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

RNA 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 RNA RNA Antisense RNAs Riboregulators  sRNAs (small regulatory RNAs) miRNAs siRNAs Riboswitches Ribozymes

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

Small regulatory RNAs (sRNAs) repress and activate (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)

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

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.

1. Engineered Riboregulators 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. Isaac et al m/nbt/journal/v22/n7 /pdf/nbt986.pdf

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)]

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. Method See next slide…

(non-coding RNA [ncRNA]) Unfolds hairpin to expose RBS 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

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

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

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

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. Tweaking

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 (pBAD promoter for taRNA) Black and white bars – GFP fluorescence Dark and light grey – taRNA concentrations

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.

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

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.

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 Rackham and Chin A network of orthogonal ribosome-mRNA pairs 2005 Image from Rackham and Chin 2005 Blue = original ribosome; purple = second ribosome. Green = original mRNA; orange= duplicate. Evolution until pairs do not interact anymore.

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 Ribosome – mRNA pairs

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 against mRNAs 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 clones, found four distinct O-mRNAs and ten distinct O-rRNA sequences Strategy

Synthesized a library of all possible RBSs and another of all possible 16S rRNA anti-RBS sequences > 10 9 unique mRNA-rRNA combinations Positive selection: Chloramphenicol resistance (CAT gene). Negative selection: uracil phosphoribosyltransferase (UPRT). Fused CAT (cat) and UPRT (upp) downstream of a constitutive promoter and RBS so the single transcript can be either positively or negatively selected.

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!

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

In-Vitro Nucleic Acid Systems 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. Inputs = nucleic acids, signals, or proteins Networks of nucleic acids = molecular automaton Outputs = nucleic acids (red), signals (green) and protein (blue).

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.

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?

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?

And now for more cell logic… 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): Rackham, Oliver, and Jason W. Chin. "A Network of Orthogonal Ribosome- mRNA Pairs." Nature Chemical Biotechnology 1.3 (2005): 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: FU_TDS_01E24-SV.pdfhttp:// FU_TDS_01E24-SV.pdf Thanks for listening!