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Year 2 - Chem > Biomolecules > Flashcards

Biomolecules Flashcards

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1
Q

What is the difference between DNA and RNA?

A

DNA has no -OH group on 2’ atom

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2
Q

What is a nucleotide and nucleoside difference?

A

Nucleoside - sugar and base

Nucleotide - sugar, base and connected to phosphate

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3
Q

What is an anti and syn nucleoside?

A
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4
Q

What is the α and β anomer of a nucleoside?

A
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5
Q

What is the config of the sugar ring?

A

Sugar ring not flat

CH2OH and nucleobase above sugar ring

2’ and 3’ OH groups below sugar ring

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6
Q

What are the base structures?

A
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7
Q

What are the watson-crick base pairs?

A
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8
Q

What is 5’ to 3’ and vice versa?

A

5’-GCAT-3’ = GCAT

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9
Q

How does antisense oligonucleotides used for therapy?

A

Antisense oligo DNA bonds to mRNA from the virus - forms duplex

Duplex bonds to RNase-H which hydrolyses mRNA to prevent translation

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10
Q

What is solid-phase synthesis of oligonucleotides?

A

Solid support on beads: controlled pore glass or polystyrene

Molecules bound to this support or resin during synthesis

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11
Q

What are the benefits of solid-phase over solution chemistry?

A

Large XS of reagents can be added to drive reactions quickly to completion

Impurities and XS reagents washed away, so less purification steps

Can be automated

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12
Q

What is the direction of the phosphoramidite method?

A

3’ to 5’ direction

Takes advantage of the more reaction 5’-OH group, as not hindered

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13
Q

What are the steps of the phosphoramidite method?

A

Step 1: activation and coupling - to 5’-OH

Step 2: capping - stops unreacted 5’ from reacting

Step 3: oxidation - P(III) to P(V)

Step 4: Detritylation - for next cycle

After finished, oligonucleotide cleaved from support, deprotected, & purified

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14
Q

What is the 1st step in the phosphoramidite method?

A

Detritylation with TFA/TCA (strong acid)

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15
Q

What is useful in the detritylation step of the phosphoramidite method?

A

The cation has a delocalised structure and is coloured
Step can be monitored by UV/Vis

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16
Q

Why is tritylation required?

A

Prevents 5’-OH polymerizing as the resin is functionalization

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17
Q

What is the 2nd step in the phosphoramidite method?

A

Activation and coupling
Mix of nucleoside phosphoramidites with tetrazole (weak acid) which reacts with a nucleoside on the resin

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18
Q

What is the 3rd step in the phosphoramidite method?

A

Capping to prevent them reacting in next coupling step
Mix of acetic acid, N-methylimidazole and pyridine
Reacts with any acetitc acid formed to prevent acid-catalysed detritylation

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19
Q

What is the 4th step in the phosphoramidite method?

A

Oxn of P(III) to P(V) using iodine, water and pyridine
As unstable to acid, so must protect

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20
Q

How do you cleave oligonucleotide from support in phosphoramidite method?

A

Succinyl linker used as can be cleaved by conc aq ammonia

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21
Q

How are synthetic oligos purified?

A

Gel filtration - separates components according to size
Also HPLC purification

Impurities include truncated oligos, cleaved PG, chemically-modded bases

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22
Q

What is depurination?

A

Side reaction in oligo synthesis, loss of purine

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23
Q

What can occur after depurination?

A

DNA chain cleavage

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24
Q

What occurs in mutagenesis?

A

DNA chain grows on thymine base

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25
Q

What is a side reaction with acrylonitrile in oligo synthesis?

A

Need a scavenger

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26
Q

What is the scavenger required for acrylonitrile?

A

Diethylamine

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27
Q

What are the steps in phosphoramidite monomer synethsis?

A

1) Protection of amines (not needed for T)
2) Tritylation of 5’ OH
3) Protected phosphorylation of 3’ OH

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28
Q

What is the 1st step of phosphoramidite monomer synthesis?

A
29
Q

What is the 2nd and 3rd steps of phosphoramidite monomer synthesis?

A

Cannot react at 3’ OH as sterically hindered

30
Q

How are nucleotides added to the synthetic resin?

A
31
Q

What are the differences of DNA and RNA synthesis?

A

2’-OH in RNA makes more difficult

t-butyldimethylsilyl group used to protect 2’-OH group, removed using TBAF at end

Longer coupling times due to steric of pg

Capping reagent changed as to negate effect of acyl exchange at protected amino groups of bases

32
Q

What occurs in RNA synthesis if 2’-OH not protected?

A

Backbone migration or cleavage

33
Q

What are the steps in RNA synthesis?

A

1) Acylation
2) Tritylation
3) TMSCl protection of 2’-OH
4) Phosphorylation of 3’-OH
5) Deprotection of 2’-OH

34
Q

What is the first step in RNA synthesis?

A
35
Q

What are the 2nd and 3rd steps of RNA sytnthesis?

A
36
Q

What is the 4th step of RNA synthesis?

A
37
Q

What is epigenetics caused by?

A

DNA with modified nucleobases which interact with proteins to regulate gene expression

38
Q

What are endo and exonucleases?

A

Endonuclease - enzyme that cleaves within dsDNA
(specific to a sequence)

Exonuclease - enzyme that cleaves from termini of ssDNA and/or dsDNA
(normally termini specific)

39
Q

What is DNA ligase?

A

Enzyme which covalently joins 5’ phosphate and 3’-OH of two dsDNAs

40
Q

What is the use of PCR?

A

Exponential amplification of dsDNA

41
Q

What are the number of copies in PCR?

A

No of copies = 2N

where N is the number of cycles (exponential)

42
Q

What are the steps of PCR?

A

Deaturation (~90C) - causes H bonds to break so single stranded

Annealing (~50C) - polymerase binds to new ssDNA on primers

Extension (~72C) - polymerase adds nucleosides to grow complementary strand and form 2xdsDNA

43
Q

What direction does DNA polymerase work for replication?

A

5’ to 3’ direction

Active site holds new nucleosides in correct direction

44
Q

How do proofing exonuclases work?

A

3’ to 5’ exconuclease remvoes incorrect bases at the end

45
Q

What types of ends do endonucleases make?

A

Always leave a 5’ phosphate and 3’ hydroxyl

46
Q

What is required for a ligase?

A

ATP required
Enzyme joins together dsDNA/dsRNA with “sticky” duplex forming ends

Can do with blunt but less efficient

47
Q

What are the steps of ligase action?

A
48
Q

How are restriction and ligation enzymes used?

A

Restriction enzymes cut DNA with sticky ends
Fragments with matching overhangs introduced
Ligase causes phosphodiester bond to be formed

49
Q

What is a vector and the transformation process?

A

Vector - plasmid

Transformation - insertion into bacteria

Single colony of bacteria will produce a single DNA clone

50
Q

What is polymerase chain assembly?

A

Larger and larger fragments of genes produced until full length template is synthesised

51
Q

How are errors in gene synthesis corrected?

A

Repair enzymes:
Cleave 2nd/3rd bond 3’ to mismatch on both strands
Single stand exonuclease then chews up overhanging ssDNA 3’ to 5’
Then polymerase chain assembly

52
Q

Why are 2 different endonuclease sites used for inserting genes into vectors?

A

Otherwise the backbone can religate and DNA can insert in both directions

53
Q

What is Gibson assembly?

A

1-step isothermal recombination of DNAs

Requires exonuclease, polymerase, and a ligase

No restriction sites required

54
Q

What is the mechanism of the gibson assembly?

A
55
Q

What is a problem with PCR?

A

Mutations are also replicated

56
Q

What is error-prone PCR?

A

Use unequal dNTPs, add Mn2+, etc.

Increases error rate

57
Q

Why is directed evolution used for asymm synthesis?

A

When completed:
* increase in yield and stereselec
* reduction in waste
* elimination of heavy metals
* reduction in cost
* avoids need for high-pressure hydrogenation equipment

58
Q

How is mRNA synthesised?

A

Process:
1) Initiation - RNA polymerase binds to promoter on DNA and forms first few bonds
2) Elongation - new strand has nucleotides added to it
3) Termination

59
Q

How can you do RNA analysis?

A

Relies on enzyme reverse transcriptase which synthesises a complementary DNA (cDNA) strand from RNA

RNAse H degrades RNA when bound to DNA

cDNA can then be amplified and analysed

60
Q

How can you complete reverse transcription?

A
  • Analyse mRNA using a poly-T tail, as m-RNA only has an A-tail
  • Random primers - used to analyse total RNA, not just mRNA
  • Gene-specific primers - used to analyse specific RNA sequences
61
Q

What is Western Blotting?

A

Used to analyse specific proteins form a cell
Can therefore detect changes in levels of specific single proteins without purification (uses antibodies)

62
Q

How does a protein recognized?

A

1 antibody bonds to protein of interest
2nd recognises primary antibody and is conjugated to the enzyme

Enzyme oxidises luminol and releases light via chemiluminescence

63
Q

How does a protein recognized?

A

1 antibody bonds to protein of interest
2nd recognises primary antibody and is conjugated to the enzyme

Enzyme oxidises luminol and releases light via chemiluminescence

64
Q

What is the mechanism for luminol chemiliminescence?

A
65
Q

What is RNA interference (RNAi)?

A

small strands of dsRNA containing an antisense strand complementary to a seqence in the target mRNA

Then RISC binds and cleaves the target mRNA

66
Q

How does CRISPR-Cas9 work?

A

Gene editing tech
Breaks ds at specific place in genome by Cas9 and guide RNA (gRNA)

Then knock-out or knock-in occurs

67
Q

What is knock-out in CRISPR?

A

Frame-shift disrupts original gene sequence
Means cannot express a protein

68
Q

What is knock-in in CRISPR?

A

New sequence gives new function

Year 2 - Chem (23 decks)

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