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PHAR 211 > Gene Silencing > Flashcards

Gene Silencing Flashcards

1
Q

What is RNA interference? (RNAi) (2)

A
  1. An accurate and potent gene silencing method
  2. Silences RNA by breaking it down or stopping translation
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2
Q

The key requirement of RNAi is?

A

dsRNA (double-stranded)

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3
Q

Several types of silencing RNAs (RNAis) have been discovered. Name them (4)

A
  1. Small interfering RNAs (siRNAs)
  2. MicroRNAs (miRNAs)
  3. shRNA (short hairpin)
  4. CRISPR
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4
Q

The key enzyme in RNAis is?

A

Dicer

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5
Q

How does dicer work?

A

Has RNAase domains - chops long dsRNA into short RNAs and they will become siRNAs, causing RNAi

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6
Q

How does RNAi work? (2)

A
  1. Is a natural cellular process that silences gene expression by promoting the degradation of mRNA
  2. It plays an impotant role in gene regulation and innate defense against invading viruses
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7
Q

What are the sources of long dsRNA? (3)

A
  1. Hairpin
  2. Complementary RNAs
  3. RNA dependent RNA polymerases
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8
Q

siRNAs have a defined structure. Describe how it is made and what the structure is

A

Dicer cleaves long dsRNA into siRNA 21-23nt dsRNA
- Symmetric 2nt 3’ overhangs
nt = nucleotide

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9
Q

Explain the mechanism of action of siRNA (4)

A
  1. The siRNA interacts with dicer and activates the RNA-induced silencing complex (RISC)
  2. The endonuclease argonaute 2 (AGO2) component of the RISC cleaves the passenger strand (sense strand) of the siRNA while the guide strand (antisense strand) remains associated with the RISC.
  3. Subsequently, the guide strand guides the active RISC to its target mRNA for cleavage by AGO2.
  4. As the guide strand only binds to mRNA that is fully complementary to it, siRNA causes specific gene silencing
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10
Q

miRNA is a class of small RNA molecules that __________ regulates gene expression

A

negatively

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11
Q

miRNA gene transcription is carried out by ___ __________ in the nucleus to give _______ _____ with a double-stranded stem-loop structure

A

RNA polymerase; primary miRNA (pri-miRNA)

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12
Q

pri-miRNA is cleaved by a microprocessor complex comprising of ______ to form _________ _____, which is a duplex that contains 70-100 nucleotides with interspersed mismatches and adopts a loop structure

A

Drosha; precursor miRNA (pre-miRNA)

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13
Q

pre-miRNA is subsequently transported by ________ _ from the nucleus to the cytoplasm where what happens? (5)

A

Exportin 5
1. Where it is further processed by Dicer into a miRNA duplex of 18-25 nucleotides
2. The miRNA duplex then associates with the RISC forming a complex called miRISC
3. The miRNA duplex is unwound, releasing and discarding the passenger strand (sense strand) - unlike in the processing of siRNA, in which the AGO2 of the RISC causes the cleavage of the passenger strand of siRNA
4. The mature single-stranded miRNA guides the miRISC to the target mRNAs.
5. The miRNA binds to the target mRNAs through partial complementary base pairing with the consequence that the
target gene silencing occurs via translational repression, degradation, and/or cleavage.

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14
Q

What are the differences between target recognition by siRNA and miRNA? (3)

A
  1. siRNA is usually fully complementary to the coding region of its target mRNA.
  2. miRNA is partially complementary to its target mRNA. Complementary binding usually occurs at the seed region (nucleotides (nt) 2–7 of the 5’ end) of miRNA and the 3’ UTR of the target mRNA.
  3. Since miRNA-mRNA recognition does not require perfect pairing, one miRNA strand can recognize an array of mRNAs, and hence miRNA has the characteristic of having multiple targets.
    - For example: miRNA-124, which is preferentially expressed in brain tissues, can downregulate 174 annotated genes.
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15
Q

What are 2 benefits of RNAi therapeutics?

A
  1. They can overcome the major limitation of traditional small drug molecules, which can only target certain classes of proteins.
  2. Even for protein-based drugs including monoclonal antibodies that are highly specific, their targets are mainly limited to cell-surface receptors or circulating proteins.
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16
Q

siRNA vs. miRNA:
Prior to Dicer processing - what do these molecules look like/what is their structure prior to Dicer?

A

siRNA:
- Double-stranded RNA that contains 30 to over 100 nucleotides
miRNA:
- Precursor miRNA (pre-miRNA) that contains 70-100 nucleotides with interspersed mismatches and hairpin structure

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17
Q

siRNA vs. miRNA:
What is the structure? (Post Dicer)

A

siRNA:
- 21-23 nucleotide RNA duplex with 2 nucleotides 3’ overhang
miRNA:
- 19-25 nucleotide RNA duplex with 2 nucleotides 3’ overhang

18
Q

siRNA vs. miRNA:
Complementarity

A

siRNA:
- Fully complementary to mRNA
miRNA:
- Partially complementary to mRNA, typically targetting the 3’ untranslated region of mRNA

19
Q

siRNA vs. miRNA:
mRNA target

A

siRNA:
- One
miRNA:
- Multiple (could be over 100 at the same time)

20
Q

siRNA (1) vs. miRNA (3):
Mechanism of gene regulation

A

siRNA:
- Endonucleolytic cleavage of mRNA
miRNA:
- Translational repression
- Degradation of mRNA
- Endonucleolytic cleavage of mRNA (rare, only when there is a high level of complementarity between miRNA and mRNA)

21
Q

siRNA (1) vs. miRNA (3):
Clinical applications

A

siRNA:
- Therapeutic agent
miRNA:
- Drug target
- Therapeutic agent
- Diagnostic and biomarker tool.

22
Q

RNAi - stability and delivery. What are the limitations? (4)

A
  1. RNAs are extremely vulnerable to serum nucleases.
  2. Although double-stranded RNA is more resistant to nuclease degradation than single-stranded RNA, naked RNAs in their unmodified forms are degraded rapidly following administration. This degradation is done by the abundant nucleases present in the bloodstream, which contributes to a short half-life in vivo.
  3. Poor stability is one of the major obstacles toward the successful application of siRNAs and miRNAs as therapeutic agents.
  4. Solution - Chemical modifications of RNA
23
Q

Poor delivery is still a major challenge in translating therapeutic siRNAs and miRNAs into the clinic. Why?

A

Both types of RNA molecules have an intracellular site of action, but their intrinsic properties, including hydrophilic nature, negative charge and high molecular weight (∼14–15 kDa), render them poorly permeable across biological membranes.

24
Q

The primary role of a delivery system is to…?

A

Facilitate the cellular uptake of siRNAs or miRNAs to their target sites

25
Q

The secondary role of a delivery system is to…?

A

Protect the nucleic acids from premature nuclease degradation, thereby reducing the need for chemical modifications, which may affect the specificity and functionality of the RNA molecules.

26
Q

Why are viral vectors used as a delivery system?

A

Lentiviruses, adenoviruses, and adeno-associated viruses (AAVs) are extremely efficient in transferring the RNA-encoding vectors into the nucleus of mammalian cells to ensure high expression of RNA. High transduction efficiency.

27
Q

Viral vectors as a delivery system - how are they designed?

A

Viruses that are used to carry therapeutic RNA are genetically engineered to remove their virulence, and their tropism can be altered by genetic manipulation of the viral capsid for targeting to specific cell types. In addition, long-term expression can be achieved by using viruses, such as lentiviruses, that can integrate into the host genome

28
Q

What are 3 limitations of viral delivery?

A
  1. Serious safety concerns - high immunogenicity
  2. Risk of insertional mutagenesis
  3. High production costs have also limited their clinical applications.
29
Q

What are the 2 main categories of non-viral vectors?

A
  1. Polymer-based
  2. Lipid-based systems
30
Q

Small molecules vs. Protein-based drugs vs. siRNA/miRNA-based drugs:
Nature of action

A

Small molecules:
- Activation or inhibition of targets
Protein-based drugs:
- Activation or inhibition of targets
siRNA/mRNA:
- Inhibition of targets

31
Q

Small molecules vs. Protein-based drugs vs. siRNA/miRNA-based drugs:
Site of target proteins

A

Small molecules:
- Extracellular and intracellular
Protein-based drugs:
- Mainly extracellular
siRNA/mRNA:
- Virtually any sites

32
Q

Small molecules vs. Protein-based drugs vs. siRNA/miRNA-based drugs:
Selectivity and potency

A

Small molecules:
- Variable
Protein-based drugs:
- Highly specific and potent
siRNA/miRNA:
- Highly specific and potent

33
Q

Small molecules vs. Protein-based drugs vs. siRNA/miRNA-based drugs:
Lead optimization

A

Small molecules:
- Slow
Protein-based drugs:
- Slow
siRNA/mRNA:
- Rapid

34
Q

Small molecules vs. Protein-based drugs vs. siRNA/miRNA-based drugs:
Manufacture

A

Small molecules:
- Easy
Protein-based drugs:
- Difficult
siRNA/mRNA:
- Easy

35
Q

Small molecules vs. Protein-based drugs vs. siRNA/miRNA-based drugs:
Stability

A

Small molecules:
- Stable
Protein-based drugs:
- Unstable
siRNA/mRNA:
- Unstable

36
Q

Small molecules vs. Protein-based drugs vs. siRNA/miRNA-based drugs:
Delivery

A

Small molecules:
- Easy
Protein-based drugs:
- Difficult
siRNA/mRNA:
- Difficult

37
Q

What is CRISPR-Cas9? (2)

A

A genome editing technique that:
1. Targets a specific section of DNA
2. Makes a precise cut/break at the target site

38
Q

CRISPR-Cas9 can do one of three things:

A
  1. Make gene nonfunctional
  2. Replace one version of a gene with another
  3. Base editing
39
Q

How does CRISPR-Cas9 work? (5)

A
  1. CRISPR-Cas9 was adapted from a naturally occurring genome editing system in bacteria.
  2. The bacteria capture snippets of DNA from invading viruses and use them to create DNA segments known as CRISPR arrays.
  3. The CRISPR arrays allow the bacteria to “remember” the viruses (or closely related ones).
  4. If the viruses attack again, the bacteria produce RNA segments from the CRISPR arrays to target the viruses’ DNA.
  5. The bacteria then use Cas9 or a similar enzyme to cut the DNA apart, which disables the virus
40
Q

What are the features of CRISPR-Cas9? (4)

A
  1. CRISPR/Cas9: Simple to make components: guide RNAs, Cas9, and DNA templates
  2. Relatively simple to introduce these into cells and early embryos.
  3. Highly specific (off-target events ?)
  4. Highly efficient
41
Q

What makes CRISPR-Cas9 versatile? (2)

A
  1. Alterations to DNA: including “indels” and deletions, insertions or substitutions from single base-pairs up to many kilobases
  2. Cas9 DNAse activity can be mutated and other proteins linked to it: including transcriptional activators or repressors to manipulate specific gene activity without altering DNA
42
Q

Sickle cell disease is caused by a mutation in the __________-____ gene found on chromosome __

A

hemoglobin-Beta; 11

PHAR 211 (10 decks)

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