Gene Silencing Flashcards
What is RNAi technology?
RNA interference (RNAi) is an accurate and potent silencing method
RNA is silenced via degradation or stopping translation by specific double strand RNA sequences
What are some types of RNAi technologies?
- Small interfering RNAs (siRNAs)
- MicroRNAs (miRNAs)
- shRNA
- CRISPR
- Others
What is the key enzyme involved in RNAi technology?
DICER breaks down long sequences of dsRNA into smaller fragments (siRNAs)
Where is the dsRNA sourced for RNAi?
Hairpin, complementary RNAs, RNA dependent RNA polymerases
This dsRNA is processed by DICER enzyme into smaller dsRNA fragments known as siRNA (21-23 nucleotides long with 3’ two-nucleotide overhangs)
What is the role of siRNA in RNAi?
siRNA interacts with dicer and activates the RISC (RNA-indicing silencing complex)
see slide 10
What is the role of RISC in RNAi?
The AGO2 component of RISC cleaves the passenger strand (sense strand) while the guide strand remains associated with the RISC
The guide strand guides the active RISC to its target mRNA for cleavage by AGO2 component of RISC
see slide 10
What is miRNA?
miRNA is a class of small RNA molecules that negatively regulate gene expression
How can miRNA contribute to gene silencing?
miRNA gene is transcribed to create primary miRNA
Primary miRNA is cleaved by Drosha (protein complex) to form precursor miRNA
Precursor miRNA is further processed by dicer
This sequence of processed pre-miRNA then interacts with RISC and looses passenger strand while guide strand is retained
The RISC-guide strand complex (miRISC) will bind to complementary (usually only partially complementary) mRNA strand and prevent translation of that sequence
Do miRISCs have a specific target?
No, unlike siRNA-mRNA recognition, miRNA-mRNA recognition does not require perfect pairing, one miRNA can recognize an array of mRNAs
Review slide 19
Are free RNA molecules relatively resistant to decompostion?
No, abundant serum nucleases contribute to short half-life in vivo
This instability is resolved through chemical modification of RNA
What are the issues for RNA delivery into the cell (site of action)?
Their intrinsic properties, including hydrophillic nature, negative charge, and high molecular weight , render them poorly permeable across biological membranes
What is the role of a delivery system in siRNA or miRNA therapeutic agents?
Facilitating the cellular uptake of siRNA or miRNA to their target sites
Protect the nucleic acids from premature nuclease degradation
What are some characteristics of viral vectors as RNA delivery systems?
- Lentiviruses, adenoviruses, and adeno-associated viruses can transfer RNA into the nucleus effectively (high transduction efficiency)
- Viral vectors have their virulence removed
- Long-term expression can be achieved by using viruses that integrate into the host genomes (ensure important genes are not unintentionally silenced)
What are some limitations associated with viral RNA delivery systems?
- Serious safety concerns (high immunogenicity)
- Risk of insertional mutagenesis
- High production costs
- Non-viral vectors are available (better safety profile and lower cost)
