1 Self-Replication versus Self-Elongation Or: How to make long oligonucleotides without enzymes,...

1

Self-Replication versus Self-ElongationSelf-Replication versus Self-Elongation

Or:Or:How to make long oligonucleotides without How to make long oligonucleotides without enzymes, primers, templates, surfaces, or enzymes, primers, templates, surfaces, or

stepwise feeding?stepwise feeding?

Oliver Thoennessen, Mathias Scheffler & G. von Kiedrowski, Ruhr-University Bochum

3rd COST D27 workshop, Heraklion, Crete, Sept. 30-Oct. 3, 2004

2

The "standard" pictureThe "standard" picture

Who agrees?

1. Self-Replication

2. Metabolism

3. Mutability

4. Some way of keeping 1-3 connected, viz. compartimentation

3

Chemical self-replicationChemical self-replication

+

katalysierteLigation

Assoziation Dissoziation

ABC C2

CCA B

spontane Ligation

ka

kb

4

Open systems, possible non-catalyzed pathwaysOpen systems, possible non-catalyzed pathways

+2 3 4

+ + + +

x 2

x 2

432 5 61

building blocks for "closed" systems,single-sided reactivivity

building blocks for "open" systems,dual-sided reactivity

templates,non-reactive

complementary

self-complementary

5

Open systems: possible template-directed pathwaysOpen systems: possible template-directed pathways

+ + + + n

6

OP

O

O

ON

N

N

N

NH2

O

O

P

O

O

O

ONH2 N

N

O

NH2

OP

O

O

O

OO N

N

N

NH

O

NH2

P

O

O

O

ONH2 N

N

O

NH2

OP

O

O

O

OO N

N

N

NH

O

NH2

P

O

O

O

ONH2 N

NH

O

O

CH3

OP

O

O

ON

N

N

N

O

O

P

O

O

O

ONH2 N

NH

O

O

CH3

NH2

PO42-NH2 CA

bzw.CAn p

bzw.TGn p

PO42-NH2TG

bzw.TAn p

PO42-NH2TA

bzw.CGn p

PO42-NH2 CG

T G

C G

C A

T A

DimerDimerbuilding blocksbuilding blocks

for an open for an open system:system:

nYRpnYRp

7

Dimer synthesisDimer synthesis

OH

OBOH

O

OP

O

O

OB'2OH

Cl S

B'1

O

OP

O

O

O

Cl

N3

P

O

O

OB2OP

O

O

OB1N3

O

S

O

O

N+

O

OMe SO2ClH

+NEt3

B1 B2

C A C G T A T G

OP

O

O

OB2O

O

P

O

O

O

OB1NH2

OP

O

O

OB2O

O

P

O

O

O

OB1N3

OP

O

O

OB2O

O

P

O

O

O

ONH2 B1

S

1. base protection2. CBr4, PPh3, LiN3, DMF

3. o-ClPhOP(OCE)O2- Et3NH+,

Efimov coupling reagents4. NEt3

A', G'

C', T

1. Efimov:

2. TBAF3. NH3

P(Ph)3

NaIO4,

NaOH

+ P(Ph)3

1. base protection2. DMT-Cl3. o-ClPhOP(OPTE)O2

- Et3NH+,

Efimov coupling reagents4. H+

8

Ligation versus Cyclisation

OHP

O

O

OA/GO

O

P

O

O

O

OC/TNH

2

NC

N NH

+

NH

NH

+

NH

O

OHP

O

O

OA/GO

O

P

O

O

O

OC/TNH

2NH

NH

+

NH

O

OP

O

OA/GO

O

P

O

O

O

OC/TNH

2

XYn p

DimerisierungA:nXYp

- EDU

P

O

O

OA/GO

O

P

O

O

O

OC/TN

H

n

OHP

O

O

OA/GO

O

P

O

O

O

OC/TN

HP

O

O

OA/GO

O

P

O

O

O

OC/TNH

2

EDCnXYp

- EDU

CyclisierungB:- EDU

O

O

O

NH

P

PO

O

OO

OO

A/G C/T

+ H+

+ H2O

EDC

EDU

- H+

A

+

B

Oligomerisierung

12-Ring

CA-Cyclus

c( XY ) = 1-10 mM Cyclisierung

c( XY ) > 20 mM Oligomerisierung

n p

n p

B:

A:

Reaktionsbedingungen:

0.2 M EDC in 0.1 M HEPES-Puffer, 2° - 30°C

9

Oligomerisation of nCGp-dimersOligomerisation of nCGp-dimers

10 15 20

0

250

500

750

1000

1250

1500

2

4 6 8 10 1216

14 221820 24

26

nCGp

120 min

240 min

3 min

60 min

Abs

orpt

ion

[mV

]

time [min]

C G

C GC GC GC G

C GC GC G

C G C G

2-mer

4-mer

6-mer

8-mer

10

Reactivity of nYRp building blocks Reactivity of nYRp building blocks

Reaktivität: nCGp >> ( nTAp nTGp ) > nCAp

2mer, 30°C

0

10

20

30

40

50

0 100 200 300 400Zeit [min]

c[m

M]

nTGp,30°C

nTAp,30°C

nCGp,30°C

nCAp,30°C

4mer, 30°C

0

1

2

3

4

5

0 100 200 300 400Zeit [min]

c[m

M]

(nTGp)2,30°C

(nTAp)2,30°C

(nCGp)2,30°C

(nCAp)2,30°C

Dimer Tetramer

11

The current "Guiness" of prebiotic polymerisationThe current "Guiness" of prebiotic polymerisation

5 10 15 20 25 30 35 400

200

400

600

4mer

60mer

40mer50mer

20mer30mer

10mer

nCGp

Abs

orpt

ion

[mV

]

time [min]

50 mM CG0.4 M EDC, 2°C

n p

5 10 15 20 250

200

400

Xmere

3 d20mer 30mer

10mer

nCGp

241 min

Abs

orpt

ion

[mV

]

time [min]

20 mM CG0.4 M EDC, 2°C

n p

nach 3 Tagenvollständige Abreaktion

der kurzen Oligos

nach 120 minOligos > 60mer

12

No template effects in reactions using No template effects in reactions using single-sided building blocks single-sided building blocks

pteG C n pG C N 3

H OC G C G O H H OC G C G O H

G C G C O H

pteG C npG C G C G C O H

H OC G C G C G C G O HH OC G C G C G C G O H

ppteG C n G C G C G C O H

pteG C npG C G C O H

H OC G C G C G O H

pteG C npG C N 3

2 + 2an 4

2 + 4an 6

2 + 6an 8

H OC G C G C G O H

ppteG C n

HO(CG)3p + nCGpte je 20 m M + Te m plat HO(CG)4

o, 2° C,0.4 M EDC / 0.1 M HEPES, Produk t: HO(CG)3

pnCGpte

0

2

4

6

8

10

0 50 100 150 200 250Zeit [min]

c[m

M]

+0% Tem plat

+10% Tem plat

+20% T em plat

+40% T em plat

13

Earlier results from Zielinski & Orgel: Earlier results from Zielinski & Orgel:

Nature 1987: Experiments on a self-replicating tetraribonucleotide analogue confirmed our "square-root law". EDC as the source of energy, efficient replication in:

GCn + pGC --> GCnpGC

J. Mol. Evolution, a few years later: No self-replication at all in a slightly different system:

CGn + pCG --> CGnpCG

Speculations about the involvement of "slidomers".

14

Efficient oligomerisation via sliding, Efficient oligomerisation via sliding, concatenation, and concatomer ligation? concatenation, and concatomer ligation?

free oligomers

straight duplexes

slidomer duplexes

concatomerC GC GC G

G CG CG C

C G C G

G C G C

C GC GC GC G C G

G CG CG CG C G C

C G C G

G C G C

C GC GC GC G

G CG CG CG C

10-mer 4-mer 8-mer6-mer 4-mer


C GC GC G

G CG CG C

C GC GC G

G CG CG C

C G C G

G C G C

C GC GC GC G

G CG CG CG C

C G C G

G C G C

C GC GC GC G C G

G CG CG CG C G C

C G C G

G C G C

C GC GC GC G C G

G CG CG CG C G C

C GC GC G

G CG CG C

C GC GC GC G

G CG CG CG C

C G C G

G C G C

duplexation

sliding

aggregation

15

How a concatomer might lookHow a concatomer might look

C GC GC G

G CG CG C

C G C G

G C G C

C GC GC GC G C G

G CG CG CG C G C

C G C G

G C G C

C GC GC GC G

G CG CG CG C



16

Better base stacking via slidomer Better base stacking via slidomer concatenationconcatenation

CG dimer GC dimer CGCG slided duplex

17

Thermodynamic data support Thermodynamic data support slided concatomersslided concatomers

Stabilitätsverhältnis von Slidomer zu Duplex inAbhängigkeit von der Bausteinanzahl, B-Form

-120

-100

-80

-60

-40

-20

0

20

40

60

0 10 20 30Anzahl der Bausteine

G° (S

lid

om

er-

Du

ple

x)/

G° (D

up

lex

)in

%

dCGCG dCGCGCG dCGCGCGCG

pGCnpGCn

G0 = 0.22 kcal·mol-1 G0 = -4.19 kcal·mol-1

G = -14.24 kcal·mol-1

G0 = -8.60 kcal·mol-1

G37°C,Total = -26.81 kcal·mol-1

G = -7.12 kcal·mol-1 G = -7.12 kcal·mol-1

Energiegewinn durch Slidomerfortsetzung

pGCnpGCn

nCGpnCGp

nCGpnCGp nCGpnCGpnCGp

pGCnpGCnpGCn

nCGpnCGpnCGpnCGp

pGCnpGCnpGCnpGCn

nCGpnCGpnCGpnCGp

pGCnpGCnpGCnpGCn pGCnpGCnpGCn

nCGpnCGpnCGp

G37°C,Total = -12.57 kcal·mol-1

18

Two possible modes of ligationTwo possible modes of ligation

GC GC

CGCGCG CGCGCG

GCGCGCGC

CG CG

GC GC

CG CGCGCG

GCGCGC GC

CGCGCG

GCGCGC

CG CG

GC GC

GCGCGCGC GC

CGCGCGCG CG

19

SimFitting supports slidomer modelSimFitting supports slidomer model

RMS = 12.5% RMS = 2.4%

n pCGCGn pC G + 7.5%, 15%, 30%

3rd order ligation slidomer model

rate parameter

k EDC hydrolysis = 4 4.93 10•-8

s -1

k 3rd order ligat. = M s1-2 -18.55 10•

-3

M s-1 -1k slidom er ass. = 10ass

6

k slidomer diss. = 2 3.24 10 s•4 -1

k slidom er ligat. = 3 2.56 M s-3 -1

20

Reaction modelReaction modelSpontanes Ligationsmodell

Nr. Reaktionsgleichung Simfit-Kurzform k

1 nCGp + nCGp + EDC → (nCGp)2 + EDU n2 + n2 + EDC → n4 + EDU k1

2 nCGp + (nCGp)2 + EDC → (nCGp)3 + EDU n2 + n4 + EDC → n6 + EDU k1






8 (nCGp)2 + (nCGp)3 + EDC → (nCGp)4 + EDU n4 + n4 + EDC → n8 + EDU k1









Erweiterung für Slidomermodell

Nr. Reaktionsgleichung Simfit-Kurzform k

(nCGp)2 + (nCGp)2 → (nCGp)2/(nCGp)2-Slidomerduplex

(nCGp)2 + n(CG)2p → (nCGp)2/

n(CG)2p- Slidomerduplex 17

n(CG)2p + n(CG)2

p → n(CG)2p/n(CG)2

p- Slidomerduplex

n4 + n4 → n4s 106

18 (nCGp)3 + (nCGp)3 → (nCGp)3/(

nCGp)3- Slidomerduplex n6 + n6 → n6s 106

19 (nCGp)4 + (nCGp)4 → (nCGp)4/(


20 (nCGp)5 + (nCGp)5 → (nCGp)5/(


21 (nCGp)6 + (nCGp)6 → (nCGp)6/(


22 (nCGp)7 + (nCGp)7 → (nCGp)7/(


23 (nCGp)8 + (nCGp)8 → (nCGp)8/(


(nCGp)2/(nCGp)2-Slidomerduplex → (nCGp)2 + (nCGp)2

(nCGp)2/n(CG)2

p-Slidomerduplex → (nCGp)2 + n(CG)2p 24

n(CG)2p/n(CG)2

p-Slidomerduplex → n(CG)2p + n(CG)2

p

n4s → n4 + n4 k2

25 (nCGp)3/(

nCGp)3-Slidomerduplex → (nCGp)3 + (nCGp)3 n6s → n6 + n6 k2·10-4

26 (nCGp)4/(


27 (nCGp)5/(


28 (nCGp)6/(


29 (nCGp)7/(nCGp)7-Slidomerduplex → (nCGp)7 + (nCGp)7 n14s → n14 + n14 k2·10-20

31 (nCGp)2/(nCGp)2-Slido + (nCGp)2/(nCGp)2-Slido + 2

EDC → (nCGp)4/(nCGp)4-Slido + 2 EDU

n4s + n4s + 2 EDC

→ n8s + 2 EDU

k3 (nCGp)2/n(CG)2p-Slido + (nCGp)2/(nCGp)2-Slido + 2

EDC → (nCGp)4/((nCGp)2(n(CG)2p))-Slido + 2 EDU

(nCGp)2/n(CG)2p-Slido + (nCGp)2/n(CG)2p-Slido + 2

EDC → (nCGp)4/(n(CG)2p)2-Slido + 2 EDU

(nCGp)2/n(CG)2p-Slido + n(CG)2p/n(CG)2p-Slido + 2

EDC → ((nCGp)2n(CG)2p)/(n(CG)2p)2-Slido + 2

EDU

n(CG)2p/n(CG)2p-Slido + n(CG)2p/n(CG)2p-Slido + 2

EDC → (n(CG)2p)2/(n(CG)2p)2-Slido + 2 EDU

32 (nCGp)2/(nCGp)2-Slido + (nCGp)3/(nCGp)3-Slido +

2 EDC → (nCGp)5/(nCGp)5-Slido + 2 EDU

n4s + n6s + 2 EDC

→ n10s + 2

EDU

k3



n4s + n8s + 2 EDC

→ n12s + 2

EDU

k3



n4s + n10s + 2

EDC → n14s +

2 EDU

k3



n4s + n12s + 2

EDC → n16s +

2 EDU

k3



n6s + n6s + 2 EDC

→ n12s + 2

EDU

k3



n6s + n8s + 2 EDC

→ n14s + 2

EDU

k3



n6s + n10s + 2

EDC → n16s +

2 EDU

k3



n8s + n8s + 2 EDC

→ n16s + 2

EDU

k3

40 2 (nCGp)2 + (nCGp)2/(nCGp)2-Slido + 2 EDC

→ (nCGp)3/(nCGp)3-Slido + 2 EDU

2 n2 + n4s + 2

EDC → n6s + 2

EDU

k3



2 n2 + n6s + 2

EDC → n8s + 2

EDU

k3



2 n2 + n8s + 2

EDC → n10s + 2

EDU

k3



2 n2 + n10s + 2

EDC → n12s + 2

EDU

k3



2 n2 + n12s + 2

EDC → n14s + 2

EDU

k3



2 n2 + n14s + 2

EDC → n16s + 2

EDU

k3

46 EDC → EDU EDC → EDU k4

21

RMS as the function of a common RMS as the function of a common slidomer equilibrium factorslidomer equilibrium factor

Geschwindigkeitskonstantendes Slidomer-Modells

k EDC-Hydrolyse = 4 4.93 10•-8

k Spontane Ligation = 1 8.55 10•-3

k Slidomer-Assoziation = 10ass6

k Slidomer-Dissoziation = 2 3.24 10•4

k Slidomer-Ligation = 3 2.56

22

"Template" addition even inhibits "Template" addition even inhibits polymerisationpolymerisation

nCGp 20 m M + Tem plat H OCGCGOH , 2°C,0.4 M EDC/0.1 M HEPES, Produkt: 8m er

0 ,0 0

0 ,0 2

0 ,0 4

0 ,0 6

0 ,0 8

0 2 0 4 0 6 0Zeit [m in]

c[m

M]

8m er, 0%T

8m er, 10%T

8m er, 20%T

8m er, 40%THOGCGCOHHOGCGCOH

pnCG HOCGCG OH HOCGCG OH

HOGCGCOHHOGCGCOH

pHOCGCG OH nCGpnCG HOCGCG OH

HOGCGCOH

Modes of inhibitionby "non-reactive" template

4mer1.

6mer2.

8mer3.

nCGp

HOCGCG OH

HOCGCGCG OH

HOCGCGCGCGOH

20m M10%, 20%, 40%

Templat+

23

Summary and possible significanceSummary and possible significance The current picture to make long prebiotic oligomers is by primer-extension on a solid support (clay) via feeding with nucleotide-phosphorimidazolides (Ferris & Orgel, "crepes scanario"). Traces of 50-mers can be detected after several weeks and daily replenishment of the imidazolides.

"Self-elongation" as an alternative picture: In the presence of the dehydration reagent EDC, the dimer nCGp yields high molecular weight oligomers (quantitatively for n >> 40) after 3 days.

"Self-elongation" and "self-replication" may be different sides of the same coin. Exactly the same reason that caused poor self-replication in a comparable system causes efficient polymerisation in our system.

Eigen, Hartman, and others have speculated that the earliest "genes" were rich in C and G, or even CG-repeats. Our experiments indicate that one may neither need templates nor surfaces to arrive at such structures.

Outlook: Co-oligomerization experiments with nYRp are expected to result in materials still rich in CG but "being doped" with other bases. Such materials may have the capacity to fold into discrete secondary structures.

1 Self-Replication versus Self-Elongation Or: How to make long oligonucleotides without enzymes,...

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