Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

Lab 3 > RNA therapeutics > Flashcards

RNA therapeutics Flashcards

1
Q

what 2 big discoveries questioned the dogna: gentic info flow from DNA -> RNA -> protein through transcription + translation?

A

catalytic RNAs
RNA interference

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

give example of catalytic RNA?

A

RNA can have catalytic enzymatic activity - ribozymes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What is RNA interference?

A

RNA molecules inhibit gene expression or translation by neutralising targeted mRNA molecules

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

what does RNA play an important role in?

A

regulating geen function.. needed to maintain cell health

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Why is RNA targeted in therapeutics?

A

RNA translation is involved in the pathogenesis of many diseases so can be used to downregulate disease.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

what do rna based therapeutics often manipulate?

A

transduction of rna signal + offer therapeutic effects

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

T/F
all rna based therapeutics are rna oligonucleotides -> polymers of nucleotides

A

True

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What determines what bases are linked together in Watson-Crick?
specificity

A

The distances between the bases.

A = U
G ≡ C

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

what are the types of RNA therapeutics? 5

A

antisense oligonucleotides ASOs

RNA interference RNAi

ribozymes

aptamers

RNA vaccines

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

how do antisense oligonucleotides work?

A

inhibit mRNA translation

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

RNAi form RNA induced silencing complex w a protein (Argonaute) how?

A

=RNA inhibitors of gene expression by degradation of mRNA

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

How do ribozymes work? catalytically active RNAs

A

cleave covalent bonds in the target mRNA molecules next to a recognition sequence = destroying RNA sequences

similar to protein ribonucleases

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

How do RNA vaccines work?

A

Introduce mRNA sequence coded for a disease specific antigen into cell to build up an immune response.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

How do aptamers work? protein binding RNAs

A

length of RNA that binds to a protein (like a small mol inhibitor)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

What is an antisense oligonucleotide? (ASO)

A

Short strand oligonucleotide that hybridises with complimentary mRNA in a sequence-specific manner via watson-crick base pairing.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

approx how many bp long are the short strand oligonucleotides used in ASOs?

A

20 bp

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

What does the entry of ASO into a cell cause?

A

protein knockdown in the cytoplasm or the nucleus

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

What does the binding of ASO to mRNA (cytoplasm) cause?

A

bind to mRNA

– triggers ribonuclease (RNase H) activity
OR
– inhibits ribosomal mRNA translation by steric hindrance.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

what happens when ASOs bind in the nucleus?

A

regulates mRNA maturation (via 3 mechanisms)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

how can ASOs regulate mRNA maturation in nucleus?

A

– inhibition of 5’ cap being formed OR
– inhibition of RNA splicing OR
– recruits ribonuclease (RNase H)

these -> knockdown of target genes so target protein knockdown by preventing mature mRNA from forming

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

what are some examples of RNA interference?

A

siRNA
RNAi

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

RNA interference- what happens if the AGO siRNA is
- perfectly complementary
- partially mismatched

A
  • mRNA cleavage
  • association-mediated repression
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

describe process of RNAi

A
  • siRNA enters cell as duplex, incorporated into Argonaute (AGO) to form part of RISC
  • in RISC, siRNA is unwound, passenger strand removed, guide strand left to find desired target RNA in cell
  • target mRNA bound in AGO then cleaved and digested/ silenced
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

whats AGO argonaute

A

protein in cells

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Q

whats RISC?

A

RNA induced silencing complex

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
26
Q

whats the difference between ASO’s and RNAi?

A

ASO delivered as ss, finds target alone

siRNA duplexes taken up by AGO (part of RISC)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
27
Q

ribozymes: catalytic RNA enzymes used to inhibit gene expression
whats their enzymatic mechanism similar to?

A

that of protein ribonucleases

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
28
Q

What is an aptamer and how does it work?

A

RNA sequence that binds to a protein, preventing it from functioning.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
29
Q

what do aptamers act similarly to?

A

small mol inhibitor

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
30
Q

when may RNA vaccines not be safe to administer?

A

for patients susceptible to autoimmune response

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
31
Q

an mRNA vaccine introduces an mRNA sequence from disease specific antigen -> px cells. what does cell do then?

A

translates this RNA to produce antigen then triggers immune response + antibodies made against this antigen
-> allows body to build up immune response to protect from original diseasw

32
Q

rna production very rapid. what does this mean for mRNA?

A

can be altered easily

33
Q

half life of RNA and implication?

A

short half life = fast degradation

34
Q

How can you design ASOs for a therapeutic beneft?

A

Can design the RNA sequence of the ASO to fit the mRNA of a specific gene

just make complementary base pairs in right sequence

35
Q

What does the strength and stability of interactions between ASOs and complementary mRNA target depend on?

A

Thermodynamic stability

Secondary structure

Proximity of hybridisation state to functional motifs on designed transcript

36
Q

why is Thermodynamic stability something to consider for designing ASOs?

A

how tightly bound is RNA to ASO?
how stable is complex?

37
Q

what about the Secondary structure should be considered for designing ASOs?

A

which sites are accessible to ASO?
what does RNA look like in 3D?

38
Q

what about ‘Proximity of hybridisation state to functional motifs on designed transcript’ should be considered for designing ASOs?

A

strategy can use Proximity to translation start factor eg AUG initiation codon or 5cap. block translation

or can block splice points in nucleus

39
Q

How do researchers increase the ‘hit rate’ in discovering therapeutic ASOs?

A

Predicting secondary structure of the target RNA.

Identification of preferable secondary local structures.

(use software)
3D shape of RNA is V important

40
Q

the ASO can be designed to bind at which 5 sites? of 10 or more consecutive nucleotides?

A

terminal end
internal loops
joint sequences
hairpin
bulges

41
Q

the bp in the nucelotides that make ASOs have great impact on stability of ASO-mRNA complex and mRNA knockdown.

what does presence of CCAC, TCCC, ACTC… motifs do?

A

increases their mRNA knockdown effects

42
Q

what does presence of GGGG, ACTG, AAA, TAA… motifs in ASO do?

A

weakens its activity

43
Q

some motifs (unmethylated CG sequences) are common in bacterial, not eukaryotic DNA so may trigger what?

A

immune response

44
Q

GGGG can form intra-strand tetraplexes, what is this?

A

single structures of 4 strands
.. which have high affinity for proteins so can also cause bad side reactions/ effects

45
Q

why is GC content strongly correlated with thermodynamic stability of the complex and RNase H activity?
in design of antisense oligonucleotides

A
  • GC bp have 3 H-bonds, while AT bp have only 2. GC stronger + more stable
  • Oligonucleotides w higher GC content more stable and less prone to degradation
  • RNase H: enzyme that degrades RNA in sequence-specific manner when it is bound to a DNA/RNA hybrid molecule.
46
Q

what is efficiency of RNase H activity influenced by?

A

length, sequence, and thermodynamic stability of the DNA/RNA hybrid.

47
Q

for a potent ASO designm binding energy of ASO and mRNA should be …

A

change in G 37 degrees more negative than
-8kcal/mol

48
Q

what is the MAIN disadvantages of RNA therapeutics?

A

their delivery to site of action

49
Q

3 disadvantages of RNA therapeutics?

A
  • enzyme degradation - activity of ribonucleases (cleavage by DNase and RNase)
  • fast excretion - unmodified oligonucleotides have a poor PK due to weak plasma binding = filtration thru the kidney
  • poor absorption/bioavailability= poor cellular uptake
50
Q

why RNA therapeutics have v poor cellular uptake?

A

typical ASO: 20bases long, charged backbone of -19

51
Q

how to chemically modify ASO in mRNA vaccines to try + decrease inflamm response/ enhance translational capacity/ increase stability of mRNA by changing untranslated regions?

A

use nucleoside-modified mRNA (eg pseudouridines)

52
Q

chem modifications of ASOs must retain ability to do what?

A

recognise their target mRNA by watson-crick bpairing

also modifications should not reduce efficiency of mRNA cleavage by RNase H (regardless of binding affinity) to use ASO in same way

53
Q

why are chem modifications needed for ASOs?

A

stabilise ASOs and make them resistant to ribonucleases

should improve ASO’s:
- PK
- PD
- retain/ improve RNase H efficiency
- reduce immunogenecity

54
Q

to what 3 sites can chem modifications be made to ASO?

A

sugar
base
ASO backbone

55
Q

in terms of PK why do you want to chemically modify ASO?

A

enhance nuclear resistance + increase tissue half life
- want to prevent nucleolytic degradation of ASO BUT NOT RNase H activity on mRNA

need enough stability too avoid metab of ASO before gets to correct cells

56
Q

what type of tox would you want to decrease when chem modifying ASOs?

A

non-sequence specific tox
- design ASO so wont hit anything off target
- small ASOs less than 20 bp

57
Q

how do you prevent ribonuclease attacking RNA therapeutics?

A

chemically modify the antisense oligonucleotide

58
Q

2 chemical modifications you can add to ASOs/ DNA/RNA?

A

Thiophosphoramidate
PNA
Phosphoramidate

59
Q

why is thiophosphoramidate used in chemical modification of ASOs?

A

improve stability, binding affinity, immunogenicity, PK, and reduce off-target effects, making them more effective as therapeutic agents.

60
Q

what are generation one modified antisense oligonucleotides?

A

PS-modified ASOs
non bridging O atom of phosphodiester bond replaced by S (phosphorothioate bond)

61
Q

what are the advantages of PS-modifications?
1st gen mASOs?

A

increase nucelase -resistance
thus increase BA (less cleavage of ASO)

62
Q

what are the disadvantages of PS-modifications?
1st gen mASOs?

A

reduces binding affinity (Tm reduces by 0.5degrees per nucleotide)
increases non-specific effects by interactions w cell surface + other cellaulr proteins

63
Q

what are generation two modified antisense oligonucleotides?

A

changes to 2’ position of sugar

eg:
2’-O-MOE
2’-O-methyl
2’-O-Fluoro

64
Q

what are generation two modified antisense oligonucleotides?
2nd gen

A

increase resistance to nucleases
tight binding between ASO and RNA

but reduce RNase H activity

65
Q

how do you overcome the disadvantages of using generation 2 modified antisense oligonucleotides?

A

ASOs w central 10-PS-modifies 2’deoxynucleotides flanked by 5 2’OMe/O-MOE nucelotides each side

66
Q

what are generation three modified antisense oligonucleotides?

A

ASOs w changed in sugar region, developed w non-charged backbone

67
Q

name a gen 3 ASO other than PNA?

A

locked nucelic acids LNAs conformationally restricted nucelotide containing a 2’-O-4’-C-methylene bridge in b-D-ribofuranosyl configuration

68
Q

what are the advantages/disadvantages of Peptide Nucleic Acid (PNA)?

A
  • higher binding affinity to complementary RNA and DNA than RNA-RNA and DNA-DNA duplexes
  • more stable towards nucelases + peptidases
  • good stability in vivo
  • can bind DS DNA -> transcriptional arrest: stop RNA made in nucleus

but: not substrates for RNase H, thus only produce effects by steric inhibition of mRNA translation

69
Q

what are the advantages/disadvantages of Locked Nucleic Acid (LNA)?

A
  • increased affinity towards target mRNA
  • resistant to nucelase degradation

but not processed by RNase H, but can be incorporated into RNA + DNA as w PNA, which then reintroduces RNase H activity

70
Q

What affects ASOs excretion from the body?

A

The interaction of ASOs with plasma proteins prevents their excretion in the urine.

  • Notably, binding to high-affinity plasma proteins does not lead to better antisense activity.

All factors need to be balanced

71
Q

what affects affinity of pp to ASOs?

A

type of chemical modifications made to ASOs.
Single stranded, PS-modified ASOs exhibit high affinity for plasma proteins and prevent excretion.

72
Q

what oligonucleotides have low pp binding?

A

Uncharged oligonucleotides
PNAs and morpholino
have low plasma protein binding and are rapidly cleared.

73
Q

T/F
ASOs DO NOT affect proteins that alr expressed, instead PREVENT protein expression

A

true
think abt mechanism

74
Q

what 2 things can ASOs target both

A

cellaulr (host) RNAs and viral RNAs

75
Q

possible to detect changed in RNA lvls how long after admin ASOs in wohle organism work 3-4hrs in cell?

A

12-24hrs

76
Q

why 2nd gen ASOs allow weekly and biweekly dosing? advantageous as allows admin by injection

A

produce effects 10-15 days after single admin

77
Q

disadvnatages of rna therapeutics?

A

RNA therapeutics, such as siRNA and antisense oligonucleotides (ASOs), have revolutionized the field of gene therapy. However, they have several limitations, including:

  • Poor stability: RNA is prone to degradation by nucleases, which limits its stability and half-life in biological fluids.
  • Limited tissue penetration: RNA is relatively large and hydrophilic, which makes it difficult to penetrate cell membranes and reach its target site.
  • Immunogenicity: RNA is recognized by the immune system as a foreign substance, which can lead to unwanted immune responses.
  • Off-target effects: RNA can interact with unintended mRNA targets, leading to off-target effects.

Lab 3 (21 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions