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- "In general, we target the coding region; however, targeting the 5′- and 3′-UTR sequences is possible"
- "It is important to note that siRNA design rules serve more as guidelines, and that sequences adhering to them may not silence and vice versa. To date, no algorithm guarantees silencing efficacy, and most recommend the user pick three to four candidates for screening."
- "To achieve faithful loading of the antisense strand, the duplex must be designed such that there is strong G–C base pairing present at the 5′-end of the sense (passenger) strand and weak A/G-U base paring at the opposing terminus"
- "We identify 22-nt target sites within the target transcript that adhere to four criteria: (1) high propensity to primarily load the antisense guide strand into RISC, (2) GC content between 20% and 70%, (3) void of restriction enzyme sites relevant to downstream applications (e.g., cloning RNAi expression cassettes into viral vector systems), (4) lacking a stretch of four continuous A or T nucleotides (i.e., AAAA or TTTT)"
- "To achieve faithful loading of the antisense strand, the duplex must be designed such that there is strong G–C base pairing present at the 5′-end of the sense (passenger) strand and weak A/G-U base paring at the opposing terminus (Khvorova et al., 2003; Schwarz et al., 2003). The RISC complex selects the strand with the weakest 5′-end thermodynamic stability, in this case, the antisense strand (Fig. 14.1). Hence, we select target sequences that have G or C nucleotides at positions 3 and 4"
- Diagram #1
- "The shRNA DNA template consists of the following from 5′ to 3′: (1) the sense sequence consisting of nucleotides 3–22 of the target site, (2) the 19-nt loop sequence (5′-CTGTAAAGCCACAGATGGG-3′) which is partially derived from the naturally occurring human miR-30 transcript, and (3) the antisense sequence which is the reverse complement of the target site (positions 3–22)"
- Unafold allows you to confirm that your sequence will form a hairpin
- Diagram #2
- Diagram #3
- "Although we select siRNA sequences based on the most significant determinants of gene silencing efficacy (i.e., strand biasing and GC content), not all sequences will be functional. Thus, we typically generate several constructs, each with unique sequences, for a given target gene"
Questions/concerns about miRNA:
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