatly enhanced enzymatic activity compared to its cysteine homologue.

Note: SECISDesign is not maintained anymore.

When using SECISDesign please cite :

Results are computed with SECISDesign version 1.0 (2009-10-13) using Turner99 energies

Overview

The following parameters are used to control the execution of SECISDesign

Furthermore, additional information is available

Original Protein Sequence

?  Protein sequence

Your sequence of amino acids of the protein in single letter code in which you wish to insert the selenocysteine. Indicate a stop by '#'.
The parameter constraints are: Only the IUPAC alphabet ACDEFGHIKLMNPQRSTVWY# (all capital) is allowed for specification. String length has to be in range (5,300). Maximally 1 line is allowed.

SECIS Design Constraints

?  Position of Selenocystein in Protein

Here, you can choose the position within your amino acid sequence where you wish to insert the selenocysteine.
The parameter constraints are: Input value has to be parsable as a Integer. The value must be greater than or equal to 1 and must be smaller than or equal to 300. Has to be within protein length range.

?  Amino Acids to Conserve

Please insert the number and the amino acid(s) of the conserved position(s) of your given amino acid sequence. Indicate a stop by #.

Examples:
97 F means, that the F is conserved at the 97th position
98 S T means, that the 98th position is conserved to S or T
The parameter constraints are: Has to be in the format 'POSITION AA1 AA2 ...' per line.

?  SECIS-Element

You can choose one of the following six SECIS-elements. A graphical depiction is given blow.

FdhF-std: The natural SECIS-element FdhF of E.coli with all bonds fixed
and some conserved positions.
FdhF-std (optional): The natural SECIS-element FdhF of E.coli with some optional bonds
and some conserved positions. The optional bonds are not fixed, but
it would be of advantage if they form. Nevertheless they are not
necessary to ensure the function of the SECIS-element.
FdhF-insert: The SECIS-element FdhF of E.coli with an additional codon
between the UGA and the actual SECIS-element. All bonds are fixed
and some positions conserved.
FdhF-insert (optional): The SECIS-element FdhF of E.coli with an additional codon
between the UGA and the actual SECIS-element. Some positions are conserved
and some bonds are optional. These are not fixed but of advantage if they form.
FdhF-delete: The SECIS-element FdhF of E.coli lacking the first codon.
All bonds are fixed and some positions conserved.
FdhF-delete (optional): The SECIS-element FdhF of E.coli lacking the first codon.
Some positions are conserved and some bonds are optional. These are not
fixed but of advantage if they form.


FdhF-std FdhF-std FdhF-insert FdhF-insert FdhF-delete FdhF-delete
(optional) (optional) (optional)
FdhF-std FdhF-std+optional FdhF-insert FdhF-insert+optional FdhF-delete FdhF-delete+optional

?  Custom structure

If you have not chosen one of the six given SECIS-elements, you can create your own one. To this end, you have to give the structure in bracket notation and the conserved nucleotides. An example for FdhF-std (optional) is given below.

       [,[[[[[{[[/((.((((....))))))\]]}]]]]]]....
       NNNNNNNNNNNSSUWSCAGGUCUGSWSSNNNNNNNNNNNNNN

The following symbols can be used to define the custom SECIS element.

( ) represents a fixed bond
G U represends a fixed bond for which G-U and U-G pairs are allowed (Please note, that "G" always represents the opening bracket and "U" represents the closing one.)
[ ] represends an optional bond (a bond that is not fixed, but of advantage if it forms, nevertheless it is not necessary to ensure the function of the SECIS-element)
. represents a fixed unbound position
/ \ represents fixed unbound positions in an interior loop
{ } represents optionally unbound positions (of advantage if they do not bind)
, represents optionally unbound positions in a bulge loop

Note: Unbound positions in interior loops (/ \) and optionally unbound positions in bulge loops (,) must be given by special characters.
The parameter constraints are: String length has to be in range (0,300). Maximally 1 line is allowed. Has to be of the alphabet '.,()[]{}GU/\' and its length has to be a multiple of 3 to encode codons.

?  Custom sequence

Sequence constraint using IUPAC RNA nucleotide alphabet {ACGTURYMKWSBDHVN} with wild-card "N". A detailed list of the codes is given below.

IUPAC nucleotide code Base
A Adenine
C Cytosine
G Guanine
U Uracil
R A or G
Y C or U
S G or C
W A or U
K G or U
M A or C
B C or G or U
D A or G or U
H A or C or U
V A or C or G
N any base
The parameter constraints are: Only the IUPAC alphabet 'ACGURYMKWSBDHVN' is allowed for specification. If provided, it has to have the same length as the structure constraint.

Similarity Scoring

?  Similarity

Chose one of the available amino acid similarity matricies to be used for substitution scoring.

?  Insertion Penalty

During the structure optimization, we allow insertions and deletions in the amino acid sequence. This is to avoid contradictions between fixed positions on the amino acid and the nucleotide level. Nevertheless, these insertions and deletions have to be penalized. The values of these penalties are given by the Insertion and Deletion Penalty and related to the similarity scores of PAM 250 and BLOSUM 62.

?  Deletion Penalty

During the structure optimization, we allow insertions and deletions in the amino acid sequence. This is to avoid contradictions between fixed positions on the amino acid and the nucleotide level. Nevertheless, these insertions and deletions have to be penalized. The values of these penalties are given by the Insertion and Deletion Penalty and related to the similarity scores of PAM 250 and BLOSUM 62.

RNAinverse (local search)

?  Search Strategy

The postprocessing is done by a local search method as implemented in RNAinverse. During each of the following strategies, single bases or base pairs are mutated.

Adaptive Walk: During this strategy, a mutation is accepted if it results in a better value of the objective function (e.g. folding probability). Therefore, the adaptive walk is also called fast local search. The search terminates if no mutation can be found which betters the objective function.
Full Local Search: This approach is similar to the adaptive walk. But during the full local search, a mutation will just be accepted if it results in a better value of the objective function AND no other mutation exists that yields a better value. The search terminates if no mutation can be found which betters the objective function.
Stochastic Local Search: The strategy of stochastic local search has a lot in common with the adaptive walk. Whereas the latter often gets stuck in local optima (sequences for which no mutation with a better value of the objective function exists), the stochastic local search is allowed to mutate to worse sequences with a fixed probability p to overcome local optima. A mutation is retained if it results in a better value of the objective function or even if the value is worse with probability p. We set p to 0.1. The search terminates after a fixed number of mutations. We set this number to 500.

?  Objective Function

During the postprocessing (local search), a second objective function is needed (in addition to the similarity) to increase the folding probability of the mRNA sequence.

One of the following functions or combinations of them can be chosen:

mfe: Minimizing the distance of the minimum-free-energy-structure of the designed sequence and the wanted structure.
nc: Minimizing the average number of incorrect paired nucleotides.
pf: Maximizing the probability of the designed sequence folding into the wanted structure.

?  Valid Similarity Fraction

During the postprocessing (local search), a new objective function is considered. Nevertheless the similarity has to be kept clearly in mind.

Therefore you can choose the fraction of the similarity to compare with, which must be kept during the local search (while optimizing the second objective function).

e.g.: Valid Similarity Fraction = 0.9 assures that, during local search, the new similarity is not allowed to be lower than 90% of the compared similarity.

?  Compared Similarity

During the postprocessing (local search), a new objective function is considered. Nevertheless the similarity has to be kept clearly in mind.

Therefore you can choose the similarity which has to be compared to the values arising during the local search.

Either you choose the start similarity (the best possible one, which arises after the first part of the algorithm), or you decide to compare your current value with the previous one.

?  Probabilities of Bases

During the postprocessing (local search), single bases or base pairs are mutated. Here, you can choose whether

Output Description

Here, a typical use case of SECISDesign is given: If you wish to express an eukaryotic selenoprotein in E.coli, this is not directly possible, since there are differences between the mechanisms for inserting selenocysteine in eukaryotic and bacterial proteins. In eukaryotes, the SECIS-element is located in the 3' UTR of the mRNA with a distance from the UGA-codon that varies from 500 to 5300 nucleotides. In bacteria, the situation is quite different. The SECIS-element is located immediately downstream the UGA-codon, which implies that the SECIS-element is in the coding part of the protein.

Thus, we have the following implications. First, an eukaroytic selenoprotein cannot directly be expressed in the E.coli system, since it requires the design of an appropriate SECIS-element directly after the UGA-position. Second, this design always changes the protein sequence. Therefore, one has to make a compromise between changes in the protein sequence and the efficiency of selenocysteine insertion (i.e. the quality of the SECIS-element).

SECISDesign searchs for similar proteins under sequential and structural constraints imposed on the mRNA by the SECIS-elements.

Let's choose the mammalian methionine sulfoxide reductase B (MsrB). If we wish to express it in E.coli, we have to change the coding mRNA such that it can form a SECIS-element and codes for a highly similar amino acid sequence.


Input (see example):

First, the sequence of amino acids of the protein, in which the selenocysteine should be inserted, is put into the Amino Acid Sequence field, e.g.:
 
     MSFCSFFGGEVFQNHFEPGVYVCAKCSYELFSSHSKYAHSSPWPAFTETIHPDSVTKC
     PEKNRPEALKVSCGKCGNGLGHEFLNDGPKRGQSRFCIFSSSLKFVPKGKEAAASQGH
  
Second, you can choose
  • the Position of Selenocysteine: the position (within your given amino acid sequence) on which you wish to insert the selenoysteine (e.g. 95)
  • the SECIS-Element you wish to insert (e.g.: FdhF-std+optional)
  • and optionally some restrictions about positions of your sequence which must not be changed (e.g.: 98 S T, which means, that the 98th position is conserved to S or T). You have to put these information into the field of the Amino Acid Conditions.

Third, the Similarity measurement, e.g.: BLOSUM62, and the values for penalizing insertions and deletions can be chosen.

Finally, you can set some parameters, which will be used during the preprocessing step of SECISDesign.


Results:

mRNA Sequence with Structure and its Probability for the SECIS-Element region after UGA stop codon

Wanted Structure:
[,[[[[[{[[/((.((((....))))))\]]}]]]]]]....
Prob.:
mRNA-Sequence without optimizing the stability of the structure:
AUUUUCUCUUCGCUACCAGGUCUGGUGCCAAAAGGAAAAGAA 
..(((((.((.((.((((....)))))).)).))))).....
.((((((.((.((.((((....)))))).)).))))))....

(0.04)
(0.19)
mRNA-Sequence after optimizing the stability of the structure:
AUCUUCUCGUCGCUACCAGGUCUGGUGCCACAAGGAGCCGAA
..(((((.((.((.((((....)))))).)).))))).....

(0.75)

The "mRNA-Sequence without optimizing the stability of the structure" is the best sequence after the first step of SECISDesign. The structure below is the wanted structure with the folding probability. If this is not the structure of minimum free energy (mfe-structure), this one is given as well. If the mfe-structure is given in green, it is valid as well. But if it is given in red, the mfe-structure is not valid concerning the wanted structure. The user might decide whether this structure of minimum free energy fits his requirements anyway. The folding probability is also given.
The "mRNA-Sequence after optimizing the stability of the structure" is the gained mRNA-sequence after the second step of SECISDesign. The structure and folding probability are given as well. Analogous to the sequence of the first step, the structure of minimum free energy might be given. The color helps the user again to identify whether this structure is valid or not.

Amino Acid Sequence (after SECIS insertion position)

Original Sequence (starting at pos. 96):
 I F S S S L K F V P K G K E 
Without optimizing the stability of the mRNA-structure:
 
 I F S S L P G L V P K G K E 
              
After optimizing the stability of the mRNA-structure:
 
 I F S S L P G L V P Q G A E 
              

The "Original Sequence (starting at pos. 96)" is the considered part of your given amino acid sequence.
The "Amino Acid Sequence without optimizing the stability of the mRNA-structure" is the resulting amino acid sequence after the first part of SECISDesign. Changed positions are given in blue.
The "Amino Acid Sequence after optimizing the stability of the mRNA-structure" is the final amino acid sequence encoded by an mRNA which has a higher probability to fold into the desired structure than the mRNA of the "Amino Acid Sequence without optimizing the stability of the mRNA-structure". Changed position are given in blue as well.

Input Examples

?  Insert SECIS in MsrB

Insert SECIS to mammalian methionine sulfoxide reductase B (MsrB). See help page for an explanation of the output.
The example's result can be directly accessed here

?  Custom SECIS in MsrB

Insert a custom SECIS (which is FdhF-std (optional)) to mammalian methionine sulfoxide reductase B (MsrB). See help page for an explanation of the output.
The example's result can be directly accessed here

List of Changes