- learn about luciferase assay
"Generation of Hairpin-Based RNAi Vectors for Biological and Therapeutic Application":
- "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"
"MicroRNA Targeting Specificity in Mammals: Determinants beyond Seed Pairing":
- "we uncovered five general features of site context that boost site efficacy: AU-rich nucleotide composition near the site, proximity to sites for coexpressed miRNAs (which leads to cooperative action), proximity to residues pairing to miRNA nucleotides 13–16, positioning within the 3′UTR at least 15 nt from the stop codon, and positioning away from the center of long UTRs"
- "Overall, consequential pairing preferentially involved Watson-Crick pairing to miRNA nucleotides 12–17, most especially nucleotides 13–16"
- "When sites were divided into functional and nonfunctional sites based on their performance on the microarray, we found that the nucleotides immediately flanking the functional sites were highly enriched for A and U content relative to the nonfunctional sites (Figure 3A, green plot). This phenomenon of high local AU content was important in the immediate vicinity of the site and then fell off quickly"
Questions/concerns about miRNA:
- "Overall hairpin length and loop size influence the efficiency of Dicer processing"–how should we design the loop?
- The specificity of base pair matching in the stem influences processing efficiency
- Which of the two strands from the stem is incorporated into the RNA-induced silencing complex?
- miRNA's are readily degraded by RNase (present ubiquitously in cells). So we should be sure to inhibit RNase when working with miRNA's; this inhibitor is available for purchase or might already be in the lab.
Pay attention to cross talk. look for foreign miRNA
BLAST NCIB
Results from miR-Synth: