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Biotechnology > Antisense RNA > Flashcards

Antisense RNA Flashcards

1
Q

Antisense Definition

A

shorter fragments of nucleic acid complimentary to the sense strand

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2
Q

Antisense function

A 
blocks mRNA (sense strand) blocks translation and expression --> reduction protein
also alter mRNA production/turnover
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3
Q

Ribozymes

A

Catalytic RNAs/enzymes
endonucleases
anti-sense elements dictate

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4
Q

Antisense can change behaviour of mRNA by (3)

A

block protein binding
disrupt RNA structure
target RNA for degradation

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5
Q

Conserved sequences

A

5’ and 3’ splice sites
branch site,
these define the limits of introns
—>recruit the splicing machinery required to remove intron and join exons

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6
Q

Step 1 of splicing

A

cut at 5’ splice –> bonds branch site

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7
Q

Step 2 of splicing

A

Cut at 3’ splice site to form ‘lariat’ (loop)

ligation of 2 exons

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8
Q

Translation mechanism

A

complex around 5’ prime end of mRNA and ribosome sub unit
eiF4E and G bind cap
eiF4G bind polyA binding protein at 3’ end - circularise mRNA

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9
Q

Circularised mRNA leads to

A

Inintiation linked to termination

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10
Q

Natural anti-sense RNA is due to

A

bi-transcription of genes
e.g. Msx1 - murine tooth development
Frq - cicardian rhythm

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11
Q

Therapeutic use of antisense

A

correct defects in expression from mutations

e.g. thalassaemia

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12
Q

Thalassaemia

A

splicing in wrong place

incorporation of introns

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13
Q

Duchenne Muscular Dystrophy

A

mutation in 23 stop codon

antisense used to create shortened dystrophin instead of truncated

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14
Q

Spinal Muscular Atrophy

A

Mutation in exon 7
exclusion of exon - enhance sequence element mutation
unstable, shorter form
mRNA
antisense brings in SF2/ASF protein blocked by mutation by binding to it

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15
Q

Most mutations

A

stop mRNA and protein being produced

unstable

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16
Q

Antisense problem

A

unstable
difficult to transfer to cell
natural mechanisms in cell block function and degrade

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17
Q

Solutions to antisense problems

A

modify ribose/backbone
change phosphate
change hydrogen

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18
Q

Morpholino

A

ring structure instead of ribose

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19
Q

Benefits of modified nucleic acids (3)

A

stability
easier delivery
not recognised as nucleic acids - not sequestered

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20
Q

Produce antisense in cell

A

by introducing gene in cell that does so

e.g. U7 sRNA

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21
Q

Ribozyme action (5)

A 
RNA ligation and cleavage
RNA virus genomic replication
Intron splicing 
cleave tRNA
peptide bond formation
22
Q 
Hammerhead ribozyme  (4)
example
function
A

plant RNA viruses
cleave individual genomic RNAs during rolling cell replication
produce long linear genome
usually cis cleavage

23
Q

Hammerhead structure

A

T junction

cleavage site at H

24
Q

Adapt hammerhead (2) to
by…
possible uses

A

to trans
sequence stems I and II to target any sequence
possibly use in cancer and HIV

25
Q

Aptamers/decoys

not antisense

A

short RNA sequence - bind specific substrate
block activity
engineered
SELEX used to select interacting molecule

26
Q

SELEX definition

A

Systematic evolution of ligands by exponential enrichment

27
Q 
SELEX procedure (5)
evolution
A 
random RNAs
bind to target
enrich 
RT into DNA template
select RNA bind tightly
28
Q

Inhibit HIV replication by aptamer

specific protein

A

TAR decoy aptamer

bind TAT protein

29
Q 
Aptamer use (4)
examples
A

target validation
diagnostic
therapeutics
screening

30
Q

RISC definition

A

RNA induced silencing complex

protein and miRNA/siRNA

31
Q

30nt pair dsRNA does not

A

not induce interferon response

32
Q

siRNA (4)

features

A

21-23 nucleotide
perfect complimentary to RNA
viral defence mech
degradation RNA - endonucleolytic cleavage

33
Q

miRNA (4)

features

A

21-23 nucleotides
imperfect complimentary
gene regulatory mechanism 33- 80%
block in translation - deadenylation, decapping and degradation

34
Q

Pri-miRNAs —> miRNA/siRNA (4)

A

longer precursor cut by Drosha
recognised and cut by Dicer
recruit Ago with other factors –>RISC
Ago drives repression of translation

35
Q

miRNA translation

A

RNA pol II and III
independent genes or within pre-mRNA introns
Multi-miRNA precursor cleaved to individual

36
Q

dsRNAs (3)

A

part immune defence against exogenous genetics
e.g. transposons and viruses
both produce dsRNA
viruses can suppress RNAi

37
Q

DICER domains

A

dsRNA binding domain
Paz domain
2 RNAse III domain - catalytic

38
Q

Paz domain of dicer

A

recognise 3’ end
preferentially 3’ overhang
used to measure where to cleave - molecular ruler principle
position RNAse III domains

39
Q

RNAse III domain dicer

A

2 active sites

cut RNA each side

40
Q

RISC function

A

directs either mRNA cleavage or translational repression
catalytic component - Argonaute
Piwi domain - cleaves mRNA

41
Q

Piwi domain of argonaute

A

resembles RNase H

PAZ domain positions it

42
Q

Dicer complex hands to

A

Argonaute complex

43
Q

Cap - dependent regulation translation (4)

A

micro RNA bind 3’ end untranslated between stop codon and polyA tail
- displace eiF4E and G
argonaute interacts with miRNA and cap
recruit enzyme for degrad

44
Q

Direct regulation translation

A

inhibition

45
Q

Indirect regulation translation

A

de-adenylation

46
Q

after miRNA regulation action

A

move to P bodies

mRNA degradation

47
Q

miRNA production linked when cell is

A

stress –>
oxidative stess
AA starvation

48
Q

siRNA and miRNA delivery

A

transfection
transcription of two complimentary RNA/loop structure
cojugation with cholesterol/liposomes/peptide, polymers/antibodies
nano particles
viral vectors
stem
aptamer conjugation - bind extracell receptor

49
Q

siRNA design

A

modified 2’ base overhangs

50
Q

shRNA can be

A

taken up by cells and used for function

51
Q

Viral delivery siRNA/miRNA

A

lentiviral delivery

or adenoviral

52
Q

Multiple treatment for HIV (3)

A

ribozyme target cell mRNA - resistant/slow progression
TAR decoy target viral protein - bind and inactivate
shRNA target viral RNAs - inhibit

Biotechnology (7 decks)

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