Introduction to RNA

Question 1

What is the transcriptome?

Answer 1

Complete set of RNA transcripts

Question 2

What are the two RNA hypotheses?

Answer 2

A. Variation is hierarchical so variation in DNA leads to variation in RNA in a linear manner. B. The combination of variation across all possible omic levels.

Question 3

How many genes are needed to make humans?

Answer 3

Estimated to be 100 000 genes but now believed to have 20 000 protein coding genes. Highlights importance of regulatory network and NC RNAs. Prompted reevaluation of the gene.

Question 4

What was discovered about the genome sequence and loci in the 2000s?

Answer 4

The number of coding loci remains fixed throughout evolution while the content of noncoding DNA increases. Implies new and unexplored functions for this part of the genome.

Question 5

What are the RNA categories?

Answer 5

Protein coding mRNAs, structural/housekeeping rRNAs, snRNAs, snoRNAs, regulatory miRNAs, lncRNAs, circRNAs.

Question 6

What is the RNA structure based on?

Answer 6

Structure based on how complementary the sequence is as RNA is single stranded. RNA is more flexible than DNA to form different structural shapes and can be used in different ways.

Question 7

What does predicting the RNA secondary structure show?

Answer 7

What the function of the RNA is.

Question 8

What are 3 examples of non coding RNAs in gene expression and cell function?

Answer 8

1. Transcription regulation by riboswitches. 2. Post-transcriptional regulation of protein synthesis. 3. Small non coding RNA to protect from exogenous nucleotides.

Question 9

What is the riboswitch?

Answer 9

The riboswitch is a ribozyme that cleaves itself in the presence of sufficient concentrations of its metabolite. Riboswitch alternate structures affect the splicing of the pre mRNA.

Question 10

What is the conventional drug strategy?

Answer 10

The ability of small molecule drugs to target active sites of proteins, so as to inhibit or alter their function.

Question 11

What is the problem with conventional drugs?

Answer 11

Only 1.5% of human genome encodes proteins and 10-14% of proteins have active binding sites that are druggable.

Question 12

What are the other options to conventional drugs?

Answer 12

Recombinant proteins (size and stability issues folding is an added problem) Drug therapeutics- DNA drugs can generate therapeutic proteins when delivered as plasmids or in viral vectors. CRISPR-Cas. RNA therapeutics largely focused on 2 approaches with multiple variants.

Question 13

What are the hurdles to overcome in RNA therapeutics?

Answer 13

Rapid degradation of exogenous RNA by RNase that are ubiquitous. Delivery of negatively charged RNA across hydrophobic cytoplasmic membrane. Strong immunogenicity of exogenous RNA.

Question 14

What is the process of RNA therapeutics?

Answer 14

Mechanistic understanding + technology development to target to drug to therapy.

Question 15

What are advantages of RNA-based drugs?

Answer 15

Ability to act on targets that are undruggable for small molecules. Rapid and cost effective compared to small molecules and recombinant proteins. Flexible for personalized treatments and can adapt to an evolving pathogen.

Question 16

What are the 3 main types of RNA therapeutics?

Answer 16

mRNA, aptamers, ASOs (RNA interference and miRNAs).

Question 17

Why are RNA vaccines advantageous?

Answer 17

If a change occurs in virus that causes the disease a new vaccine needs to be made. RNA vaccines can be generated much quicker. Targeting the correct organ with the RNA vaccine is the difficult step.

Question 18

What are mRNAs?

Answer 18

The intermediate between the coding genomic DNA and the encoded proteins.

Question 19

What did Wolff et al do?

Answer 19

Use exogenous mRNA to induce expression of protein in vivo. Injected synthetic mRNA encoding luciferase, chloramphenicol acetyltransferase or beta-galactosidase into mouse skeletal muscle and detected proteins translated from these RNAs at injection site.

Question 20

What does synthetic RNA consist of?

Answer 20

Single stranded open reading frame flanked by untranslated regions and has a 5’ cap for translation and a 3’ poly(A) tail for stability.

Question 21

Why does exogenous mRNA show no risk of integration into the genome?

Answer 21

It is translated into protein in cytoplasm and degrades within minutes to hours.

Question 22

What is replacement therapy?

Answer 22

Where mRNA is administered to the patient to compensate for a defective gene/protein. Or to supply therapeutic proteins.

Question 23

What is mRNA vaccination?

Answer 23

mRNA encoding specific antigens is administered to elicit protective immunity.

Question 24

What is mRNA cell therapy?

Answer 24

mRNA is transfected into cells ex vivo to alter cell phenotype or function and then these cells are delivered into the patient.

Question 25

What are the three companies working on mRNA cell therapy?

Answer 25

Moderna, CureVac, BioNTech.

Question 26

What is a current example of mRNA cell therapy?

Answer 26

CV9202 cancer vaccine in clinical trials: a self-adjuvating RNA vaccine targeting six antigens commonly expressed in non-small cell lung cancer.

Question 27

What is the solution to difficulty with RNA targeted delivery?

Answer 27

Novel vehicles that will deliver the RNA drug to the site of therapeutic action facilitating entry of RNA drug into cytoplasm for effect.

Question 28

What are three delivery options for mRNAs?

Answer 28

Lipid based nanoparticles, polymer nanomaterials, carbon and gold nanomaterials.

Question 29

What are lipid-based nanoparticles?

Answer 29

Liposomes formed when phospholipids are dispersed in aqueous phase. Spherical vesicles with at least one phospholipid bilayer enclosing an aqueous core. Drug with surface modifications to mimic cell membrane composition. mRNA combined with cationic lipids.

Question 30

What are polymer nanomaterials?

Answer 30

Synthetic compounds made of a handful of base units that come together to form complex structures. Usually synthetic polymers with a long shelf life. Can encapsulate hydrophilic and hydrophobic compounds and proteins.

Question 31

What are carbon and gold nanoparticles?

Answer 31

Gold nanoparticles, quantum dots, nanographene oxide, carbon nanotubes are each synthesized nanostructures that have the capacity to harbour RNA and protect it from degradation before delivery to target.

Question 32

What are antisense oligonucleotides ASOs?

Answer 32

Short single stranded DNA, phosphorothioate DNA, RNA analogs conformationally restricted nucleosides (LNA locked nucleic acids) complementary to a certain region of RNA that are meant to target.

Question 33

What are examples of ASOs?

Answer 33

RNase H-dependent ASO. Enzymes that hydrolize RNA strand of duplexes. Knockdown gene expression. RNase H-independent (steric)ASOs inhibit translation or splicing.

Question 34

What is a clinical use of ASOs?

Answer 34

Nusinersen treats spinal muscular atrophy (caused from deletions or mutations in survival of SMN1 motor gene. Mutation doesn’t produce enough SMN.) Nusinersen modulates splicing of SMN2 so as to increase the transcript containing exon 7 to produce SMN2. This results in expression of a small amount of a full length SMN protein.

Question 35

What are aptamers?

Answer 35

Short single stranded nucleic acids that bind to a variety of targets from its tertiary structure instead of sequence. One approved aptamer drug. Pegaptanib 27 based selected against VEGF to treat age related macular degeneration. Prevents protein receptor interaction and prevents formation of blood vessels under retina causing vision lost.