Synthesis Flashcards
How are nucleic acids made?
- nucleosides undergo chemical synthesis to form oligonucleotides
- oligonucleotides undergo enzymatic synthesis to form polynucleotides
What are oligonucleotides (oligos) /polynucleotides (genes)
- number of bases
- what they are used for
oligonucleotides
- < 200 bases
- PCR, profiling, diagnosis, sequencing
polynucleotides
- biotechnology
- protein expression
- vaccines
What are chemical synthesis disconnections?
- 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)
What is solid phase synthesis of oligonucleotide
- 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
Why is a bead used in solid phase synthesis of oligonucleotides?
- 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
Is solid phase synthesis of oligonucleotides done manually or automatically?
- it is done automated in benchtop machine in lab
- automation allows us to scale this up
What is synthetic cycle of solid phase synthesis oligonucleotides?
1 - couple then wash
2 - cap then wash
3 - oxidise then wash
4 - deprotect then wash
What happens in coupling stage of solid phase synthesis of oligonucleotides?
- 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 %
What happens in the capping stage of solid phase synthesis of oligonucleotides?
- 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
What happens if the unreacted strands aren’t capped during the second stage of solid phase synthesis of oligonucleotides?
- leads to deletion mutations
- primer may not bind
- error will be carried forwards
- other biochemistry altered
What happens during oxidation stage of solid phase synthesis of oligonucleotides?
- iodine is oxidising agent
- THF solvent used
- water and pyridine also required
- P=O bond obtained and pyridium iodide formed as side product
What happens during deprotection stage of solid phase synthesis of oligonucleotides?
- 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
What are two ways to purify oligonucleotides?
What does this involve?
- PAGE purification
- HPLC purification
- failed strands have to be removed in either one of these two ways
What is PAGE purification?
- 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
What is HPLC purification?
- 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
What is required to get oligonucleotide to gene?
- 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
What are two forms of gene synthesis?
- ligation
- polymerase cycling assembly
What is ligation in gene synthesis?
- 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
What is polymerase cycling assembly in gene synthesis?
- 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
How is polymerase used in body?
- involved in making and fixing DNA
- it identifies end of DNA that needs fixing and adds nucleotides to 3’ end of DNA
After gene synthesis how do we purify and scale up?
- 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
How to produce RNA vaccines?
- 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
