Transcript RNA Synthetic Biology

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
Isaac et al 2004
http://www.nature.co
m/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.

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)]
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…
Modular:
crRNA can be
inserted upstream
of any gene
 Can change
levels of cisrepression 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])
Same idea, different figure
pyrimidine-uracilnucleotide-purine
Images from Isaac 2004, Engineered riboregulators
enable post-transcriptional control of gene expression
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
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.
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)
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.
Rackham and Chin
A 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
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 (Oribosome-O-mRNA) pairs that permit robust translation
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 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
Positive selection: Chloramphenicol
resistance (CAT gene).
Negative selection: uracil
phosphoribosyltransferase (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.
A Follow-Up Study - Logic Gates
 Can multiple orthogonal ribosomes simultaneously
function in the same cell?
Yes!
 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
Application
Good for creating synthetic, orthogonal
cellular pathways
Cell logic applications
In-Vitro Nucleic Acid Systems
•Inputs =
nucleic acids,
signals, or
proteins
•Networks of
nucleic acids =
molecular
automaton
•Outputs =
nucleic
•Luminescence-linked riboregulator detector for genotyping - acids (red),
distinguish between different input nucleic acid alleles.
signals
•A molecular automaton constructed from DNA and enzymes, (green) and
protein (blue).
used to ‘diagnose’ mRNA of disease-related genes in vitro.
•Tic tac toe (boolean network)
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?
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 RibosomemRNA 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/5FU_TDS_01E24-SV.pdf
And now for more cell logic…
Thanks
for
listening!