Synthesis

Question 1

How are nucleic acids made?

Answer 1

• nucleosides undergo chemical synthesis to form oligonucleotides
• oligonucleotides undergo enzymatic synthesis to form polynucleotides

Question 2

What are oligonucleotides (oligos) /polynucleotides (genes)
- number of bases
- what they are used for

Answer 2

oligonucleotides
- < 200 bases
- PCR, profiling, diagnosis, sequencing

polynucleotides
- biotechnology
- protein expression
- vaccines

Question 3

What are chemical synthesis disconnections?

Answer 3

• usually break bonds between carbon and other atoms
• phosphate linkage is a good site since then each base can be added step-by-step (target C - O bond where O on phosphate group)

Question 4

What is solid phase synthesis of oligonucleotide

Answer 4

• introduce gene to bead (glass or polystyrene) one at time onto surface of bead until we have the primary structure
• then cleave from bead at end and we have nucleic sequence that we can do what we want with
• can scale up with multiple beads

Question 5

Why is a bead used in solid phase synthesis of oligonucleotides?

Answer 5

• to perform each step in solution would require a purification step at each stage
• easier to put on bead so do not have to purify at each stage
• by attaching one end of strand to solid support the excess reagents and side products can simply be washed away
• beads have little cavities on surface of bead to maximise surface area to be able to have as many interactions as possible

Question 6

Is solid phase synthesis of oligonucleotides done manually or automatically?

Answer 6

• it is done automated in benchtop machine in lab
• automation allows us to scale this up

Question 7

What is synthetic cycle of solid phase synthesis oligonucleotides?

Answer 7

1 - couple then wash
2 - cap then wash
3 - oxidise then wash
4 - deprotect then wash

Question 8

What happens in coupling stage of solid phase synthesis of oligonucleotides?

Answer 8

• attachment of nucleosides
• use DMT as protecting group to prevent further reactions and terminate everything
• the efficiency of this reaction defines how well we are at making target gene
• if 1 in 200 beads fail then yield of 50 bases is only 78.2 %
• if 20 in 200 beads fail then yield of 50 bases is only 0.6 %

Question 9

What happens in the capping stage of solid phase synthesis of oligonucleotides?

Answer 9

• must catch failed strand as do not want to produce something we don’t want
• might be dangerous protein so important to do capping to delete mutations and catch them at a time it is still relevant
• capping = killing unreacted strands
• 1 coupling failure every 200 strands

Question 10

What happens if the unreacted strands aren’t capped during the second stage of solid phase synthesis of oligonucleotides?

Answer 10

• leads to deletion mutations
• primer may not bind
• error will be carried forwards
• other biochemistry altered

Question 11

What happens during oxidation stage of solid phase synthesis of oligonucleotides?

Answer 11

• iodine is oxidising agent
• THF solvent used
• water and pyridine also required
• P=O bond obtained and pyridium iodide formed as side product

Question 12

What happens during deprotection stage of solid phase synthesis of oligonucleotides?

Answer 12

• removal of DMT using dichloroacetic acid in dichloromethane
• as do not want this on final synthetic of base pairs
• trityl cation is stabilised by resonance – making it easy to remove
• deep yellow/orange colour used to measure coupling efficiency

Question 13

What are two ways to purify oligonucleotides?

What does this involve?

Answer 13

• PAGE purification
• HPLC purification
• failed strands have to be removed in either one of these two ways

Question 14

What is PAGE purification?

Answer 14

• we know number of base pairs we want
• run a gel and find length that we want for primary structure
• find this section in gel, cut out or take it out (freeze and squeeze)
• older approach

Question 15

What is HPLC purification?

Answer 15

• allows us to select whatever we want depending on column
• normal phase - polar molecules will stick to column
• reverse phase - non-polar molecules will stick to column
• leave DMT (trityl) group on - very hydrophobic so any group with it on is easy target
• easy to separate full length strands from failed
• once separated needs to be held at a stable pH in solution and then freeze dried for storage as solid

Question 16

What is required to get oligonucleotide to gene?

Answer 16

• maximum length can chemically synthesise from oligonucleotide is 200 bases and typical length of chemically synthesised oligonucleotide is 50 bases so need to do a lot to get full length gene
• need to get full length gene…
• …at commercial scale
• …into RNA (we create DNA but we need RNA)
• …stable enough to be effective in the body

Question 17

What are two forms of gene synthesis?

Answer 17

• ligation
• polymerase cycling assembly

Question 18

What is ligation in gene synthesis?

Answer 18

• use enzyme ligase to bridge between pairs to join fragments together to make a single gene (ligase bridges between phosphate groups)
• need to do PCR amplification to increase size
• good technique as not many errors (controlled and precise) as all bases of final gene is chemically synthesised
• suitable for <2k base genes as relies on chemical synthesis
• only certain limited number of uses of how it can be applied at this level

Question 19

What is polymerase cycling assembly in gene synthesis?

Answer 19

• get fragments to offset together with gaps of 20-30 bases that aren’t artificially synthesised
• enzyme polymerase fills in the gaps between fragments
• as we have slightly less control we get more errors e.g. base pairs that are wrong
• have to cut off point of an error with enzymes (cut of enzymatic fragmentation at error side)
• melt, anneal, and polymerase to produce target gene
• less chemical synthesis so longer genes are possible >2k base genes but more problems and potentials for error

Question 20

How is polymerase used in body?

Answer 20

• involved in making and fixing DNA
• it identifies end of DNA that needs fixing and adds nucleotides to 3’ end of DNA

Question 21

After gene synthesis how do we purify and scale up?

Answer 21

• insert target gene into plasmid
• transform into bacteria
• plate and grow
• sequence colonies (ones that have managed to grow without inserting lots of defects)
• not too good at being reproducible so must check got what need
• extract relevant DNA from positive colonies to give us target DNA of what we are interested in

Question 22

How to produce RNA vaccines?

Answer 22

• linearise and trim plasmid DNA to get linear DNA
• linear DNA undergoes transcription to form RNA
• put RNA in shell of lipids to protect it from body
• RNA is what goes into body - to reprogram cells to allow us to fight stuff e.g. covid