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! In vitro selection and SELEX!Prac%caluses.BrianLarson

PortlandStateUniversity

DepartmentofChemistry

Bclarson@pdx.edu

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Invitroselec%on

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WetChemistry

Invitroselec%on

•  recreate/parameterizethehistoricalOoL

•  developanar%ficiallivingsystem

SELEX(Systema%cEvolu%onofLigandsbyExponen%alEnrichment)

•  Developaptamersextremelyhighbindingaffinitytoavarietyoftargetligands

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SELEX

Selec%onforligand

binding

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Testtubeevolu%on

•  Threestepstoinvitroevolu%on –  Diversifica%on

 –  Selec%on –  Amplifica%on

•  Selec%onrelieson

handleormolecularmarker

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Cataly%cenrichment

•  Word

G2 G4

G6 G8

Lehman & Joyce (1993) Nature 361: 182–185

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Cataly%crepertoire

Chen, Li, & Ellington (2007)

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a 190-nt ribozyme that can polymerize up to 20 nt

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polymerasechemistry:classIligaseribozyme

b201 ligase (Bartel & Szostak, 1993)

14 rounds of 

in vitro selection

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Johnstonet al 

. (2001)Science

 292

, 883-896.

In vitro selection of the original replicase ribozyme (2001)

class I ligase

ribozyme

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Johnstonet al 

. (2001)Science

 292

, 883-896.

In vitro selection of the original replicase ribozyme (2001)

class I ligase

ribozyme

replicase-14

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Johnstonet al 

. (2001)Science

 292

, 883-896.

template extension by replicase-14

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Zaher & Unrau (2007) RNA 13, 1017-1026.

In vitro selection of an improved replicase ribozyme

replicase-14

water-in-oil

emulsions

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Zaher & Unrau (2007) RNA 13, 1017-1026.

In vitro selection of an improved replicase ribozyme

replicase-20

up to 20 nt, with 3–4-

fold more accuracy

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RoleofMetalIonsinanRNAWorld

•  Abio%cchemical

organiza%ondoesnot

becomelostuponthe

emergenceoflife•  Inves%gatethemetalion

toleranceofcataly%c

RNA

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DSLRibozyme

•  Designedusing3Dmolecularmodelingsfrom

naturallyoccurringRNAmo%fs

•  Amodularcataly%cunitwasaddedtoaselffolding

scaffold

Y. Ikawa et al. Proc. Natl. Acad. Sci. USA 101 (2004) 13750–13755.

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•  10roundsofinvitroselec%onfroma

randomsetof30nucleo%des

•  106-foldrateenhancement

•  MetalIonandpHdependent

DSLRibozyme

S Horie et al . Biochemical and Biophysical Research Com. 339 (2006) 115–121

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DSNL-0LigaseDesign

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DSNL-0Characteriza%on

8.37.57.0 8.37.57.0 8.37.57.0MgCl2

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

8.3 7.5 7

     %     S

     L     i    g    a     t    e     d

pH

MgCl2

MnCl2

CoCl2

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

MgCl2 MnCl2 CoCl2

     %     S

     L     i    g    a     t    e     d

Salt

pH8.3

pH7.5

pH7.0

MnCl2CoCl

2

Substrate

Liga%onProduct

•  MetalIonDependenceof

DSNL-0Ribozyme

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Progress:Gene%cvariability

•  Introducegene%cdiversitythroughmutagenicPCR

 –  ErrorRateof6pernucleo%deposi%on

•  Probabilityofintroducingk muta%onsforasequenceoflengthn

mutagenizedwithanerrorrateε

 –  P(k,n,ε)=(n!/[(n–k)!k!])εk (1–ε)n–k

0

2

4

6

8

10

12

14

16

18

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

     P    e    r    c

    e    n     t    a    g    e    o     f     P    o    p    u     l    a     5    o    n

Numberofmuta5onspermolecule

Distribu5onofMuta5ons

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Selec%on

•  StartSelec%onforligasevariantsthatareac%veinanunusualmetal(Co2+,Mg2+)andeffec%velycatalyzeliga%onatanop7mal pH.

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Progress:Selec%on

•  Posi%veselec%onathighpH(8.3)versusposi%veselec%onatlowpH(7.0)

G0 DSNL-0 Variants

High pH

Co2+ Mg2+ 

Low pHPosi%veselec%on

Mg2+ Co2+ Metalion

dependence

Nega%veselec%onHydrolysis HydrolysisHydrolysis Hydrolysis