Synthetic biology

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


Tools for transcriptional control Natural transcriptional regulators : LacI , TetR and GAL4. Programmable transcription regulators : Zinc finger, TALEs and CRISPR-based regulators.


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.


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


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


Multiple enzymes localize on RNA scaffolds


Scaffolding approaches


Co-localized enzymes on RNA scaffold Increase succinate production


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.


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


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


Strains dependent tyrosine production


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

Synthetic biology

Science

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