synthetic biology
TRANSCRIPT
Synthetic biology :Synthetic biology :Using synthetic RNAs as scaffold and regulatorUsing synthetic RNAs as scaffold and regulator
Presented By : Rajni
M.Sc. 2nd year
Synthetic biology
Design and construction of new biological parts, devices , and systems using genetic information.
Re-design of existing, natural biological systems for a useful application.
How is Synthetic Biology Different?
Synthetic biology uses four principles not typically found in genetics, genomics, or molecular biology:
Abstraction
Modularity
Design and Modeling
Standardization
Recent advances in the field of synthetic biology, particularly in the programmable control of gene expression at multiple levels of regulation, have increased the ability to efficiently design and optimize biological systems to perform designed tasks.
Synthetic biology research tools
Lienert et.al. Nat Rev Mol Cell Biol. (2014)
Tools for signalling pathway engineering
Control different cellular functions.
Rerouting of signals can be done :
• Modified binding site of cell surface of membrane receptors
• Modified intracellular domain of membrane receptors.
• Cytosolic protein also involve in the intracellular signalling Lienert et.al. Nat Rev Mol Cell Biol.
(2014)
Tools for protein turnover regulation
Altering the protein stability
protein stability depend upon several factors;
• Length of peptide sequence.• Occurrence of specific
amino acid that can be phosphorylated.
Proteins can be actively degraded through the ubiquitylation pathway
Lienert et.al. Nat Rev Mol Cell Biol. (2014)
Tools for transcriptional control Natural transcriptional regulators : LacI , TetR and GAL4. Programmable transcription regulators : Zinc finger, TALEs and CRISPR-based regulators.
Lienert et.al. Nat Rev Mol Cell Biol. (2014)
Tools for genome engineering
Recombinases catalyze the recombination of a pair of short target sequences.
Nucleases fused to DNA-binding factors such as ZFNs ,TALE and CRISPR-based system and induce a double-strand break.
Lienert et.al. Nat Rev Mol Cell Biol. (2014)
RNA Molecule
Synthetic RNA RNA is an information bearing molecule. RNA form dynamic structure via base-pairing
RNA component of
Ribosomes(rRNA)
RNA component of
telomerase (TERC)
Variety of non coding RNA
(ncRNAs)
Because of dynamic structure and predictable base pairing synthetic RNA can used as molecular scaffold and regulator.
Synthetic RNAs as scaffold
RNA scaffolds are synthetic noncoding RNA molecules.
Engineered RNA molecules serve as a more versatile, rationally programmable alternative to protein based scaffolding strategies , allowing a new level of access and control over spatial organization of proteins.
Tradeoffs when designing synthetic RNAs
Choice to assemble discrete or periodic
structuresGeometry Choice of the
targeted pathway.
Discrete structures are smaller and their self-assembly is difficult. Characterized by molecular weight.
Periodic structure are polydisperse Characterized by imaging.
contact dependent chemical reactions are more likely to benefit from scaffolding than reactions with diffusible substrates chemicals reactions.
Any considerable changes in yield can result from small variations in the length and the orientation of the aptamer (scaffolded enzymes)
General workflow for designing RNA scaffold
Choose aptamer sequences
Terminator Promoter Aptamer Restriction site
Design scaffold secondary structure and use RNA designer to compute a sequence
RNA scaffold optimization
Synthesize RNA scaffold
Clone RNA scaffold into expression system
Induce scaffold expression
In vivopull-down
In vitroassemblyqRT–PCR
Expression analysis
Target proteins onto the RNA scaffold
Delebecque et.al. Nat. Protoc. (2012)
Use of synthetic RNAs as scaffold
RNA scaffolds are used in many areas in which the spatial organization of biomolecules is desirable.
RNA scaffolds use for co-localization of enzymes.
RNA scaffolding was first demonstrated in vivo with a contact-dependent electron transfer reaction in a hydrogen-production pathway.
Co-localization plays a key role in the directional control of metabolic fluxes toward specific products in cells.
Reactions catalyze by RNAs scaffold
Sachdeva et.al. Nucleic Acids Res. (2014)
Affects of length and Orientation of aptamers
Sachdeva et.al. Nucleic Acids Res. (2014)
Maximal alkane production with 14 &16 bp stem
Synthesis was near-maximal with 13- to 17- bp stems, maximal with 14- and 16-bp stems, and minimal with a 15-bp stem
Model for two maximal configurations ofintermediate flux channeling
On varying the anti-BIV-TAT aptamer stem loop length, different rotational conformations of the BIV-Tat-AAR moiety are possible, relative to the PP7-ADO dimer
Sachdeva et.al. Nucleic Acids Res. (2014)
Multiple enzymes localize on RNA scaffolds
Sachdeva et.al. Nucleic Acids Res. (2014)
Scaffolding approaches
Sachdeva et.al. Nucleic Acids Res. (2014)
Co-localized enzymes on RNA scaffold Increase succinate production
Sachdeva et.al. Nucleic Acids Res. (2014)
Intermediates can be channeled toward desired product formation on RNA scaffolds with different aptamer.
Synthetic RNAs as regulator
Synthetic RNAs can regulate gene expression. Synthetic RNAs mimicking of biological regulatory RNAs.
RNAs regulators
CRISPR-Cas system
sRNAs
Riboswitches
Attenuators
Toehold switches
Cis-acting regulating elements
Trans-acting regulatory elements
Riboswitches Riboswitches are RNA motifs that bind to small molecules
that lead to a conformational change in a hairpin and regulate gene expression or enzymatic activity of a ribozyme
Myhrvold & Silver. Nat. Str. Mol. Bio.(2015)
Regulation of T-cell proliferation by Riboswitch
Chen et.al. PNAS (2010)
CRISPR- Cas based system
Gilbert et.al. Cell(2013)
Transcription Attenuators
Attenuators are RNA hairpin structures that regulate gene expression by prematurely terminating transcription.
Myhrvold & Silver. Nat. Str. Mol. Bio.(2015)
Natural antisense-RNA transcriptional control
Melissa K.et.al Nucleic Acids Res. (2013)
Chimeric antisense-RNA transcriptional control
Melissa K.et.al Nucleic Acids Res. (2013)
Toehold switches Toehold switches are short synthetic RNAs that act via
strand displacement to activate gene expression by opening hairpins designed to impede translation
Myhrvold & Silver. Nat. Str. Mol. Bio.(2015)
Translation repression
Green et.al. Cell (2014)
Small regulatory RNA (sRNA) sRNAs are short (50–250 nt) noncoding RNA molecules that
regulate mRNA translation in bacteria through base-pairing sRNAs are targeted to mRNAs by the protein Hfq and
trigger their degradation
Na et.al. Nat. Biotch. (2013)
Metabolic engineering for tyrosine production
Na et.al. Nat. Biotch. (2013)
Strains dependent tyrosine production
Na et.al. Nat. Biotch. (2013)
Synthetic Biology rewire biological systems by modifying and recombining existing genetic elements and creating entirely new genetic parts
Natural versatility and our ability to predict, makes RNA an ideal tool in synthetic biology
RNA scaffold can co-localize multiple enzymes to enhance yields of sequential metabolic pathways
RNA also act as regulator to control expression of genes
Take Home Message
References-1
Cameron Myhrvold & Pamela A Silver. Using synthetic RNA as scaffold and regulator Nat. Struct. Mol. Bio. (2015) 22: 8-10.
Florian Lienert et al. Synthetic biology in mammalian cells: Next generation research tools and therapeutics. Nat. Rev. Mol. Cell Bio. (2014) 15: 95–107.
Gairik Sachdeva et al. In vivo co-localization of enzymes on RNA scaffolds increases metabolic production in a geometrically dependent manner. Nucleic Acids Res. (2014) 42:9493–9503.
Alexander A. Green et.al. Toehold Switches: De-Novo-Designed Regulators of Gene Expression. Cell (2014) 159: 925–939.
Luke A. Gilbert et al. CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes. Cell (2013)154: 442–451.
Na et.al Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs Nat. Biotech (2013) 31: 170-174.
Melissa K. Takahashi and Julius B. Lucks. A modular strategy for engineering orthogonal chimeric RNA transcription regulators. Nucleic Acids Res. (2013) 41, No: 7577–7588.
Camille J Delebecque et al. Designing and using RNA scaffolds to assemble proteins in vivo.Nat. Protoc. (2012) 7:1797-1807.
Yvonne Y. Chena et al. Genetic control of mammalian T-cell proliferation with synthetic RNA regulatory systems. PNAS (2010) 107:8531–8536
References-2