Powerpoint for .pptx - MSU chemistry

Utility of Synthetic Riboswitches in Probing

and Reprogramming Bacterial Behavior!

November 13, 2013!Elizabeth Santos!

Computer-Programmable Bacteria!

Andrianantoandro, E.; Basu, S.; Karig, D.; Weiss, R. Mol. Syst. Biol. 2006, 2, 1-14. !

Inspiration from Nature: Oil Eating Bacteria!

Redmond, M.; Valentine, D. Proc. Natl. Acad. Sci. 2012, 109, 20292-20297.!Cleveland, C. Deepwater Horizon oil spill. The Encyclopedia of Earth [Online] Nov. 1, 2013. http://

www.eoearth.org/view/article/161185/!

Deepwater Horizon oil spill! •  68,000 square miles!•  4,900,000 barrels of oil!

Inspiration from Nature: Oil Eating Bacteria!

Head, I.; Jones, M.; Roling, W. Nat. Rev. Microbiol. 2006, 4, 173-182.!Van Beilen, J.; Funhoff, E. Appl. Microbiol. Biotechnol. 2007, 74, 13-21.!

Alcanivorax (alkane degrader)!

Time (hours)!0 1 10 100 1,000 10,000!

OHalkane-1-monooxygenase

O2

Engineered Bacterial Systems!

Can we reprogram organisms to autonomously perform complex tasks?!

Wang, S.; Ghosh, A.; Bongio, N. Stebbings, K.; Lempe, D. Proc. Natl. Acad. Sci. 2012, 109, 12734-12739.!First Successful Laboratory Production of Human Insulin Announced. Genentech, San Francisco, Sep. 6, 1978. &

Poforelc, D. $1 M grant will help researchers develop “smart insulin”. MedCity News, March 14, 2013.!

Health & Medicine! Biosynthesis!

Preventing Mosquitos from Transmitting Malaria!

Artemisinin-based combination therapies

(ACTs)!ex. Coarsucam!

NCl

HN

OH

N

O

O

O

H

H

H OO

OOH

O

Ranson, H.; N’Guessan, R.; Lines, J.; Moiroux, N.; Nkuni, Z.; Corbel, V. Trends Parasit. 2011, 27, 91-98.!Mutabingwa, T. Acta Tropica. 2005, 95, 305-315. Hiskey, D. Why Mosquito Bites Itch. TodayIFoundOut [Online]

Nov. 6, 2013. http://www.todayifoundout.com/index.php/2010/08/why-mosquito-bites-itch/!

•  300-500 million people contract the disease annually!

•  ~1.2 million people die of malaria annually!

•  Caused by parasitic Plasmodium!

Prevention!

Preventing Mosquitos from Transmitting Malaria!

Riehle, M.; Moreira, C.; Lampe, D.; Lauzon, C. Int. J. Parasitol. 2007, 37, 595-603.!

Isolate Panteo agglomerans!

Antimalaria proteins!

Transmit malaria!Introduce new bacteria into

mosquito!(3)!

(1)!

Engineer the bacteria!

(2)!Mosquitos produce toxic

protein to parasite!

(4)!

Biosynthesis of Insulin!

First Successful Laboratory Production of Human Insulin Announced. Genentech, San Francisco, Sep. 6, 1978. & Poforelc, D. $1 M grant will help researchers develop “smart insulin”. MedCity News, March 14, 2013.!

plasmid!(small piece of DNA)!

Cut plasmid by

enzyme!

Insert human insulin gene!

Insert plasmid into

bacteria!

Bacteria divide & produce insulin!

Outline!

Programmable Bacteria!Probing for Small Molecules! Reprograming Bacteria!

Prokaryotic Gene Regulation!

Proteins! RNA!

Engineered Biological Systems!

Control of Gene Expression in Prokaryotes!

Transcription!

PDB ID: 2LZV; PDB ID: 3C44; PDB ID: 4I9S!Pearson, H. Nature. 2006, 441, 398-401.!

Translation!

DNA! RNA! Protein!

Gene: “A locatable region of genomic sequence, corresponding to a unit of inheritance, which is associated with regulatory regions, transcribed regions and/or other functional sequence regions.”!

Transcriptional control!

Translational control!

Transcription!

DNA! RNA!

Transcription & Translation. Cold Spring Harbor Laboratory [Online] Nov. 9, 2013. http://www.dnalc.org/resources/3d/12-transcription-basic.html!

Transcriptional Control!Positive Control!Regulatory protein triggers transcription!

Negative Control!Regulatory protein shuts down transcription!

Image adapted from Regulation of Gene Expression. [Online] Oct. 13, 2013. http://www.bio.miami.edu/dana/250/250S12_11.html!

Promoter! Operator!

Promoter! Operator! Promoter! Operator!

Activator!

Repressor!

Transcription ‘On’!

Transcription ‘Off’!

RNA Poly.!

Promoter! Operator!

RNA Poly.!

Activator!

Repressor!

Tryptophan Operon!

Tryptophan absent: Enzymes needed to synthesize tryptophan are produced.!

Tryptophan present: Enzymes used to synthesize tryptophan are not produced.!

Regulation of Gene Activity. [Online] Oct. 20, 2013. http://classes.midlandstech.edu/carterp/Courses/bio101/chap15/chap15.htm!

Summary of Transcriptional Control!

Typically a regulatory protein that can sense the level of the product binds to the DNA to affect the expression of the relevant enzymes!

Promoter! Operator! Structural Genes!

Regulatory Protein!

RNA Poly.!

Regulation of Genes Involved in Synthesis and Uptake of Vitamins!

OHSN+

NH2N

N

Thiamin !Vitamin B1!

OH

N NH

O

NOH

HO OH

N O

Riboflavin!Vitamin B2!

Promoter! Operator! Structural Genes!

Regulatory Protein!

RNA Poly.!

N NNN

Co

NH

O

O

OHO

HO N

NCONH2

O

NH2

O

NH2

H2NOCH2NOC

OHOHO

NN

NNNH2

H2NOC

POO

Miranda-Rios, J.; Navarro, M.; Soberon, M. Proc. Natl. Acad Sci. 2001, 98, 9736-9741.!Nou, X.; Kadner, R. Proc. Natl. Acad. Sci. 2000, 97, 7190-7195.!

Adenosyl cobalamin!

Vitamin B12!

Stormo, G.; Ji, Y. Proc. Natl. Acad. Sci. 2001, 98, 9465-9467. !

Is transcription ONLY controlled by regulatory proteins? Or are their

other regulatory elements???!

Adenosyl cobalamin bound riboswitch!

PDB ID: 4FRN!Nahvi, A.; Sudarsan, N.; Ebert, M.; Zou, X.; Brown, K.; Breaker, R. Chem. Biol. 2002, 9, 1043-1049.!

Johnson, J.; Reyes, F.; Polaski, J.; Batey, R. Nature. 2012, 492, 133-137.!

•  Discovered in 2002!•  Major classes!

–  Purines and derivatives!–  Protein coenzymes!–  Amino acids!

Discovery of RNA Based Gene Control!

N NNN

Co

NH

O

O

OHO

HO N

NCONH2

O

NH2

O

NH2

H2NOCH2NOC

OHOHO

NN

NNNH2

H2NOC

POO

Gene Regulation!RNA Based!

RNA! Switch!

Translation!

RNA! Protein!Small

molecule!

Riboswitch!Are we increasing or decreasing the

production of proteins??!

Diverse Metabolites Bind to RNA!

adenosylcobalamin1!

1Nahvi, A.; Sudarsan, N.; Ebert, M.; Zhou, X.; Brown, K. Chem. Biol. 2002, 9, 1043-1049.!2Winkler, W.; Nahvi, A.; Breaker, R. Nature. 2002, 419, 952-956.!

3Mandal, M.; Boese, B.; Barrick, J.; Winkler, W.; Breaker, R. Cell. 2003, 113, 577-586.!4Sudarsan, N.; Wickiser, J.; Nakamura, S.; Ebert, M.; Breaker, R. Genes Dev. 2003, 17, 2688-2697.!

N

N

NS

NH2

OP OOH

O P OHOH

OThiamine PyroPhosphate2!

N

HN

NH2N

HN

O

guanine3!

NH2

H2NO

OH

lysine4!

N NNN

Co

NH

O

O

OHO

HO N

NCONH2

O

NH2

O

NH2

H2NOCH2NOC

OHOHO

NN

NNNH2

H2NOC

POO

PreQ1 Riboswitch!

6.4 Å!

PDB ID: 3Q50, 3Q51!

Free state in green (dark purple) and metabolite-bound state in red (light purple)!

A14!

Jenkins et al. J. Biol. Chem. 2011, 286, 24626-24637.!

HN

N NH

O

H2N

NH2

Carbon!

Nitrogen!Oxygen!

Phosphorous!

PreQ1 Riboswitch Binding Pocket!Metabolite-Bound State!Free State!

U6!

A14!C15!

A29!

U6!

A14!

C15!

A29!

2.9 Å!

3.1 Å!

3.1 Å!2.7 Å!3.1 Å!3.3 Å!

Nitrogen! Oxygen!Phosphorous!

Jenkins et al. J. Biol. Chem. 2011, 286, 24626-24637.!

HN

N NH

O

H2N

NH2

PreQ1 Riboswitch Metabolite-Bound State!

U6!

A14!

C15!

A29!

2.9 Å!

3.1 Å!

3.1 Å!2.7 Å!

Nitrogen! Oxygen!Phosphorous!

N

NH2

O

N

N

N N

O

HN

NH2H

H H

N

N

NN

N H

H

HN N

O

O

The conformational change affects translation…!

U6!

C15!

A29!

Translation!

RNA! Protein!

Protein Synthesis Animation. Youtube [Online] Nov. 9, 2013. http://www.youtube.com/watch?v=5iS4CRPPDus!

Mechanisms of Natural Riboswitches!Translational Control!

Nudler, E.; Mironov, A. Trends Biochem. Sci. 2004, 29, 11-17.!Barrick, J.; Breaker, R. Genome Biology. 2007, 8, R239 .!

RBS! AUG!

AUG!

Gen

e Ac

tivat

ion!

RBS! AUG!

AUG!

Gen

e Re

pres

sion!

M ! !Small molecule!RBS! !Ribosomal Binding Site “RBS”!

M!

M! M!

M!

RBS!

RBS!

5’! 3’!

5’! 3’!

5’! 3’!

5’! 3’!

Riboswitch Components!

Winkler, W.; Nahvi, A. Breaker, R. Nature. 2002, 419, 952-956.!Can bacterial chemotaxis be reprogrammed? Biotweet [Online] Oct 31, 2013. http://2011.igem.org/Team:WITS-

CSIR_SA/Project/Concept!

RBS! !Ribosomal Binding Site “RBS”!

AUG UAA

RBS

5' 3'

Riboswitch Coding Region

Design of Synthetic Riboswitches!

Jaschke, A. FEBS Lett. 2012, 586, 2106-2111.!

RBS! ! Ribosomal Binding Site “RBS”!

We need an RNA sequence to bind the target molecule…!

AUG UAA

RBS

5' 3'

Riboswitch Coding Region

Tuerk, C.; Gold, L. Science. 1990, 249, 505-510.!Ellington, A.; Szostak, J. Nature. 1990, 346, 818-822.!

Zimmerman, B.; Bilusic, I.; Lorenz, C.; Schroeder, R. Methods. 2010, 52, 125-132.!

Systematic Evolution of Ligands by EXponential Enrichment “SELEX”!

Add RNA pool (1014)

Wash unbound RNA molecules

Analyze the bound

sequences

Selected aptamer with high binding

affinity

Target molecule

RNA sequence

Outline!



Proteins! RNA!


Probing DNT by a Synthetic Riboswitch!

Ehrentreich-Förster, E.; Orgel, D.; Krause-Griep, A.; Cech, B.; Erdmann, V.; Bier, F.; Scheller, F.; Rimmele, M. Anal. Bioanal. Chem. 2008, 391, 1793-1800.!

Davidson, M.; Harbaugh, S.; Chushak, Y.; Stone, M.; Loughnane, N. ACS Chem. Bio. 2013, 8, 234-241. !

RBS!AUG TEV!

‘Off’! ‘On’!

RBS! AUG TEV!

NO2O2N

Synthetic!Riboswitch!

TEV!PDB ID: 1Q31!

5’! 3’! 5’! 3’!

An Aptamer for 2,4,6-Trinitrotoluene (TNT)!

Ehrentreich-Förster, E.; Orgel, D.; Krause-Griep, A.; Cech, B.; Erdmann, V.; Bier, F.; Scheller, F.; Rimmele, M. Anal. Bioanal. Chem. 2008, 391, 1793-1800.!

U! C!C!A!

A!

U!C!G!A!G!

C!U! U!

U!

U!U!

U!U!U!

U!U!

U!

U!

U!U!U!

C!C!

C!C!

C!

C!

C!

C!C!

C!C!C!

C!C!

C!C! C!

C!C!

C!

C!

C!

A!A!

A!A!A!A!

A!A!A!

A!

A!

A!

G!G!

G! G!G! G!

G!G!

G!

G!

G!G! G!

G!

G!G!

A!G! C!

G!G! A!

G!

G!G!

A!

!!!!!!!!

!!!!

!!!!

!!!!

!!!! !!

!!!!

!!

!!!!!!!!!!!!!!!!!!!!

NO2O2N

NO2

NO2O2N

! Represents base pairing !

Kd = 1 x 10-8 M!

Design of the Riboswitch Library!


UA

U CCA

A

UC

GAGCU U

U

UU

UUUU

U

U

U UU

C

CCC

CC

C

CC

CCC

CC

CC C

CC

C

C

C

AAAA

AA

AA

A

A

A

GG

G GG G

GG

G

GG G GG

G

G

AG C

GG A

GG

G

A

!!!!!!!!

!!!!

!!!!!! !!

!!!!!!

!!!!!!!!!!!!!!!!!!!!

N30!U A AGGUAACAA

CAAGAUG

3’!5’!

Aptamer!Linker!Ribosomal Binding Site!Start Codon!

AUG UAA

RBS

5' 3'

Probing DNT by a Synthetic Riboswitch!

Ganesan, S.; Ameer-beg, S.; Ng, T.; Vojnovic, B.; Wouters, F. Proc. Natl. Acad. Sci. 2006, 103, 4089-4094.!Harbaugh, S.; Kelley-Loughnane, N.; Davidson, M.; Narayanan, L.; Trott S.; Chushak, Y.; Stone, M.

Biomacromolecules. 2009, 8, 1055-1060. !

RBS!AUG TEV!


RBS! AUG TEV!

NO2O2NCl

eava

ge S

eq!

How can we know if the RS is working??!

TEV!

5’! 3’! 5’! 3’!

eGFP-quencher FRET Pair!

Clea

vage

Seq

uenc

e!

TEV!

eGFP!PDB ID: 2Y0G!

REACh!PDB ID: 3V3D (eYFP)!

Ganesan, S.; Ameer-beg, S.; Ng, T.; Vojnovic, B.; Wouters, F. Proc. Natl. Acad. Sci. 2006, 103, 4089-4094.!Meulenaere, E.; Asselberghs, I.; Wergifosse, M.; Botek, E.; Spaepen, S.; Champagne, B.; Vanderleyden, J.;

Clays, K. J. Mater. Chem. 2009, 19, 7514-7519. !

eGFP-quencher FRET Pair!

eGFP! quencher!

Excitation!10-15 sec!

Internal Conversion!

10-12 sec!

Fluorescence!10-9 sec!

Donor Energy Transfer!

No emission!!

eGFP! quencher!

FRET = Förster Resonance !Energy Transfer!

FRET Requirements!

Spectral Overlap!

Broussard, J.; Rappaz, B.; Webb, D.; Brown, C. Nature Prot. 2013, 8, 265-281. !

0"

0.05"

0.1"

0.15"

0.2"

0.25"

0.3"

0.35"

0.4"

0.45"

450! 500! 550!

Inte

nsity!

Wavelength (nm)!

488! 509! 510!

eGFP !Ex !488 nm!! !Em !509 nm!

REACh !Ex !510 nm!

Distance 1 - 10 nm!

Visualization of DNT Dependent Riboswitch Activation!


RBS!AUG TEV!


RBS! AUG TEV!

NO2O2N

eGFP!

0 1 2 3 4 5 6!

Clea

vage

Seq!

After Adding DNT: Cell Lysates!(hours)!

Outline!



Proteins! RNA!


Reprogramming Bacteria to Seek and Destroy Atrazine!

1.  Recognize!Design an RNA sequence!

2.  Seek!Incorporate the aptamer into a riboswitch!

3.  Destroy!!Degrade the atrazine into a nontoxic product!

N

N

N

Cl

NNH H

The Herbicide Atrazine!

•  Most heavily used herbicide in the United States!•  Persistant environmental pollutant!•  Attractive target for interacting with RNA!

Benefits. Syngenta [Online] Nov. 1, 2013. http://www.atrazine.com/Benefits/atrazine_benefits.aspxAtraMain.aspxAckerman, F. Int. J. Occup. Environ. Health. 2007, 4, 437-445.!

Winchester, P.; Huskins, J.; Ying, Acta Paediatr. 2009, 98, 664-669.!

H bond donor!H bond acceptor!

N

N

N

Cl

NNH H

Concentration of Atrazine in Streams (2009)!

Watershed Regressions for Pesticides (WARP) Atrazine Model. USGS [Online] Nov 6, 2013. http://infotrek.er.usgs.gov/warp/ !

*EPA drinking water standard 3 ppb*!

< 0.001 ppb! < 0.001 – 0.038! 0.039 – 0.150!

< 0.151 – 1.026! > 1.026! Estimate Unavailable!

Chemical Feminization in Frogs!

Hayes, T.; Khoury, V.; Narayan, A.; Nazir, M. Park, A. Brown, T.; Adame, L.; Chan, E.; Buchholz, D.; Stueve, T.; Gallipeau, S. Proc. Natl. Acad. Sci. 2010, 107, 4612-4617.!

•  Reduce testosterone levels!

•  Decreased fertility!

•  Produce viable eggs!

3’!

Design of an Atrazine-Sensitive Riboswitch that Regulates Chemotaxis in E. coli!

Kirby, J. Nat. Chem. Biol. 2010, 6, 398-399.!Sinha, J.; Reyes, S.; Gallivan, J. Nat. Chem. Biol. 2010, 6, 464-470.!

Selection!

N

N

N

Cl

NH

NH

Atrazine!Random

RNA Pool!

Adapt aptamer as riboswitch!

RBS!AUG!

‘Off’!

N

N

N

Cl

NH

NH

‘On’!

RBS!AUG!

5’! 3’!

5’!

E. Coli Chemotaxis!

The cheZ gene product controls flagellar motion.!

Kuo, S.; Koshland, D. J. Bacteriol. 1987, 169, 1307-1314.!An overview of E. coli chemotaxis. [Online] Oct. 15, 2013. http://chemotaxis.biology.utah.edu/Parkinson_Lab/

projects/ecolichemotaxis/ecolichemotaxis.html!Swimming ecoli. Youtube [Online] Nov. 2, 2013. http://www.youtube.com/watch?v=AKHbqOD_OQU!

E. Coli Chemotaxis!

The cheZ gene product controls flagellar motion.!

N

N

N

Cl

NH

NH

Kuo, S.; Koshland, D. J. Bacteriol. 1987, 169, 1307-1314.!An overview of E. coli chemotaxis. [Online] Oct. 15, 2013. http://chemotaxis.biology.utah.edu/Parkinson_Lab/

projects/ecolichemotaxis/ecolichemotaxis.html!

N

N

N

Cl

NH

NH

Riboswitch Selection by Observing Physical Movement!

Topp, S.; Gallivan, J. ChemBioChem. 2008, 9, 210-213.!Sinha, J.; Reyes, S.; Gallivan, J. Nat. Chem. Biol. 2010, 6, 464-470. !

Pick cells that do not move!

Pick cells that do not move!

Pick cells that do move!

RBS! AUG!

‘On’!

RBS!AUG!

‘Off’!

cheZ!cheZ!

- atrazine! - atrazine! + atrazine!

Atrazine Degradation Pathway!

Souza, M.; Sadowsky, M.; Wackett, L. J. Bacteriol. 1996, 178, 4894-4900.!Scott, C.; Jackson, C.; Coppin, C.; Mourant, R.; Hilton, M.; Sutherland, T.; Russell, R.; Oakeshott, J. Appl.

Environ. Microbiol. 2009, 75, 2184-2191.!

N

N

N

Cl

NH

NH

H2O HCl

N

N

N

OH

NH

NH

atzA

atrazine hydroxyatrazine

N

NN

Cl

NH

NH

O

OD327

Fe2+O

ON328

N OHS331

HH

H

N

N

N

HN NH

O

OD327

Fe2+

ON328

N OS331

HH

OCl H

H

N

N

N

HN NH

O

OD327

Fe2+

ON328

N OHS331

HH

OH

Not toxic!!!!

Mandelbaum, R.; Wackett, L.; Allan, D. Appl . Environ. Microbiol. 1993, 59, 1695-1701.!Mandelbaum, R.; Allan, D.; Wackett, L. Appl. Environ. Microbiol. 1995, 61, 1451-1457.!

Agar plates of the atrazine-degrading isolate showing clearing zones from the degradation of 1,000 ppm of atrazine. A) 48 h after inoculation; B) 72 h after inoculation.!

N

N

N

NH

NH

Cl

N

N

N

NH

NH

OHAtzA

H2O HCl

Visualization of Atrazine Degradation (without Riboswitch)!

Visualization of Reprogrammed E. coli Cells!

Sinha, J.; Reyes, S.; Gallivan, J. Nat. Chem. Biol. 2010, 6, 464-470.!Adler, J. Science. 1966, 153, 708-716.!

Adler, J. Science. 1969, 166, 1588-1597.!

N

N

N

NH

NH

Cl

N

N

N

NH

NH

OHAtzA

H2O HCl

No AtzA!

AtzA!

" High throughput!" Inexpensive!" Able to generate riboswitch with desirable

phenotype!

Goal: Recognize, Seek & Destroy!

In vitro Selection!

N

N

N

Cl

NH

NH

Atrazine!Random

RNA Pool!~1013-1015!

In vivo Selection!

N

N

N

Cl

NH

NH

Sinha, J.; Reyes, S.; Gallivan, J. Nat. Chem. Biol. 2010, 6, 464-470. !Smuc, T.; Ahn, I.; Ulrich, H. J. Pharm. Biomed. Anal. 2013, 81-82, 210-217. !

Summary!

" Bacterial gene expression is well understood!–  Regulation by protein cofactors!–  Regulation by riboswitches!

" Synthetic riboswitches can be used to probe and reprogram bacterial cell behavior!

!

RBS!hidden!

AUG! RBS!accessible!

AUG!

RNA ‘OFF’ SWITCH!

gene!gene!

Protein Production!

RNA ‘ON’ SWITCH!Small

molecule (M)! M!

Conformational Change!

5’! 3’! 5’! 3’!

Acknowledgements!

"  Dr. Borhan!"  Dr. Huang!"  Chrysoula!"  Labmates: Ipik, Tanya, Arvind, Kumar, Bardia,

Carmin, Hadi, Nastaran, Eddie, Yi, Calvin, Wei, Jun, Ding, Meisam, Camille!

"  Family and Friends!

THANK YOU FOR YOUR ATTENTION.!

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