RNA Synthetic Biology

1. RNA Synthetic Biology Farren J Isaacs, Daniel J Dwyer, & James J Collins Nature Biotechnology May 2006 iGEM 2010 Journal Club 7/7/2010

2. Any sequence  diverse 2° structure and function • Interact with proteins, metabolites, other nucleic acids • Levels of modulation: • Transcription • Translation • Cis = same molecule • Trans = another molecule • Work mostly in bacteria and yeast RNA

3. RNA RNA RNA • Antisense RNAs • Riboregulators • sRNAs (small regulatory RNAs) • miRNAs • siRNAs • Riboswitches • Ribozymes

4. Controlling Gene Expression - overview • Antisense RNAs - silence expression by targeting specific mRNA sequences (physically obstruct machinery)

5. Small regulatory RNAs (sRNAs) repress andactivate(unlike antisense RNAs) bacterial gene expression in trans by base pairing with target RNAs • Chaperone proteins (Hfq) prevent sRNA degradation by RNAses; mediate mRNA – sRNA binding. • Stress response (heat, cold, oxidative)

6. Single-stranded microRNAs (miRNA) formed from cleavage of hairpin RNAs • Bind to 3’UTR region of mRNA • Mostly gene silencing; each miRNA  repress many mRNAs. • Possible positive regulation. • Conserved

7. Riboswitches contain aptamer domain sites— Highly specific pockets in the 5′ UTR of the mRNAs that bind ligands  conformational change in RNA structure  change in gene expression. Unlike ribozymes, use only changes in DNA conformation, no catalytic activity.

8. 1. Engineered Riboregulators Isaac et al 2004 http://www.nature.com/nbt/journal/v22/n7/pdf/nbt986.pdf • Regulate expression by interfering with ribosomal docking at RBS. • Goal: create a modular post-transcriptional regulation system that works with any promoter or gene. • In contrast to endogenous riboregulators - limited to specific transcriptional and regulatory elements.

9. Gene Repression • ‘Old’ way: antisense RNA (trans-acting) • ‘New’ way: form hairpin in 5′ UTR of mRNA  sequester RBS to inhibit translation initiation. [cis-repressed RNA (crRNA)]

10. Method • taRNA and crRNA • taRNA is regulated by PBAD (inducible), so can determine when translation is allowed • Gene expression is off when there is crRNA upstream of the gene (no taRNA is in the system). • taRNA present gene expression is turned back on. See next slide…

11. Modular: • crRNA can be inserted upstream of any gene • Can change levels of cis-repression and trans-activation with different promoters (tried with PLAC also) driving expression of taRNA and crRNA transcripts Unfolds hairpin to expose RBS (non-coding RNA [ncRNA])

12. Same idea, different figure pyrimidine-uracil-nucleotide-purine Images from Isaac 2004, Engineered riboregulators enable post-transcriptional control of gene expression

13. Measure GFP levels at controlled induction levels of taRNA • linear dependence between taRNA concentration and GFP expression. • Rapid response (GFP within 5 min of taRNA activation) • Tunable gene expression activation Blue – normal GFP Green – with taRNA and crRNA Red – with crRNA only Black – no GFP gene Image from Isaac 2004

14. What components enable this repression?To find out… • Compared activity of four crRNA variants with different degrees hairpin (stem sequence) complementarity in 5′-UTR with GFP reporter • Complementarity  98% of repression • Less complementarity in hairpin  less repression

15. Tweaking • Induced rational changes: • Alter GC content and size of the cis-repressed stem • Varied number of base pairs that participate in intermolecular pairings • incorporating RNA stability domain on the taRNA. • Increasing GC content in crRNA stem and having more base pairs participating in the taRNA-crRNA intermolecular interaction improved activation 8X (24 bp design) to 19X (25 bp design) from the crRNA repressed state.

16. Specificity • Designed four taRNA-crRNA riboregulator pairs. • To determine “orthogonality”, tested all 16 taRNA-crRNA combinations (4 cognate, 12 noncognate combos) • taRNA-crRNA interactions that expose the RBS require highly specific cognate RNA pairings Black and white bars – GFP fluorescence Dark and light grey – taRNA concentrations (pBAD promoter for taRNA)

17. A Note on Modularity • crRNA construct added to the gene needs to contain the RBS unless the gene's RBS is close enough to the complement to bind to it. • Small changes to a RBS can result in large changes in transcription rate • If the original RBS is not close enough to the complement in the crRNA and you want to keep the original transcriptional rate and level – need to redesign.

18. Application • Probe or modify translational dynamics of natural networks • Tool for studying isolated network components. • Generate translationally based reversible knockouts

19. Future – Engineered Riboregulators Two challenges: • Integrate rational design and evolution-based techniques to generate new and enhanced (e.g., ligand-modulated) riboregulation • More versatile; limited with inducible promoters • Eukaryote and mammalian cells – more tightly regulated/specific events and mechanisms. • Interfere with eukaryotic initiation factors that direct ribosomal subunits to mRNA. Similar to engineered prokaryotic version.

20. Rackham and ChinA network of orthogonal ribosome-mRNA pairs 2005 2. Engineered ribosome-mRNA pairs • Goal: Reduce interference with ribosome assembly, rRNA processing and cell viability • Rational design + directed evolution to manipulate ribosome-mRNAs specificities Blue = original ribosome; purple = second ribosome. Green = original mRNA; orange= duplicate. Evolution until pairs do not interact anymore. Image from Rackham and Chin 2005

21. Ribosome – mRNA pairs • Orthogonality is a way to eliminate pleiotropic effects. • Tailored interaction of ribosome-mRNA pairs so an engineered ribosome could translate only its engineered mRNA pair and not any endogenous mRNA • A native E. coli ribosome would not be able to initiate translation on an engineered mRNA • Developed two-step pos/neg selection strategy to evolve orthogonal ribosome-orthogonal mRNA (O-ribosome-O-mRNA) pairs that permit robust translation

22. Strategy 1. Select for mRNA sequences that are not substrates for endogenous ribosomes • mRNA library into E. coli • grew in presence of 5-FU to select againstmRNAs that could translate UPRT. • Viable cells had orthogonal mRNAs incompatible with endogenous ribosomes. 2. Transformed with library of mutant ribosomes and grown in chlor+ media • So only ribosomes that translate orthogonal mRNA pairs were selected for. • From 1011 clones, found four distinct O-mRNAs and ten distinct O-rRNA sequences

23. Positive selection: Chloramphenicol resistance (CAT gene). Negative selection: uracilphosphoribosyltransferase (UPRT). • Synthesized a library of all possible RBSs and another of all possible 16S rRNA anti-RBS sequences • > 109 unique mRNA-rRNA combinations Fused CAT (cat) and UPRT (upp) downstream of a constitutive promoter and RBS so the single transcript can be either positively or negatively selected.

24. A Follow-Up Study - Logic Gates • Can multiple orthogonal ribosomes simultaneously function in the same cell? • Combined several orthogonal pairs in a single cell • Constructed set of logical AND/OR gates: • AND gate: separately cloned the genes for two fragments—α and ω—of lacZ onto distinct O-mRNAs so that the expression of both genes is required for lacZ expression. • β-galactosidase signal detected only when O-mRNAs with α and ω coexpressed with respective O-ribosomes Yes!

25. Application • Good for creating synthetic, orthogonal cellular pathways • Cell logic applications

26. In-Vitro Nucleic Acid Systems • Inputs = nucleic acids, signals, or proteins • Networks of nucleic acids = molecular automaton • Outputs= nucleic • acids (red), signals (green) and protein (blue). • Tic tac toe (boolean network) • Luminescence-linked riboregulator detector for genotyping -distinguish between different input nucleic acid alleles. • A molecular automaton constructed from DNA and enzymes, used to ‘diagnose’ mRNA of disease-related genes in vitro.

27. Molecular Automaton • Input module recognizes specific mRNA levels • Computation module implements a stochastic molecular automaton • two automata (detect mRNA), one for a positive diagnosis and one for a negative diagnosis • Output module releases a short single-stranded DNA molecule or antisense drug • Pos diagnosis automaton  drug antisense molecule • Neg diagnosis automaton  drug suppressor • Together, fine control of drug concentration by determining ratio between drug antisense and drug suppressor molecules.

28. Future • RNA switches with multiple functional domains to generate stimulus-specific functional responses - already started on this, as mentioned earlier • Rapid response times • Sense biological and environmental stimuli • Computational design; experimental validation • Increase precision, number and functional complexity of molecular switches and automata. • In vitro  in vivo – integrate more systems into cellular environments, eliminate pleiotropic effects. • Synthetic genomes?

29. General points • RNA is very versatile • Engineer systems • Probe natural networks • Characterization is just as important as figuring out a novel approach • Importance of being able to distinguish between engineered organisms and wildtype?

30. Other References • Isaacs, Farren J., Daniel J. Dwyer, Chunming Ding, Dmitri D. Pervouchine, Charles R. Cantor, and Jaes J. Collins. "Engineered Riboregulators Enable Post-transcriptional Control of Gene Expression." Nature Biotechnology 22.7 (2004): 841-47. • Rackham, Oliver, and Jason W. Chin. "A Network of Orthogonal Ribosome- mRNA Pairs." Nature Chemical Biotechnology 1.3 (2005): 159-66. • Rackham, O. & Chin, J.W. Cellular logic with orthogonal ribosomes. Journal of the Americal Chemical Society 127, 17584–17585 (2005). • Stojanovic, M.N. & Stefanovic, D. A deoxyribozyme-based molecular automaton. Nature Biotechnology 21, 1069–1074 (2003). • About the upp negative screen: http://www.invivogen.com/PDF/5-FU_TDS_01E24-SV.pdf And now for more cell logic… Thanks for listening!