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MODULE 3 – Day 3
Design siRNAs to knock down the expression of four
genes that influence how cells respond upon exposure
to DNA damaging agents; ATM, ATR, EXO1 and AAG
Each group will design an siRNA sequence to knock down each of the four human genes listed
below. i.e., every group will design siRNAs for all four sequences. Each gene is shown with its
full name, common abbreviation, mRNA accession number, and the base pair region to be
targeted.
1.) Ataxia telangiectasia mutated (ATM)
NM_000051
bp 8700-8800
2.) Ataxia telangiectasia and Rad3 related (ATR)
NM_001184
bp 4450-4550
3.) 3-alkyladenine-DNA glycosylase (AAG, a.k.a. MPG)
NM_002434
bp 700-800
4.) Exonuclease 1 (EXO1)
NM_003686
bp 1500-1600
The design of the siRNA will be based on Tuschl et al.’s criteria
(http://www.mpibpc.gwdg.de/abteilungen/100/105/sirna.html).
These criteria have been incorporated into a search algorithm that was developed by a company
called Ambion. Ambion’s website contains a search engine that will find multiple candidate
sequences for each gene. You will identify candidate sequences that fall within the given base
pair region for each gene. You will then perform BLAST searches to determine which candidate
sequence has the highest selectivity for your gene of interest. For your interest the home page of
Ambion is shown below.
1) Finding the mRNA sequence of the gene target.
a.
b.
c.
d.
e.
f.
Go to the NCBI homepage: http://www.ncbi.nlm.nih.gov/.
Enter the accession number for your gene of interest.
Make sure “Nucleotide” is selected from the left hand pull-down menu.
Click on the Go button.
Click on the link for your gene of interest.
Display the mRNA sequence in FASTA format by selecting “FASTA” from the
pull-down menu at the upper left next to the display button. Click on the Display
button.
2) Finding candidate siRNA sequences.
a. Go to the Ambion siRNA design tool website:
http://www.ambion.com/techlib/misc/siRNA_finder.html.
b. Copy and paste the mRNA sequence for your gene of interest into the mRNA
sequence box.
i. NOTE: Paste ONLY the FASTA sequence in – the program cannot
recognize numbers and letters other than A, T, G, and C as being wrong.
ii. Paste the ENTIRE mRNA sequence in FASTA format in. Do not only
paste in a limited range of nucleotides. If you do, your results will not
match those of the rest of the class.
c. Select end my siRNAs with TT.
d. Select “all G/C contents” for G/C content maximum.
e. Click on the Submit button.
3) Selecting an siRNA sequence.
a. Only consider the target sequences that fall within the given base pair range for
your gene of interest. (“Position in gene sequence” is listed for each sequence).
b. Eliminate sequences that fall outside the 30-70% G/C content range.
c. BLAST the remaining sequences.
i. Click on the BLAST search link under the target sequence.
ii. In the BLAST window, scroll down to “options for advanced blasting”
and limit the search so that you are only screening Homo Sapiens entries
by selecting “Homo Sapiens [ORGN]” from the dropdown menu.
iii. You do not need to change any of the options in the “format” section.
iv. Click BLAST.
4) Analyzing BLAST results:
a. The BLAST results show “sequences producing significant alignments”. You
want to make sure that all of the sequences showing up as good matches are either
your gene of interest or some derivative of your gene of interest.
i. Find all sequences that have a “Score” of 42 bits, corresponding to
“Identities = 21/21 (100%)”. The scores can be found in the list of
“sequences producing significant alignments”. Scroll down past this list
to the “alignments” section for more detailed information about each
sequence.
ii. For each one, answer the following questions:
1. Is it your gene of interest? *
2. Is it a transcript variant of your gene of interest?
3. Is it a clone, BAC, or contig of your gene of interest? **
iii. If you answered NO to ALL of the questions above then the target
sequence you just BLASTed has significant homology to another gene and
cannot be used.
iv. If you answered YES to at least ONE of the above questions then the
sequence is a possible candidate for use.
v. HINTS:
* The same gene may go by different names. Look at the original
NCBI summary of your gene of interest from step 1 above. Under
“features: gene” some synonyms for your gene may be listed.
* If you think it might be the same gene but cannot tell, you can do an
alignment. Go to: http://www2.igh.cnrs.fr/bin/align-guess.cgi. Paste
the FASTA sequences of your gene of interest (the same mRNA
sequence that you used for your search on ambion’s website) and the
suspected match into the first and second sequence fields and click
“perform align”. Check for a high identity score (>90%).
** To determine if the BAC or clone is from the correct chromosome,
you can check the genomic location of your gene. Look at the original
NCBI summary of your gene of interest from step 1 above. Click on
“Link” in the upper right hand corner, and select “Map Viewer”. The
chromosome on which your gene of interest is located should be
highlighted in the list of chromosomes at the top. The chromosome
from which the BAC or clone was obtained is usually listed in the
BAC or clone description, OR use map viewer, as above.
b. After completing the above steps, it is likely that you will still have several
candidate target sequences. You will decide which one is best by looking at the
next highest matches in the BLAST search for each target sequence.
i. Eliminate sequences that show a 20/20 or 19/19 identity with a gene that is
not your gene of interest. Use the three criteria in (4aii) to determine this.
ii. Select the target sequence with the lowest homology to any gene that is
not your gene of interest. Compare the next highest matches that are not
21/21, 100% identity matches. Are these matches to your gene of interest?
If not, we would like for our target siRNA sequence to have LOW
homology to these other genes. For example, siRNA target sequence #1
shows 18/18 (score=36) as the next highest match to a gene that is not our
gene of interest. siRNA target sequence #2 shows 17/17 (score=34) as the
next highest match to a gene that is not our gene of interest. siRNA #2
would therefore be a better match than siRNA #1, since it has LOWER
homology to other genes. Likewise, a target siRNA sequence with 16/17
identity match to a different gene is preferred over one with 17/17 identity
match to a different gene, because it has lower homology to other genes.
iii. If you are left with more than one target sequence after eliminating all
those with greater homology to other genes, select the target sequence that
gives the least number of matches to other genes at the next highest match
level after 21/21 (e.g. 15/15).