Antisense Technology: Theory and Therapy Flashcards
What is antisense technology?
- Synthesize strand of nucleic acid (DNA/RNA) that will bind mRNA produced by gene that causes disease and inactivate it, effectively turing the gene “off”
- Essentially inhibit the protein before it is synthesized
What is the advantage of antisense technology over traditional drugs?
- Oligonucleotides are manufactured quickly
- Sensitive, selective, effective, less toxic
- Longer responses (clonal expansion may take longer to produce clinical disease once mRNA is inhibited) therefore lower doses
What is the definition of antisense?
- Single stranded oligonucleotide (nucleic acid analog), 15-20 aa in length with sequence complementary to specific target mRNA region called “sense” sequence
- When antisense oligonucleotide and mRNA bind it decreases translation
Describe normal transcription/translation
- Antisense DNA transcribed to pre-mRNA
- pre-mRNA –> mRNA
- mRNA –> ribosomes for translation to protein
What is one way to prevent transcription in a cell?
- Prevent transcription by DNA targeted agent, such as an antisense agent
- Binds DNA and prevents transcription to pre-mRNA
How can you prevent the formation of mature mRNA?
- Block pre-mRNA
- Can use any oligonucleotide-based agent
- Needs to match pre-mRNA sequence
How can you prevent translation based on interfering with mRNA?
- Block the formation of proteins by adding antisense agent to mRNA
- Stops formation of ribosomal complex, acts as a steric blocker
How can you prevent translation using an enzyme?
- Use RNAse to digest mRNA
- Most important antisense mechanism
- Any oligonucleotide, substrate for nuclease, targeting RNA may destabilize it
- Normal function of RNAses (oligonucleotides) for specific RNase response must be carefully construction
What are the two groups of RNAses?
1) RNase H
2) Double-strand RNAses (RNAse III)
What is RNase H?
- Ribonuclease that cleaves RNA in a DNA/RNA duplex to produce ssDNA
- RNAse H is a non-specific endonuclease and catalyzes the cleavage of RNA via a hydrolytic mechanism
Describe the steps used for RNAse H to break down mRNA
1) Antisense ONT binds target RNA to form heteroduplex substrate
2) RNase H binds via its binding domain at 3’ antisense ONT/ 5’ RNA pole and cleaves target RNA ~7bp from binding site
3) Target mRNA is degraded, whereas antisense ONT stays intact, allowing it to form heteroduplex substrate for induction of RNase H cleavage
What are double-stranded RNAses (or RNAse III)?
- Type of ribonuclease that digests dsRNA
- Part of (dsRNS)-Dicer family of RNAse
- Cuts pre-microRNA at a specific site and transforms it into miRNA that is actively involved in regulation of transcription and lifetime of mRNA
What are the three new tools to identify new antisense mechanisms, other than RNAse H?
1) ds-RNAses
2) RNAi
3) siRNA
What is the cell’s normal mechanism to turn off a gene?
- Cell will synthesize miRNA
- Binds to mRNA and turns off gene
What is the siRNA antisense mechanism?
1) ds-ONT enters cytoplasm
2) Helicase separates sense and antisense strands of ONT
3) RISC associates with antisense ONT
4) Antisense ONT binds target mRNA, forming sense-antisense duplex
5) Nuclease component (RNAIII) of RISC degrades target mRNA
Overall, this inhibits target mRNA expression
What is the RISC complex?
- RNA interfering silencing complex
- Short dsRNA produced endogenously by DICER or introduced to cell exogenously
- Two strands of dsDNA are separated by an ATP dependent helicase
- One strand of dsDNA remains as RISC, other strand is displaced
- Mature RISC includes single strand of RNA which acts as guide sequence for specific cleavage of targeted mRNA
What is RNAi (RNA interference)?
- Conserved biological response to dsRNA
- Natural mechanism for sequence-specific gene silencing
- Many types of small silencing RNAs have been discovered, including siRNAs (small interfering) and miRNAs (micro)
- Process of RNAi can be moderated by either siRNA or miRNA but there are differences between the two
What are the differences between siRNA and miRNA?
- miRNA = 19-25nt; endogenous; imperfect match with target; mechanism is through translation repression
- siRNA = 19-21nt; exogenous; exact match to target; mechanism is through mRNA cleavage
How do you make dsRNA?
- mRNA is initially single-stranded
- Replicate RNA from mRNA template using RNA replicase
- Two RNA strands are held together by hydrogen bonds
- Now have dsRNA
What happens to dsRNA?
- dsRNA is cut by Dicer enzyme
- This leads to formation of either siRNA (21-23nt long) or miRNA
- Cut leads to two overhanging nucleotides at end of strands
How does RNAi work?
1) Process dsRNA into 21-23nt fragments using Dicer (produces siRNA)
2) Antisense strand of siRNA guides cleavage - this is guided by base complementarity of the siRNA, RISC targets mRNA for degradation
What is the mechanism of miRNA?
1) DNA sequence –> pri-miRNA (ssRNA)
2) Hairpin around miRNA - sequence in pri-miRNA - a signal for ds-ribonuclease (Drosha) to produce pre-miRNA
3) Exportin 5 (carrier protein) leads to nuclear export of pre-miRNA
4) Dicer enzyme cleaves pre-miRNA - releases mature miRNA, which associates with RISC
5) RISC-miRNA complex blocks translation by mRNA by binding to complementary sequence
What are some applications for RNAi?
- Clarify gene function in cellular processes
- Find enhancers/suppressors of phenotypes
- Understand biology of different cell lines
- Cell and tissue engineering
- Support miRNA target identification
- Validate drug therapies
- Therapeutics
How is antisense gene therapy different from RNA interference?
- Antisense technology destroys target mRNA by recruiting enzyme RNAse H
- RNAi recruits Dicer enzyme
- RNAi molecules are twice as large as antisense ONTs because they are double-stranded, not single-stranded
