Yuan et al, 05) Thus, the seed region first identifies the target mRNAs, and subsequently form perfect basepairing with intended target mRNA and induce RNAi by Ago2Our results confirm previous reports that strength of basepairing in the siRNA seed region is the primary factor determining the efficiency of offtarget silencing However, the degree of downregulation of offtarget transcripts with shared seed sequence is not necessarily similar, suggesting that there are additional auxiliary factors that influence the silencing potential siRNA recognition of the target mRNA is conferred by the "seed region", a six nucleotide stretch corresponding to positions 27 on the antisense siRNA strand After the siRNA seed region anneals, the catalytic RNase H domain of Argonaute then subjects perfectly complementary mRNA sequences 10 nucleotides from the 5' end of the incorporated siRNA
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Seed region of sirna- The stability between seed region and target mRNA is a determinant of the efficacy of siRNA offtarget effects 33 Thus, the high stability of The various modifications at the 2′position of the pentose sugar of siRNA are not required for RNAi ,21 and target recognition 15 DNA is modified with hydrogen at C2′, and the 5′ onethird of each RNA strand is capable of replacement with DNA without substantial loss of RNAi activity 15 Consistent with our previous report, 15 DNA replacement at the seed region of the
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Previous investigations and GenScript experiment data show that siRNA "off target" transcript silencing mediated by seed region sequence is widespreed "seed region" of siRNA is analogous to seed region of microRNA GenScript resourchers found that size of seed region plays a major part in off target predictionPairing between the hexamer seed region of a small interfering RNA (siRNA) guide strand (nucleotides 27) and complementary sequences in the 3' UTR of mature transcripts has been implicated as an important element in offtarget gene regulation and false positive phenotypes To better understand the association between seed sequences and offtarget profiles we performedSeed region enrichment was confirmed in offtarget transcripts modulated by siRNA targeting the glucocorticoid receptor To investigate how these sites contribute to offtarget recognition of F71, we employed CXCL5 transcript as model system because it contains five F71 seed sequence motifs with single base mismatches We show by transient transfection of reporter gene
In the siRNA seed region makes it possible to overcome the sequence limitations of siRNA for reducing seeddependent offtarget effects through different molecular mechanisms reduction of basepairing stability by DNA modification, and steric hindrance by OMe modification SiRNA Modifications Avoid OffTarget Effect 19 Several recent studies suggest that the main source of siRNAmediated offtarget gene silencing could be the complementation between the 'seed' region of siRNAs (nucleotides 2–8 of theSeed region The chemical modifications of deoxyribonucleic acid (DNA), PS, and DNA−PS were introduced into all of the seven nucleotides of the seed region of the siRNA guide strand (Figure 2b) In addition, 3 (positions 4−6), 5 (positions 3−7),
As the offtarget effects were identified based on perfect complementarity to the seed region (positions 2–8) and both terminal nucleotides of siRNA are known to be incorporated into Ago protein (positions 1 and 21), the potential basepairing between siRNA and offtarget mRNA was determined via positions 9– (ie 3' region) The number of GC and AU basepairsSelection of Chemical Modifications in the siRNA Seed Region That Repress OffTarget Effect RNA interference mediated by small interfering RNA (siRNA) has been widely used as a procedure to knock down the expression of an intended target gene with perfect sequence complementarity However, siRNA often exhibits offtarget effects on genes with partial sequence complementaritiesFAQ ID 2262 miRNAs regulate the gene expression by binding to the mRNA The seed sequence is essential for the binding of the miRNA to the mRNA The seed sequence or seed region is a conserved heptametrical sequence which is mostly situated at positions 27 from the miRNA 5´end Even though base pairing of miRNA and its target mRNA does not match perfect, the "seed
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The seed region comprises 6 nucleotides in positions 2–7 of the antisense siRNA strand of the siRNA duplex Matches such as these can contribute to downregulation of unintended targets due to the siRNA mimicking the action of an miRNA siRNA designed at QIAGEN is analyzed for 3' UTR/seed region complementarity using a proprietary set of 3' UTR sequences derived from theIn addition, it is clear that unintended genes with complementarities only in the seed region (positions 28) are also downregulated by offtarget effects siRNA efficiency is mainly determined by the WatsonCrick basepairing stability formed between the siRNA seed region and target mRNA siRNAs with a low seedtarget duplex melting temperature (T(m)) have little or no seedThe seed region is known to be situated on the surface of Ago in a quasihelical form to serve as the entry or nucleation site for small RNAs in the RISCs (Ma et al, 05;
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Lane 1, siRNA with a dsDNA seed duplex Lane 2–lane 13, siRNA with a dsDNAmodified seed duplex and two additionally DNAsubstituted base pairs in the nonseedduplex region DNA substitutions at positions 13 and 14 and of the 3′ overhang of the guide strand changed little, if any, RNAi activity (lanes 6, 12, 13)Chemical modifications of 2'Omethyl (2'OMe) and locked nucleic acid (LNA) of the nucleotides in the seed region (positions 28) of the small interfering RNA (siRNA) guide strand significantly reduced seedmatched (SM) offtarget effects The siRNA
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