Biomolecules Flashcards
What is the difference between DNA and RNA?
DNA has no -OH group on 2’ atom
What is a nucleotide and nucleoside difference?
Nucleoside - sugar and base
Nucleotide - sugar, base and connected to phosphate
What is an anti and syn nucleoside?
What is the α and β anomer of a nucleoside?
What is the config of the sugar ring?
Sugar ring not flat
CH2OH and nucleobase above sugar ring
2’ and 3’ OH groups below sugar ring
What are the base structures?
What are the watson-crick base pairs?
What is 5’ to 3’ and vice versa?
5’-GCAT-3’ = GCAT
How does antisense oligonucleotides used for therapy?
Antisense oligo DNA bonds to mRNA from the virus - forms duplex
Duplex bonds to RNase-H which hydrolyses mRNA to prevent translation
What is solid-phase synthesis of oligonucleotides?
Solid support on beads: controlled pore glass or polystyrene
Molecules bound to this support or resin during synthesis
What are the benefits of solid-phase over solution chemistry?
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
What is the direction of the phosphoramidite method?
3’ to 5’ direction
Takes advantage of the more reaction 5’-OH group, as not hindered
What are the steps of the phosphoramidite method?
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
What is the 1st step in the phosphoramidite method?
Detritylation with TFA/TCA (strong acid)
What is useful in the detritylation step of the phosphoramidite method?
The cation has a delocalised structure and is coloured
Step can be monitored by UV/Vis
Why is tritylation required?
Prevents 5’-OH polymerizing as the resin is functionalization
What is the 2nd step in the phosphoramidite method?
Activation and coupling
Mix of nucleoside phosphoramidites with tetrazole (weak acid) which reacts with a nucleoside on the resin
What is the 3rd step in the phosphoramidite method?
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
What is the 4th step in the phosphoramidite method?
Oxn of P(III) to P(V) using iodine, water and pyridine
As unstable to acid, so must protect
How do you cleave oligonucleotide from support in phosphoramidite method?
Succinyl linker used as can be cleaved by conc aq ammonia
How are synthetic oligos purified?
Gel filtration - separates components according to size
Also HPLC purification
Impurities include truncated oligos, cleaved PG, chemically-modded bases
What is depurination?
Side reaction in oligo synthesis, loss of purine
What can occur after depurination?
DNA chain cleavage
What occurs in mutagenesis?
DNA chain grows on thymine base
What is a side reaction with acrylonitrile in oligo synthesis?
Need a scavenger
What is the scavenger required for acrylonitrile?
Diethylamine
What are the steps in phosphoramidite monomer synethsis?
1) Protection of amines (not needed for T)
2) Tritylation of 5’ OH
3) Protected phosphorylation of 3’ OH
What is the 1st step of phosphoramidite monomer synthesis?
What is the 2nd and 3rd steps of phosphoramidite monomer synthesis?
Cannot react at 3’ OH as sterically hindered
How are nucleotides added to the synthetic resin?
What are the differences of DNA and RNA synthesis?
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
What occurs in RNA synthesis if 2’-OH not protected?
Backbone migration or cleavage
What are the steps in RNA synthesis?
1) Acylation
2) Tritylation
3) TMSCl protection of 2’-OH
4) Phosphorylation of 3’-OH
5) Deprotection of 2’-OH
What is the first step in RNA synthesis?
What are the 2nd and 3rd steps of RNA sytnthesis?
What is the 4th step of RNA synthesis?
What is epigenetics caused by?
DNA with modified nucleobases which interact with proteins to regulate gene expression
What are endo and exonucleases?
Endonuclease - enzyme that cleaves within dsDNA
(specific to a sequence)
Exonuclease - enzyme that cleaves from termini of ssDNA and/or dsDNA
(normally termini specific)
What is DNA ligase?
Enzyme which covalently joins 5’ phosphate and 3’-OH of two dsDNAs
What is the use of PCR?
Exponential amplification of dsDNA
What are the number of copies in PCR?
No of copies = 2N
where N is the number of cycles (exponential)
What are the steps of PCR?
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
What direction does DNA polymerase work for replication?
5’ to 3’ direction
Active site holds new nucleosides in correct direction
How do proofing exonuclases work?
3’ to 5’ exconuclease remvoes incorrect bases at the end
What types of ends do endonucleases make?
Always leave a 5’ phosphate and 3’ hydroxyl
What is required for a ligase?
ATP required
Enzyme joins together dsDNA/dsRNA with “sticky” duplex forming ends
Can do with blunt but less efficient
What are the steps of ligase action?
How are restriction and ligation enzymes used?
Restriction enzymes cut DNA with sticky ends
Fragments with matching overhangs introduced
Ligase causes phosphodiester bond to be formed
What is a vector and the transformation process?
Vector - plasmid
Transformation - insertion into bacteria
Single colony of bacteria will produce a single DNA clone
What is polymerase chain assembly?
Larger and larger fragments of genes produced until full length template is synthesised
How are errors in gene synthesis corrected?
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
Why are 2 different endonuclease sites used for inserting genes into vectors?
Otherwise the backbone can religate and DNA can insert in both directions
What is Gibson assembly?
1-step isothermal recombination of DNAs
Requires exonuclease, polymerase, and a ligase
No restriction sites required
What is the mechanism of the gibson assembly?
What is a problem with PCR?
Mutations are also replicated
What is error-prone PCR?
Use unequal dNTPs, add Mn2+, etc.
Increases error rate
Why is directed evolution used for asymm synthesis?
When completed:
* increase in yield and stereselec
* reduction in waste
* elimination of heavy metals
* reduction in cost
* avoids need for high-pressure hydrogenation equipment
How is mRNA synthesised?
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
How can you do RNA analysis?
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
How can you complete reverse transcription?
- 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
What is Western Blotting?
Used to analyse specific proteins form a cell
Can therefore detect changes in levels of specific single proteins without purification (uses antibodies)
How does a protein recognized?
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
How does a protein recognized?
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
What is the mechanism for luminol chemiliminescence?
What is RNA interference (RNAi)?
small strands of dsRNA containing an antisense strand complementary to a seqence in the target mRNA
Then RISC binds and cleaves the target mRNA
How does CRISPR-Cas9 work?
Gene editing tech
Breaks ds at specific place in genome by Cas9 and guide RNA (gRNA)
Then knock-out or knock-in occurs
What is knock-out in CRISPR?
Frame-shift disrupts original gene sequence
Means cannot express a protein
What is knock-in in CRISPR?
New sequence gives new function
