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“Viral Smart Bombs”

“Viral Smart Bombs”. Joe Levine April 27, 2007 Caltech iGEM Team Brainstorm. Outline. Why viruses for iGEM? Project outline: targeting viruses to specific cells. Technical point: An in vivo aptamer selection scheme to increase specificity. Viruses?. Pro:

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“Viral Smart Bombs”

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  1. “Viral Smart Bombs” Joe Levine April 27, 2007 Caltech iGEM Team Brainstorm

  2. Outline • Why viruses for iGEM? • Project outline: targeting viruses to specific cells. • Technical point: An in vivo aptamer selection scheme to increase specificity.

  3. Viruses? • Pro: • Certain systems (l, M13, T7) are well understood. • Both molecular biology and biochemistry well characterized. • Wide variety of useful mutants available. • Short life cycle, short iteration times? • Even used for undergraduate lab classes (e.g. MIT 20.109, http://openwetware.org/wiki/20.109) • Con: • Hard to visualize • Usually assayed through interaction with other organisms • Is there an interested resident faculty virus expert? • Opinion: The relative ease and speed of viral manipulations may fit the limitations of iGEM, but lack of local expertise is a real worry.

  4. Virus Life Cycle

  5. Outline • Why viruses for iGEM? • Project outline: targeting viruses to specific cells. • Technical point: A completely in vivo aptamer selection scheme to increase specificity.

  6. Project Overview: Targeting Viruses Specifically & Synthetically • Can we engineer viruses to target cells expressing specific proteins or mRNA’s? • mRNA’s: easier? • Proteins: foreign proteins, or post translationally modified (phosphorylated, prions, etc)? • Virus infects and lyses cells containing target molecule. • A construct in the virus prevents it from killing other cells.

  7. Nucleic Acid Based Sensors? B Target mRNA A B A* (RBS) • Detecting mRNA’s: • Stem loop invasion • Tuneable specificity • Designable1? • Detecting other molecules: • Aptamers2,3,etc • Protein conformers, foreign molecules, etc. • In vivo functioning in the presence of confounding molecules may require in vivo selection B*/2 A* A* B*/2 A A* (RBS) Viral mRNA Covalent modification Prions • Isaacs et al. 2004. • Ellington and Szostak, 1990. • Bayer & Smolke, 2005.

  8. The plan and system • E. coli makes a sensible initial host cell • Well studied phages: l, T7, M13 naturally target E. Coli • Easy to induce varying expression of heterologous target. • Picking targets of increasing difficulties: • 1st stage: A well defined mRNA (GN?) • 2nd stage: A heterologous mRNA • 3rd stage: An easy heterologous protein (lysozyme?) • 4th stage: A difficult protein (phosphorylation state of two component response regulator?) • Hopefully goals #1, and maybe #2, achievable. #3 would be outstanding. I would be absolutely shocked to see #4 over the summer.

  9. What specific genes to block? • Early viral life cycle genes • The antiterminator ‘N’ in l • DN virus strains do not infect1 • Other strains (M13, T7) might have similar candidates • These should prevent the virus from beginning its developmental life cycle • Maybe target aptamers to later stage genes, to allow aptamers time to mature

  10. Possible problems • Proteins may be a challenging substrate for aptamers to recognize. • Specificity (false positives) will be a major issue. • How will the devices work with the virus? • Can we address these problems with in-vivo selection schemes?

  11. Selecting aptamers in vivo • Aptamers are typically selected using in vitro assays. • These selection methods do not mimic in vivo constraints: • Lots of background molecules • Unsure if random proteins will trigger aptamers. • A selection scheme that mimics cellular environment might help.

  12. 8 8 8 “In Vivo SELEX” – Positive Selection • Important caveat: Nvirus << Ncell, else cells will get infected by multiple viruses and unsuccessful aptamers will piggyback on successful ones. Lysis by successful infectors 8 8 8 Infect 8 8 8 8 Purify phage, repeat if desired.

  13. 8 8 8 8 8 8 8 “In Vivo SELEX” – Negative Selection • Want to select for viruses that do not infect cells. This is hard. • Penalize aptamers opening in the absence of ligand. Gene remains repressed Lysis N Repression non-specifically relieved N N Promoter Counterselection kills cells Counterselection N Repressing structure E. Coli counterselection: sacB (sucrose sensitivity), rpsL (streptomycin sensitivity)?

  14. % selectsim.m % % selection dynamics. % Nij is a vector of the number of viruses that are of type ij, % with: % i: 0/1 if aptamers don't/do open in the present of ligand % j: 0/1 if aptamer don't/do open in the absence of ligand % % Mijk is a number describing the multiplier that virus type ij goes % through on round k. ij are the same indices as above, k is 0 for negative % selection and 1 for positive selection. Nrounds = 100; Ratio = 5; M000 = Ratio; % aptamers not opening in either case multiply during negative selection. M001 = 1; % aptamers not opening in either case do not multiply during positive selection. M010 = 1; % aptamers opening only in the absence of ligand do not multiply during negative selection. M011 = 1; % aptamers opening only in the absence of ligand do not multiply during positive selection. M100 = Ratio; % aptamers opening only in the presence of ligand multiply during negative selection. M101 = Ratio; % aptamers opening only in the presence of ligand multiply during positve selection. M110 = 1; % aptamers opening all the time do not multiply during negative selection. M111 = Ratio; % aptamers opening all the time do multiply during positive selection. N00 = zeros(1,Nrounds); N00(1) = 0.33; N01 = zeros(1,Nrounds); N01(1) = 0.33; N10 = zeros(1,Nrounds); N10(1) = 0.01; N11 = zeros(1,Nrounds); N11(1) = 0.33; for j = 2 : Nrounds if mod(j,2) % even round = negative selection N00_new = M000*N00(j-1); N01_new = M010*N01(j-1); N10_new = M100*N10(j-1); N11_new = M110*N11(j-1); total = N00_new + N01_new + N10_new + N11_new; N00(j) = N00_new/total; N01(j) = N01_new/total; N10(j) = N10_new/total; N11(j) = N11_new/total; else N00_new = M001*N00(j-1); N01_new = M011*N01(j-1); N10_new = M101*N10(j-1); N11_new = M111*N11(j-1); total = N00_new + N01_new + N10_new + N11_new; N00(j) = N00_new/total; N01(j) = N01_new/total; N10(j) = N10_new/total; N11(j) = N11_new/total; end end plot(1:Nrounds,N00,1:Nrounds,N01,1:Nrounds,N10,1:Nrounds,N11) legend('N_{00}','N_{01}','N_{10}','N_{11}')

  15. Sketch of selection dynamics? Populations oscillate on alternate rounds due to alternating selection types.

  16. Lots of Details to Work Out!!! • Which virus to use • Culture conditions • How exactly to clone into them. • Etc…? • Thanks for your attention!

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