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If you would like to see the example inputs and outputs of each design template, please go to each template page and click the "submit this design" button; you can browse an example output (the design results page) after the design finished.

Explanation for design inputs

S+ notation of target secondary structures and structure/sequence motifs

Characters for secondary structure specification in S+ notation.

Characters	Description
U u . _	Loop region.
[ ] ( ) < > { }	Duplex. Four types of brackets are available.
?	Any structure is allowed.
*	Any structure is allowed.
+	Forming a base pair.
@	Forming an intramolecular base pair.
^	Forming an intermolecular base pair.
/ &	The boundary between two RNAs.
Alphabets (a-wyzA-WYZ)	Both capital and non-capital letters are available except for "u", "U", "x" and "X". Alphabets are used to specify secondary structure motifs and sequence constraints. Further explanation is described below (Specification of structure motif & sequence constraint).

Each integer number following one of the above characters indicates the number of nucleotide length of the character (e.g. "_3" means the loop region with three nucleotides; "[5" specifies a duplex with a length of 5 bp; NOTE: the downstream nucleotides of the duplex denoted as "]", it is not necessary to specify the length of the duplex, since it is adequate to specify the integer following the upstream one.).

Specification of structure motif & sequence constraint: A set of a structure motif and a sequence constraint is assigned to each alphabet via two lines (e.g., A="(((", and A="aug"); the structure line is mandatory; if necessary, the sequence constraint line follows the structure line. The sequence constraint line can be omitted (if omitted, "N" is assigned to each nucleotide position of the motif). In the secondary structures of the motif specification, only the standard dot-bracket notation, Only .[]()<>{}, and ?*+@^ are allowed (the integer number and alphabets are not allowed in the motif structure specification). wild card for the integer number (this wild card is not available for the brackets), the value of the “x” (or "X") is automatically determined in accordance with the whole length of the sequence(s).

Character	Description
x X	A wild card for the integer number (this wild card is not available for the brackets), the value of the “x” (or "X") is automatically determined in accordance with the whole length of the sequence(s). NOTE: At least one target structure that does not contain "x" (or "X") is needed for each sequence.

Examples of S+ notation:
Examples 1 & 2
Examples 3 & 4
NOTE: The target structure (the first line of the Example 3) written in S+ notation in Figure 3 of the MODENA web server paper (Int. J. Mol. Sci.2021,22, 2720) has an unnecessary closing bracket. The first line of Example 4 should be replaced by the following line.
U2 [4 U3 ] U A & [5 U a ] U [2 U3 ] U

The design parameters common in all design templates
G+C content constraint: MODENA designs the RNA sequences within the range of G+C content specified here. The user specifies the central value and the half of the range. Population size: The user can change the population size of the genetic algorithm in MODENA. NOTE: While a larger population size can give a better design performance, it will take much longer running time. Initial random number: The user can change the initial random number of the evolutionary algorithm in MODENA. Prohibited sequence motifs: The user can specify undesirable sequence motifs. Multiple motifs are prescribed with comma such as CGUCGU,AUUUAA Prohibited polynucleotide tracts: The user can specifies undesirable occurrence of nucleotide tracts (e.g. AAAAAA and CCCC). Prohibited and allowed nucleotide types (PolyA, PolyC, and so on) of the prohibited tracts are also specified using the dropdown menu.

The design parameters common in all design templates

G+C content constraint: MODENA designs the RNA sequences within the range of G+C content specified here. The user specifies the central value and the half of the range.

Population size: The user can change the population size of the genetic algorithm in MODENA. NOTE: While a larger population size can give a better design performance, it will take much longer running time.

Initial random number: The user can change the initial random number of the evolutionary algorithm in MODENA.

Prohibited sequence motifs: The user can specify undesirable sequence motifs. Multiple motifs are prescribed with comma such as CGUCGU,AUUUAA

Prohibited polynucleotide tracts: The user can specifies undesirable occurrence of nucleotide tracts (e.g. AAAAAA and CCCC). Prohibited and allowed nucleotide types (PolyA, PolyC, and so on) of the prohibited tracts are also specified using the dropdown menu.

Designing RNA-RNA interaction switch

The following three lines (in Step 3 of the template) are mandatory.

Line 1: Target joint secondary structure in the bracket notation (target 1), where a "." indicates a loop nucleotide and () or [] indicates a base pair. Pseudoknots within each sequence are not allowed. MODENA explores the sequence space to design the RNA sequences whose optimal structure (predicted by RactIP or RNAcofold -d2) has the structure specified here.

Line 2: Target secondary structures of isolated two RNAs in the bracket notation (we call this target 2). Pseudoknots are not allowed.

Line 3: A sequence constraint in IUPAC nucleotide codes. A,C,G,U,T,M,R,W,S,Y,K,V,H,D,B,N can be used (a T is converted into U). Capital and non-capital letters can be used. The functionally important sequence motifs can be specified here.

Designing interacting two RNAs
The following two lines (in Step 3 of the design template) are mandatory. Line 1: A target secondary structure in the bracket notation, where a "." indicates a loop nucleotide and () or [] indicates a base pair. Pseudoknots within each sequence are not allowed. MODENA explores the sequence space to design the RNA sequences whose optimal structure (predicted by RactIP or RNAcofold -d2) has the structure specified here. Line 2: A sequence constraint in IUPAC nucleotide codes. A,C,G,U,T,M,R,W,S,Y,K,V,H,D,B,N can be used (a T is converted into U). Capital and non-capital letters can be used. The functionally important sequence motifs of aptamer or ribozyme can be specified here.

Designing interacting two RNAs

The following two lines (in Step 3 of the design template) are mandatory.

Line 1: A target secondary structure in the bracket notation, where a "." indicates a loop nucleotide and () or [] indicates a base pair. Pseudoknots within each sequence are not allowed. MODENA explores the sequence space to design the RNA sequences whose optimal structure (predicted by RactIP or RNAcofold -d2) has the structure specified here.

Line 2: A sequence constraint in IUPAC nucleotide codes. A,C,G,U,T,M,R,W,S,Y,K,V,H,D,B,N can be used (a T is converted into U). Capital and non-capital letters can be used. The functionally important sequence motifs of aptamer or ribozyme can be specified here.

Inverse folding of a single RNA
The following two lines (in Step 3 of the design templates) are mandatory. Line 1: A target secondary structure in the bracket notation , where a "." indicates a loop nucleotide and () indicates a base pair. Pseudoknots are not allowed. MODENA explores the sequence space to design the RNA sequences whose optimal structure (predicted by RNAfold -d2) has the structure specified here. Line 2: A sequence constraint in IUPAC nucleotide codes. A,C,G,U,T,M,R,W,S,Y,K,V,H,D,B,N can be used (a T is converted into U). Capital and non-capital letters can be used. The functionally important sequence motifs of aptamer or ribozyme can be specified here.

Inverse folding of a single RNA

The following two lines (in Step 3 of the design templates) are mandatory.

Line 1: A target secondary structure in the bracket notation , where a "." indicates a loop nucleotide and () indicates a base pair. Pseudoknots are not allowed. MODENA explores the sequence space to design the RNA sequences whose optimal structure (predicted by RNAfold -d2) has the structure specified here.

Explanation for designed results
G+C(%): The guanine and cytosine content of the sequence(s) in percent. Str.dist.: The structure distance for the designed sequence. If this value is equal to 0, MODENA successfully designed an RNA sequence which folds into the specified target structures. If some nucleotide positions have secondary structures different from those specified by the submission page, this value becomes > 0. Ene.diff. (kcal/mol): The free energy difference between the target 1 and target 2. Seed (kcal/mol): The free energy value of the designed interaction seed. Prob.: The Boltzmann probability of the first target structure Designed sequences & predicted secondary structures: Designed RNA sequences and their predicted secondary structures predicted are tabulated here. The upper secondary structure corresponds to target 1, and the lower structure corresponds to target 2. The minimum free energy (MFE) predicted by the secondary structure prediction method is mentioned on the right hand side of each secondary structure. For the "Inverse folding of a single RNA", in addition, the energy difference between the MFE and the target structure is also displayed at the right-most side. The other designed features: At the lines below the designed sequence(s) and predicted structure(s), the other designed properties such as the interaction seed for RNA-RNA switch, prohibited motif occurrence in the designed sequence(s) are displayed. Post-processing results. For the designed sequences from the RNA-RNA switch design, this web service predicts the free energy values of homodimers as a post-processing. If this values are high, an undesirable homodimer may form in vitro/in vivo.

Explanation for designed results

G+C(%): The guanine and cytosine content of the sequence(s) in percent.

Str.dist.: The structure distance for the designed sequence. If this value is equal to 0, MODENA successfully designed an RNA sequence which folds into the specified target structures. If some nucleotide positions have secondary structures different from those specified by the submission page, this value becomes > 0.

Ene.diff. (kcal/mol): The free energy difference between the target 1 and target 2.

Seed (kcal/mol): The free energy value of the designed interaction seed.

Prob.: The Boltzmann probability of the first target structure

Designed sequences & predicted secondary structures: Designed RNA sequences and their predicted secondary structures predicted are tabulated here. The upper secondary structure corresponds to target 1, and the lower structure corresponds to target 2. The minimum free energy (MFE) predicted by the secondary structure prediction method is mentioned on the right hand side of each secondary structure. For the "Inverse folding of a single RNA", in addition, the energy difference between the MFE and the target structure is also displayed at the right-most side.

The other designed features: At the lines below the designed sequence(s) and predicted structure(s), the other designed properties such as the interaction seed for RNA-RNA switch, prohibited motif occurrence in the designed sequence(s) are displayed.

Post-processing results. For the designed sequences from the RNA-RNA switch design, this web service predicts the free energy values of homodimers as a post-processing. If this values are high, an undesirable homodimer may form in vitro/in vivo.