RNAi片段siRNA设计原则

RNAi target selection rules :

2. Targeted regions on the cDNA sequence of a targeted gene should be located 50-100 nt downstream of the start codon (ATG).
4. Search for sequence motif AA(N₁₉ )TT or NA(N₂₁ ), or NAR(N₁₇ )YNN, where N is any nucleotide, R is purine (A, G) and Y is pyrimidine (C, U).
6. Avoid targeting introns, since RNAi only works in the cytoplasm and not within the nucleus.
8. Avoid sequences with > 50% G+C content.
10. Avoid stretches of 4 or more nucleotide repeats.
12. Avoid 5URT and 3UTR, although siRNAs targeting UTRs have successfully induced gene inhibition.
14. Avoid sequences that share a certain degree of homology with other related or unrelated genes.

How to obtain a cDNA sequence for target selection

Before finding a RNAi target on the gene of your interest, first you have to get its mRNA sequence or sequence accession number as some siRNA design tools can take accession number as input. It is recommended to use the gene's RefSeq from NCBI , since the RefSeq represents non-redundant, curated and validated sequences. RefSeq mRNA sequences have unique accession numbers which start with NM or XM, followed by 6 digits. For example, NM_123456 (curated mRNA sequence) or XM_0123456 (model mRNA sequence predicted by genome sequence analysis). There are several ways of querying RefSeq.

2. Search LocusLink by gene name or symbol at http://www.ncbi.nlm.nih.gov/LocusLink/ . Once the locus of your gene is found, scroll down to the "NCBI Reference Sequence (RefSeq)" section and look for mRNA.
4. Search Entrez Gene at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene , and select the right gene of desired organism. Once the page for the gene is shown, scroll down to the "NCBI Reference Sequence (RefSeq)" and look for mRNA.
6. Search Nucleotide database using Entrez query tool at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide and use Entrez Limits settings to restrict your query to the RefSeq database only
   • select "RefSeq" from the "Only from" menu, this restricts the query to the RefSeq collection
   • select "mRNA" from the "Molecule" menu, this restricts the query to mRNA RefSeq records

Homology search
The RNAi targeted region on the mRNA sequence of a gene should not share significant homology with other genes or sequences in the genome, therefore, homology search is essential to minimize off-target effects. Although most siRNA design tools provide BLAST option, some simply use NCBI BLAST tools which sometimes are quite slow. Here are some BLAST tools for homology search.

• NCBI Blast tool: Nucleotide-nucleotide BLAST (blastn) or Search for short, nearly exact matches
• Blat tool on UCSC Genome Website http://genome.ucsc.edu/cgi-bin/hgBlat
• Ensembl Blast http://www.ensembl.org/Multi/blastview

Examples of RNAi target selection

References

1. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001 May 24;411(6836):494-8.
2. Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 2001 Jan 15;15(2):188-200.

更多信息:

• siRNA Design Guidelines (Ambion) - Using siRNA for gene silencing is a rapidly evolving tool in molecular biology. There are several methods for preparing siRNA, such as chemical synthesis, in vitro transcription, siRNA expression vectors, and PCR expression cassettes. Irrespective of which method one uses, the first step in designing a siRNA is to choose the siRNA target site. This guidelines for choosing siRNA target sites are based on both the current literature, and on empirical observations by scientists at Ambion. Using these guidelines, approximately half of all siRNAs yield>50% reduction in target mRNA levels.

• siRNA Design Rules (Protocol Online) - General siRNA design rules and rational siRNA design.

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General Guidelines

2. siRNA targeted sequence is usually 21 nt in length.
4. Avoid regions within 50-100 bp of the start codon and the termination codon
6. Avoid intron regions
8. Avoid stretches of 4 or more bases such as AAAA, CCCC
10. Avoid regions with GC content <30% or > 60%.
12. Avoid repeats and low complex sequence
14. Avoid single nucleotide polymorphism (SNP) sites
16. Perform BLAST homology search to avoid off-target effects on other genes or sequences
18. Always design negative controls by scrambling targeted siRNA sequence. The control RNA should have the same length and nucleotide composition as the siRNA but have at least 4-5 bases mismatched to the siRNA. Make sure the scrambling will not create new homology to other genes.

Tom Tuschl's rules

2. Select targeted region from a given cDNA sequence beginning 50-100 nt downstream of start condon
4. First search for 23-nt sequence motif AA(N₁₉ ). If no suitable sequence is found, then,
6. Search for 23-nt sequence motif NA(N₂₁ ) and convert the 3' end of the sense siRNA to TT
8. Or search for NAR(N₁₇ )YNN
10. Target sequence should have a GC content of around 50%

A = Adenine; T = Thymine; R = Adenine or Guanine (Purines); Y = Thymine or Cytosine (Pyrimidines); N = Any.

Rational siRNA design

By experimentally analyzing the silencing efficiency of 180 siRNAs targeting the mRNA of two genes and correlating it with various sequence features of individual siRNAs, Reynolds et al at Dharmacon, Inc identified eight characteristics associated with siRNA functionality. These characteristics are used by rational siRNA design algorithm to evaluate potential targeted sequences 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.

A sum score of 6 defines the cutoff for selecting siRNAs. All siRNAs scoring higher than 6 are acceptable candidates.

~undefinedTm = 79.8 + 18.undefinedlog₁₀ ([Na⁺ ]) + (58.4 * GC%/100) + (11.8 * (GC%/100)² ) - (820/Length)

For example, the Tm can be calculated as follows for the siRNA UUCUCCAGCUUCUAAAAUA

Tm = 79.8 + 18.undefinedlog₁₀ (0.05) + (58.4 * 31.6/100) + (11.8 * (31.6/100)² ) - (820/19)

Tm = 32.19

There are two siRNA design tools which implement this siRNA design algorithm: one is offered by Dharmacon, Inc; the other is a downloadable Excel template, written by Maurice Ho at http://boz094.ust.hk/RNAi/siRNA.

References

2. Elbashir SM et al. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 411:494-498.
4. Elbahir SM et al. (2001). Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J. 20:6877-6888.
6. Elbashir SM et al. (2002). Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods. 26:199-213.
8. 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.
10. http://www.basic.northwestern.edu/biotools/oligocalc.html
12. Maurice Ho, Rational siRNA Design

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