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RNA Synthetic Biology. Farren J Isaacs, Daniel J Dwyer, & James J Collins Nature Biotechnology May 2006. iGEM 2010 Journal Club 7/7/2010. Any sequence  diverse 2° structure and function Interact with proteins, metabolites, other nucleic acids Levels of modulation: Transcription

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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

slide2

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 rna
RNA RNA RNA
  • Antisense RNAs
  • Riboregulators
    • sRNAs (small regulatory RNAs)
  • miRNAs
  • siRNAs
  • Riboswitches
  • Ribozymes
controlling gene expression overview
Controlling Gene Expression - overview
  • Antisense RNAs - silence expression by targeting specific mRNA sequences (physically obstruct machinery)
slide5

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)
slide6

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
slide7

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
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.
gene repression
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)]
slide10

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…

slide11

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])

slide12

Same idea, different figure

pyrimidine-uracil-nucleotide-purine

Images from Isaac 2004, Engineered riboregulators enable post-transcriptional control of gene expression

slide13
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
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
slide15

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

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

slide21

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
slide22

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
slide23

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.

a follow up study logic gates
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!

application25
Application
  • Good for creating synthetic, orthogonal cellular pathways
  • Cell logic applications
slide26

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.
molecular automaton
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
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
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?
slide30

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!