Guide strand

Strand selection Asymmetry rule Strand with less stable paired 5 end is preferentially selected as guide strand... 

Strand selection and general siRNA functionality Nucleotide specificity Strands with U, alternatively A, at the 5 end have higher affinity for Ago proteins. Guide strands should start with U or A, passenger strands with G or C... 

Structural studies on Ago proteins revealed that the so-called MID domain binds the 5 end of the guide strand (39,40). Because of its central localization in the Ago protein, this domain has been named MID domain. Crystal co-structures and Kj measurements of the MID domain in combination with all four nucleotides at the 5 end revealed that uridine (U) binds with the highest affinity to the MID domain, adenosine (A) with a slightly reduced affinity, and cytosine (C) and guanosine (G) with more than tenfold less affinity (41). Therefore, siRNA guide (antisense) strands should ideally contain a U or A at the 5 end. C and G should be avoided. For the passenger (sense) strand 5 end, C and G should be selected in order to minimize strand incorporation (Fig. la). Based on our own unpublished data the nucleotide specificity is not only a tool to manipulate strand selection but siRNA strands with U or A at the 5 end show also a higher absolute affinity for Ago proteins and therefore are more likely to be potent siRNAs (see Notes 2 and 3). 

Our unpublished results indicate that asymmetry rule and nucleotide specificity can be similarly strong parameters for guide strand selection. Each parameter can overrule the other one, always depending on the individual siRNA. Therefore both parameters should be ideally combined in order to design highly asymmetric siRNAs. 

Strand-specific 5 -0-methylation of siRNA duplexes controls guide strand selection and targeting specificity. RNA 14 263-274... 

Rand TA, Petersen S, Du F et al. Argonaute2 cleaves the anti-guide strand of siRNA during RISC activation. Ce//2005 123 621-629. 

Many off-target effects associated with RNAi are caused not by homology to the coding sequences of other genes, but rather by short stretches of 6 to 7 bps within the 5 end of the guide strand... 

Analysis of 1000 siRNA sequences known to non-specilically degrade mRNA showed a bias for complementation to 3 UTRs within positions 2 to 7 of their guide strands (Jackson et al. 2006). Regardless, experimental design must take care to recapitulate phenotypes observed within an RNAi experiment through repetition (i.e more than two potent siRNA sequences yield the same phenotype) or through rescue (i.e., the phenotype can be restored by re-expression of the gene of interest). 

Fig. 7.6. Guide and passenger strand loading into RISC in the cytoplasm and nucleus. Normalized cross-correlation in the cytoplasm filled boxes) and nucleus open boxes) of EGFP-Ago2 and Cy5 labeled passenger (a) or guide strand (b) of siTK3 for several time points after injection. The bottom graphs display the corresponding cross-correlation curves...

Fig. 2 The RNAi mechanisms. In the cytoplasm, dsRNA is cleaved into 22nt fragments called small interfering RNAs (siRNAs) by the RNase III t5rpe enzyme Dicer. The siRNAs are unwound and serve either as primers for RdRP (left) in some taxa (e.g., plants and nematodes) or, more universally, as guide strand in the RISC (right). RdRP creates additional dsRNA, amplifying the substrate for Dicer. RISC cleaves the target RNA in the region bound by the siRNA guide.

Various components of RISC have been identified in different organisms, but the key constituent, the nuclease or Sheer, is an Argonaute protein. Argo-nautes share two motifs the PAZ domain, which binds to the 3 end of the siRNA guide strand,and the catalytic Piwi domain, which structurally resembles an RNase... 

Hofstetter C P, Schwarz E J, Hess D, et al. (2002). Marrow stromal cells form guiding strands in the injured spinal cord and promote recovery. Proc. Natl. Acad. Sci. USA. 99 2199-2204. 

In the cell, nucleic acids are involved in the storage of genomic information and the production of proteins. However, other functions of nucleic acids, especially RNA, have been discovered. For example, ribosomal RNAs not only fold the structure of the ribosome but play an important role in protein synthesis (Moore and Steitz, 2002), and the RNA-induced silencing complex (RICS) is composed of a guide strand of siRNA or microRNA and proteins to control gene expression through RNA interference (Filipowicz, 2005). Those RNAs and proteins work cooperatively to achieve complicated tasks. This means that if we could engineer a nucleic acid-protein complex, we could construct more functional biosensor systems. 

---