Team:SYSU-Software/Models2

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Algorithm——SiRNA designer& Riboswitch designer

siRNA designer:

Tom Tuschl's rule (for cDNA)

1. Select targeted region from a given cDNA sequence beginning 50-100 nt downstream of start codon

2. Look for sequence motif AA+N19+TT first. If there is no suitable sequence look 23-nt sequence motif NA+N21 and convert the 3' end of the sense siRNA to TT

3. Or search for NAR+N17+YNN

PS: Target sequence should have a GC content of around 50%

R means A/G, and Y means T/C.

Rational siRNA design(for mRNA)

Evaluate potential candidates and assign scores to them, sequences with higher scores will have higher chance of success in RNAi.

The table below lists the 8 criteria and the methods of score assignment.

Criteria	Description	Score
Criteria	Description	Yes	No
1	Moderate to low (30%-52%) GC Content	1 point
2	At least 3 A/Us at positions 15-19 (sense)	1 point /per A or U
3	Melting temperature-Tm*<20℃	1 point
4	A at position 19 (sense)	1 point
5	A at position 3 (sense)	1 point
6	U at position 10 (sense)	1 point
7	No G/C at position 19 (sense)		-1 point
8	No G at position 13 (sense)		-1 point

The "anti-sense" strand is the siRNA strand that is complementary to the target mRNA and that will be binding to the mRNA.

The melting temperature’ of a siRNA candidate can be calculated by the formula showed below:

wA, xT, yG, zC are separately the number of A, T, G, C in a siRNA candidate.

All siRNA candidates scored higher than 6 are acceptable.

Riboswitch designer:

The designer is based on a special promoter working in eukaryotes - internal ribosome entry site (IRES). We design the upstream sequence that can bind to IRES part so the second structure of IRES can be transformed by the ligand.

Up regulated:

Target sequence is assembled by five parts: MS-SL, aaIRES (anti-anti-IRES), aptamer, aIRES (anti-IRES), IRES.

The IRES, aIRES, aaIRES parts are settled, we get aptamer sequence according to customer’s quests from aptamer database and the MS-SL is paired to the combination part of aaIRES and aptamer. Then adjust the MS-SL part’s length to fit it’s free energy to -11.7 kcal/mol.

Down regulated:

The differences from down regulated to up regulated riboswitch are the order between aptamer and aaIRES, the sequence of MS part. The new structure is showed below:

We set MS to “CCUCU” under experimental data and the aptamer still comes from aptamer database.

Algorithm about free energy calculation comes from Turner’s team:

Hairpin loops:

In this equation, n (5) is the number of nucleotides in loop, the terminal mismatch parameter is the sequence-dependent term for the first mismatch stacking on the terminal base pair.

Watson-Crick Helices:

Specific data can be found on web site:

http://rna.urmc.rochester.edu/NNDB/turner04/index.html

References:

1.Elbashir SM et al. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 411:494-498.

2.Elbahir SM et al. (2001). Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J. 20:6877-6888.

3.Elbashir SM et al. (2002). Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods. 26:199-213.

4.Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, Khvorova A. Rational siRNA design for RNA interference. Nat Biotechnol. 2004 Mar;22(3):326-30.

5.Ogawa, A. (2011). Rational design of artificial riboswitches based on ligand-dependent modulation of internal ribosome entry in wheat germ extract and their applications as label-free biosensors. RNA (New York, N.Y.), 17(3), 478-88. doi:10.1261/rna.2433111

6.Ogawa, A. (2012). Rational construction of eukaryotic OFF-riboswitches that downregulate internal ribosome entry site-mediated translation in response to their ligands. Bioorganic & medicinal chemistry letters, 22(4), 1639-42. Elsevier Ltd. doi:10.1016/j.bmcl.2011.12.118

7.Turner, D. H. & Mathews, D. H. (2009). NNDB: The nearest neighbor parameter database for predicting stability of nucleic acid secondary structure. Nucleic Acids Research. 38, D280-D282.

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-         <h1 id="doctitle" class="pagetitle"><span lang="EN-US">Algorithm——iRNA
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 		designer</span>&amp; <span lang="EN-US">Riboswitch designer</span></h1>
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w:h-ansi=&quot;Cambria Math&quot;/&gt;&lt;wx:font wx:val=&quot;Cambria Math&quot;/&gt;&lt;w:sz w:val=&quot;28&quot;/&gt;&lt;w:sz-cs w:val=&quot;28&quot;/&gt;&lt;/w:rPr&gt;&lt;m:t&gt;]&lt;/m:t&gt;&lt;/m:r&gt;&lt;/m:e&gt;&lt;/m:nary&gt;&lt;/m:oMath&gt;&lt;/m:oMathPara&gt;&lt;/w:p&gt;&lt;w:sectPr wsp:rsidR=&quot;00000000&quot; wsp:rsidRPr=&quot;006447FA&quot;&gt;&lt;w:pgSz w:w=&quot;12240&quot; w:h=&quot;15840&quot;/&gt;&lt;w:pgMar w:top=&quot;1440&quot; w:right=&quot;1800&quot; w:bottom=&quot;1440&quot; w:left=&quot;1800&quot; w:header=&quot;720&quot; w:footer=&quot;720&quot; w:gutter=&quot;0&quot;/&gt;&lt;w:cols w:space=&quot;720&quot;/&gt;&lt;/w:sectPr&gt;&lt;/wx:sect&gt;&lt;/w:body&gt;&lt;/w:wordDocument&gt;">
@@ Line 481: / Line 480: @@
 				<ul>
 				<li>
-						<a href="https://2012.igem.org/Team:SYSU-Software/">Main Page</a>
+						<a href="https://2012.igem.org/Team:SYSU-Software">Main Page</a>
 					</li>
 					<li>
@@ Line 487: / Line 486: @@
 					</li>
 					<li>
-						<a href="/documentation-install">Video</a>
+						<a href="https://2012.igem.org/Team:SYSU-Software/SecondEdition">Second Edition</a>
+					</li>
+					<li>
+						<a href="https://2012.igem.org/Team:SYSU-Software/Accomplishment">Accomplishment</a>
 					</li>
@@ Line 499: / Line 501: @@
 				<ul>
 					<li>
-						<a href="/
+						<a href="https://2012.igem.org/Team:SYSU-Software/Download">Source Code</a>
-documentation-user-guide-main-reminderfox-window">Source Code</a>
-					</li>
-					<li>
-						<a href="/documentation-user-guide-options-and-preferences">Algorithm-FBA</a>
 					</li>
 					<li>
@@ Line 513: / Line 511: @@
 					<li>
-						<a href="/documentation-user-guide-notifications">Email</a>
+						<a href="https://2012.igem.org/Team:SYSU-Software/Contact">Email</a>
 					</li>
@@ Line 526: / Line 524: @@
 				<ul>
 					<li>
-						<a href="">Questions</a>
+						<a href="https://2012.igem.org/Team:SYSU-Software/Faq">Questions</a>
 					</li>
 					<li>
-						<a href="">Troubleshooting</a>
+						<a href="https://2012.igem.org/Team:SYSU-Software/Faq">Troubleshooting</a>
 					</li>
 					<li>
-						<a href="">Uninstalling</a>
+						<a href="https://2012.igem.org/Team:SYSU-Software/Uninstalling">Uninstalling</a>
 					</li>
 					<li>
 						<a href="">Contact Us</a>
-					</li>
-				</ul>
-			</div>
-		</div>
-		<div>
-			<h5>E-mail Integration</h5>
-			<div>
-				<ul>
-					<li>
-						<a href="/documentation-email-messages">Messages</a>
-					</li>
-					<li>
-						<a href="/documentation-email-invitations">Invitations</a>
 					</li>
 				</ul>
@@ Line 592: / Line 576: @@
    <div class="wrapper clear">
      <div id="footer-about" class="footer-column">
-       <h2>About</h2>
+       <h2>&nbsp;&nbsp; About</h2>
                    <ul>
 <li>
-<a href="/developers">Developers</a>
+<a href="https://2012.igem.org/Team:SYSU-Software/Team">Developers</a>
 </li>
 <li>
-<a href="/help/">Contact Us</a>
+<a href="https://2012.igem.org/Team:SYSU-Software/Contact">Contact Us</a>
 </li>
 </ul>
              </div>
      <div id="footer-flickr" class="footer-column">
-                               <h2>Links</h2>
+                               <h2>&nbsp;&nbsp; Links</h2>
                <ul>
                  <li><a href="http://www.sysu.edu.cn">SUN YAT-SEN University</a></li>
-                 <li><a href="http://lifescience.sysu.edu.cn/main/default/index.aspx">SYSU LifeSCience</a></li>
+                 <li><a href="http://lifescience.sysu.edu.cn/main/default/index.aspx">SYSU LifeScience</a></li>
                </ul>
                      </div>
      <div id="footer-search" class="footer-column">
-       <h2>Facebook</h2>
+       <h2>&nbsp;&nbsp;&nbsp; Facebook</h2>
      </div>