This presentation is about the use of RNA in different ways like in synthetic form to regulate the gene expression and also used as an scaffold to increase the metabolite production.

1. Synthetic biology :Synthetic biology :
Using synthetic RNAs as scaffold and regulatorUsing synthetic RNAs as scaffold and regulator
Presented By :
Rajni
M.Sc. 2nd
year

2. 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.

3. 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

4. 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.

5. Synthetic biology research tools
Lienert et.al. Nat Rev Mol Cell Biol.

6. 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)

7. 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)

8. 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)

9. 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.

10. RNA Molecule

11. 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.

12. 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.

13. Tradeoffs when designing synthetic RNAs
Choice to assemble
discrete or periodic
structures
Geometry
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)

14. General workflow for designing RNA scaffold
Choose aptamer sequences
Terminator Promoter AptamerRestriction site
Design scaffold secondary structure and use RNA designer to
compute a sequence
RNA scaffold optimization
Synthesize RNA scaffold

15. Clone RNA scaffold into expression system
Induce scaffold expression
In vivo
pull-down
In vitro
assembly
qRT–PCR
Expression analysis
Target proteins onto the RNA scaffold
Delebecque et.al. Nat. Protoc. (2012)

16. 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.

17. Reactions catalyze by RNAs scaffold
Sachdeva et.al. Nucleic Acids Res. (2014)

18. Affects of length and Orientation of aptamers
Sachdeva et.al. Nucleic Acids Res. (2014)

19. 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

20. Model for two maximal configurations of
intermediate 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)

21. Multiple enzymes localize on RNA scaffolds
Sachdeva et.al. Nucleic Acids Res. (2014)

22. Scaffolding approaches
Sachdeva et.al. Nucleic Acids Res. (2014)

23. 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.

24. Synthetic RNAs as regulator
 Synthetic RNAs can regulate gene expression.
 Synthetic RNAs mimicking of biological regulatory RNAs.

25. RNAs regulators
CRISPR-Cas system
sRNAs
Riboswitches
Attenuators
Toehold switches
Cis-acting regulating
elements
Trans-acting regulatory
elements

26. 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)

27. Regulation of T-cell proliferation by Riboswitch
Chen et.al. PNAS (2010)

28. CRISPR- Cas based system
Gilbert et.al. Cell(2013)

29. Transcription Attenuators
 Attenuators are RNA hairpin structures that regulate gene
expression by prematurely terminating transcription.
Myhrvold & Silver. Nat. Str. Mol. Bio.(2015)

30. Natural antisense-RNA transcriptional control
Melissa K.et.al Nucleic Acids Res. (2013)

31. Chimeric antisense-RNA transcriptional control
Melissa K.et.al Nucleic Acids Res. (2013)

32. 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)

33. Translation repression
Green et.al. Cell (2014)

34. 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)

35. Metabolic engineering for tyrosine production
Na et.al. Nat. Biotch. (2013)

36. Strains dependent tyrosine production
Na et.al. Nat. Biotch. (2013)

37.  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

38. 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.

39.  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
References-2