RNA Synthetic Biology

Farren J Isaacs, Daniel J Dwyer, & James J Collins

Nature BiotechnologyMay 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 RNAsRiboregulators

sRNAs (small regulatory RNAs)miRNAssiRNAsRiboswitchesRibozymes

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 RNAsChaperone 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 2004

http://www.nature.com/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 crRNAtaRNA is regulated by PBAD (inducible), so can

determine when translation is allowedGene 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

SpecificityDesigned 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.

ApplicationProbe or modify translational dynamics of

natural networks Tool for studying isolated network components.

Generate translationally based reversible knockouts

Future – Engineered RiboregulatorsTwo 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 ChinA 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 1011 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> 109 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!

ApplicationGood 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 = nucleicacids (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 ReferencesIsaacs, 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

Thanks for

listening!