BIOS 390 Midterm exam I

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BIOS 390 Midterm exam I

• mechanism of mRNA vaccine

–Answer mRNA molecules in liposomes, uptaken by muscle cells, antigen-presenting start expressing encoded sequence. Interact with T cells and eventually produce antibodies

• mRNA vaccine design considerations

–Answer why –Answer goal is to make the viral protein (central dogma) what sequence –Answer comes from virology (e.g. covid spike protein –Answer interface between protein and receptor) how to deliver –Answer mRNA is not the storage form of information, will get translated

• advantages of mRNA vaccines

–Answer safety –Answer non-infectious, minimize risk of inser- tional mutagenesis (doesn't insert into genome the same way a DNA vector would) efficacy –Answer RNA modifications make RNA more stable, effective in vivo delivery production –Answer rapid, inexpensive, scalable manufacturing

• mRNA modifications to improve mRNA vaccine

–Answer -better prep can selectively degrade dsRNA to avoid recognition and removal by target cell (had side effects) 1 / 3

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-pseudocytidine reduces ability to be recognized as foreign mRNA

• ID Now PCR

–Answer -Isothermal –Answer uses reverse transcriptase instead of thermocycling to denature -reverse transcriptase has strand displacing ability -nicking endonuclease cuts one strand downstream of specific sequence 1) primer anneals to target sequence 2) RNA template and reverse transcriptase --DNA extension 3) Single strand of DNA template without RNA left 4) Reverse primer anneals, polymerase extends to make dsDNA 5) nicking enzyme nicks

• Reverse transcriptase role in isothermal PCR

–Answer Has strand displacing ability RNAse activity –Answer digests RNA strand of DNA/RNA hybrids

• elements needed for DNA synthesis

–Answer template, primer, dNTP, DNA polymerase

• need for primer for DNA

–Answer DNAP can't initiate de novo, useful to design amplifi- cation of specific target reduces complexity of assembling genome by gragments

• primer

–Answer template junction –Answer -complememtary -no overhanging 3' end (need 3'OH) 2 / 3

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-can have 5' overhang (can be useful to add restriction sites, mutation etc) -can have mismatch in middle -sometimes can have mismatch at 3'

• general features of DNAPs

–Answer Directionality Specificity Fidelity Processivity

• DNAP directionality

–Answer 5'-3' 3D structure of polymerase resembles right hand (catalysis in fingers/palm, thumb for processivity) 2-metal ion mechanism of catalysis in palm of hand

• DNAP specificity

–Answer -can differentiate whether incoming base matches template via conformational change -incorrect base pairs don't fit into active site -discriminate rNTP by steric hindrance w/discriminator amino acids

• O-helix of DNA polymerase

–Answer in closed conformation, O-helix swings down. Ty- rosine participates in aromatic base stacking, lys and arg interact with beta/gamma phosphates of NTP to catalyze phosphodiester bond

• DNAP fidelity

–Answer Most have exonuclease activity (i.e. can remove nucleotides) 3'-5' exonuclease activity increases fidelity by ability to remove incorrect nucleotides error rate 1/10^3 w/o 3-5 exo vs. 1/10^6, 1/10^7 w/

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