RNA-centered applications

Question 1

Explain how small RNAs can inhibit gene expression.

Answer 1

If a small RNA has a sequence that base pairs with part of an mRNA, it will prevent the translation = no protein is made and no gene expression

Question 2

Explain the basis for the specificity of these small RNAs – how they recognize and interact with a specific mRNA (and not a different one encoding a different protein).

Answer 2

1. The small RNA can signal enzymes to degrade the mRNA.
2. The small RNA can get in the way of the translation process.

Question 3

Give an example of a clinical use of small RNAs in which they inhibit the production of a toxic protein. State at what stage of protein production they act.

Answer 3

1. RNA-based therapeutics (small inhibiting RNA and Antisense RNA) lower the
   production of the transthyretin
   protein, = reduce the odds of the protein misfolding and forming an amyloid structure
2. RNA binds to the mRNA = degraded and no protien produced

Question 4

Explain how small RNAs can act as gene editors and give the acronym name of this process.

Answer 4

1. DNA is cut and edited at a specific site in the sequence. The DNA is repaired naturally (with
   errors), resulting in gene inactivation or a
   new sequence is added at the break = allows flexibility in changing a genome sequence.
2. CRISPR/ Clustered regularly interspaced short palindromic repeats

Question 5

Describe the sequence of events that takes place when this gene editing process occurs in a cell.

Answer 5

1. A guide RNA (gRNA) is designed and synthesized so its sequence matches a specific target location in the genome.
2. The guide RNA base-pairs with the target DNA sequence at the correct location.
3. Once bound, the Cas9 enzyme cuts both strands of the DNA at that site.
4. The cell repairs the DNA break using its natural repair mechanisms.
5. During repair, the gene can be inactivated, modified, or replaced.
6. If additional DNA is provided, it can be inserted into the break site, allowing specific changes to the genome.

Question 6

Describe the role of the protein involved in CRISPR and its name.

Answer 6

CRISPR-associated protein 9 (CAS9) is
an enzyme that associates with the guide
RNA and cuts DNA

Question 7

GIve an example of the clinical use of an mRNA.

Answer 7

1. The mRNA molecules that encode specific
   genes can be used to produce the encoded proteins.
2. such as vaccines

Question 8

Explain the problem with unmodified mRNA for therapeutic use and name the modification that was used to make the mRNA effective.

Answer 8

1. synthetic mRNA molecules without
   base modifications are recognized as
   prokaryotic.
2. Your body recognizes it as a pathogen, and an immune response prevents mRNA arrival at the target cell and production of therapeutic
   protein

Question 9

Give a step-by-step description of COVID vaccine production

Answer 9

1. Scientists design an mRNA sequence that codes for the SARS-CoV-2 spike protein.
2. The mRNA is modified (e.g., with methylpseudouridine) to appear non-threatening to the immune system.
3. A 5′ cap and 3′ poly-A tail are added to make the mRNA stable and functional.
4. The mRNA is packaged inside lipid nanoparticles for protection and delivery.

Question 10

Give a step-by-step description of COVID vaccine administration

Answer 10

1. The vaccine (lipid nanoparticle with mRNA) is injected into the body.
2. Cells take up the mRNA from the nanoparticles.
3. Cells use the mRNA to produce the spike protein antigen.
4. The immune system recognizes the protein and mounts a response, building protection against COVID-19.