Prebiotic RNA Synthesis by Montmorillonite Catalysis: Significance of Mineral Salts Prakash C. Joshi New York Center for Astrobiology & Department of Chemistry.

Prebiotic RNA Synthesis by Montmorillonite Catalysis: Significance of Mineral Salts

Prakash C. JoshiNew York Center for Astrobiology &

Department of Chemistry and Chemical BiologyRensselaer Polytechnic Institute

Troy, NY 12180, USA

“RNA World Hypothesis”

• The “RNA World” hypothesis proposes that RNA was the central biopolymer in the first life on the Earth and DNA and protein evolved from it.

• RNA possesses both catalytic property and stores genetic information.

Walter Gilbert Thomas Cech Sidney AltmanCarl Woese

Prebiotic RNA Synthesis

Most theories of the origin of biological organization have suggested that for a functional RNA, chain length in the range of 40-60 monomers

are needed to make a genetic system viable.

Joyce, G.F & Orgel, L.E, The RNA World, Cold Spring Harbor Laboratory Press, 1993, pp 1-25

RNA Structures consisting of one, two or three stem-loop elements*

a, Single stem-loop elements (12 – 17 nucleotides)

b, Pseudoknot (18– 40 nucleotides)

c, An RNA consisting of triple stem-loop structure containing 40-60 nucleotides have the potential to fold into catalytic structures, or ribozymes.

*Joyce, G.F & Orgel, L.E, The RNA World, Cold Spring Harbor Laboratory Press, 1993, pp 1-25

Small trans-aminoacylating RNA complexes*

• Small transamino- acylating RNA complexes. (A) C3 RNA. (B) Intermediate trans complexes. (C) Final GUGGC/GCCU complex

(Michael Yarus Lab)

*Turk R M et al. PNAS, 2010, 107, 4585-4589

Model Reactions of Activated Nucleotides on Montmorillonite

A U G C

ImpNNa+

Na+-Montmorillonite

pH 6-9, 25oC,3 days

(pN)2 - (pN)n

where N = A, U, G, or C

Why Catalysis?

• Contemporary biochemical reactions require catalysts (enzymes) for biopolymer formation and a similar situation may have existed in the primitive Earth as well.

• Mineral and metal ions are most likely the only candidates to have served as catalyst for the formation of biopolymers required to initiate the formation of early life because of their large abundance on Earth.

What is Montmorillonite ?

• Main constituent of volcanic ash weathering product, Bentonite.

• Discovered in Montmorillon (France) in 1847.

• Reactive surface:800m2/g• A 2:1 layer silicate clay in

which two tetrahedral sheets sandwich a central octahedral sheet.

Montmorillon

Montmorillonite are 2:1 layer silicates that have a wide range of chemical composition:

(Ca,Na)0.3(Al,Fe,Mg)2(Si,Al)4O10(OH)2.nH2O

Site of Reaction: Clay Inter-layers

C lay p la te le ts stackedC lay p late le ts

stacked (Front V iew)

N ucleotide species

Analysis of RNA Oligomers by HPLC

0 Time, minutes 25

2C 2L 3 4 5

11¯¯

HPLC separates oligomers based on the number of negative charge. The monomer has 2 negative charges. Addition of each nucleotide adds up an additional negative charge. Products are eluted accordingly on an Ion Exchange Column with:Buffer A: 0.04M Tris, Buffer B: 0.04M Tris, 0.2M NaClO4 at pH 8.

ImpA with 1M NaCl ImpA with 1M NaCl and and montmorillonitemontmorillonite

Determination of Chain-length ofDetermination of Chain-length of RNA Oligomers RNA Oligomers

MALDI Mass Spectral AnalysisMALDI Mass Spectral Analysis AutoradiographyAutoradiography

n=17n=17

n=33n=33

↓↓

32P-labelledProducts Separated byPAGE→

Estimated maximumoligomer length around 55 nucleotides

A Comparison of Oligomerization Between Activated Ribo- and deoxyribo-Nucleotides on Na+-Montmorillonite

Montmorillonite

pH 8, 25oC,3 days

(pA)2 - (pA)n

where base = Adenine

RNA Synthesis: Yes

With or without

Montmorillonite

pH 8, 25oC,3 days

(pdA)2

DNA Synthesis: NO

Chain length of Oligomers of 15 mM ImpA formed at varying pH of Volclay

Volclay

(pH)I II* II** III IV V VI VII VIII IX X

3.0 97.1 1.4 0.01 -

4.0 94.2 4.3 0.11 -

5.0 87.2 10.0 1.0 0.07 -

7.0 29.5 47.2 13.2 4.8 1.9 0.8 0.4 0.1 0.04 0.02 -

9.0 14.9 59.4 11.5 5.2 2.7 1.4 0.9 0.3 0.1 0.06 0.02

10.5 25.3 48.0 13.6 6.3 2.8 1.4 0.7 0.3 0.1 0.01 -

Control 96.4 1.1 0.01

Origins of Life and Evol. Biosph, 36: 2006, 343-361

Catalytic action of Montmorillonite from different sources

Montmorillonite I II* II** III IV V VI VII VIII IX

Belle Fourche (SD) 29.8 45.1 14.6 5.60 2.40 1.00 0.50 0.20 0.10 0.03

Little Rock (Arkansas) 54.5 31.8 10.0 2.40 0.60 0.14 0.01 -

Chambers (Arizona) 95.0 4.40 0.11 0.01 0.002 -

Otay (California) 97.7 2.28 0.60 -

Volclay (Am. Coll. Corp) 29.5 47.2 13.2 4.80 1.90 0.84 0.36 0.12 0.04 0.02

Joshi et al., J. Am. Chem. Soc. 131, 2009, 13369-13374

Chain length→

I II II III IV V VI VII VIII IX X

Clay↓ Cyclic Linear

C-bed 14.1 58.4 12.2 5.80 3.00 1.70 1.04 0.50 0.21 0.07 Trace

F-bed 92.9 2.30 0.07 0.02 Trace

A-bed 22.2 48.0 14.3 5.80 3.00 1.40 0.74 0.23 0.05 0.01 Trace

Catalytic Activity of Belle Fourche Clay Collected from Different Beds

a to m ic p ro p o rtio n s

Excellen tG oodPoor

F e* 5M g * 5

Optimum Al41 – 48%

Optimum Mg4.8 – 6.2%

Optimum Fe5 – 6%

Al-Fe-Mg ternary diagram showing a tight cluster of catalytic

montmorillonites

Influence of Salt Concentration inRNA Oligomer Chain Length

Joshi & Aldersley (2013) J. Mol. Evolution: 76, 371-379.

ImpA + Na+-montmorillonite Reaction: Formation of Products at Varying Concentrations of NaCl

Examples of linear and cyclic dimer structures

Unwanted

a) D, D-3’,5’-ApA; b) D, D-2’,5’-UpU; c) D, D-c-3’,5’-ApA

Preferred natural regio-isomer

Unwanted

Elongation

Intercalators as Means to Supress Cyclization and Promote Polymerization of Base-pairing of Oligonucleotides

Nicholas Hud, GIT

Horowitz et al. PNAS 2010;107:5288-5293

Proflavin

A, Strand cyclization and its prevention; B, Nicked duplex resulting from intercalation

Effect of Hydrophilic and Hydrophobic Mineral Salts On Montmorillonite-Catalyzed RNA Synthesis

Nature* Reagent Chain length**Hydrophilic LiCl4 12

(NH2)2C=NH 12

Hydrophobic Na2SO4 12

LiCl 12None LiBr 12

CsCl 10*Kool & Breslow (1988) J. Am. Chem. Soc., 110, 1596-1597**Determined by HPLC analysis

Role of Mineral Anions and Cations on Montmorillonite-Catalyzed RNA Synthesis

Salt Oligomer length* Salt Oligomer length*

LiCl 12 LiI 9NaCl 11 NaI 9KCl 7 KI 5LiClO4 12 NaCl 11

NaClO4 9 NaBr 10

KClO4 6 NaI 9

Li+ > Na+ > K+ Cl- > B- > I-

*Determined by HPLC analysis

Chiral Selection in RNA Chiral Selection in RNA

• Ribose component of RNA exists in two stereoisomeric forms (D and L) that are mirror images of each other.

• Only D-ribose is present in naturally occurring RNA.• The question is how chiral selection was introduced into the

prebiological system?

D-ImpU

D-ImpA

L-ImpU

L-ImpA

*** * *** *

Homochiral Selection

Quaternary Reactions of D, L-ImpA and D, L-ImpU on Na+-Montmorillonite

(pN)2 to (pN)11

(as determined by HPLC

where N = D/L-A, D/L-U)

Na+-Montmorillonite

pH 7, 25oC, 3 days

Dimers formed by the Quaternary reaction of D, L-ImpA + D, L-ImpU on Montmorillonite

(1) UppU (2) D, D & L, L-c-A3’pU3’p (3) Uridine (4) D, L & L, D-c-A3’pA3’p (5) 3’, 5’-c-UMP (6) D, D & L, L-c-A3’pA3’p (7) D, D & L, L-U2’pU (8) Adenosine (9) D, L & L, D-U2’pU (10) D, D & L, L-U3’pU & D, D & L, L-A2’pU (11) D, L & L, D-U3’pU (12) D, L & L, D-A2’pU (13) D, D & L, L-A2’pA (14) D, D & L, L-A3’pU (15) D, L & L, D-A3’pU(16) D, L & L, D-A2’pA (17) D, D & L, L-A3’pA (18) D, L & L, D-A3’pA

Ion exchange HPLCReverse-phase HPLC

Fractioncollection

Homochiral SelectionHomochirality of oligomers in a quaternary reaction of

D, L-ImpA with D, L-ImpU on Na+-montmorillonite

Homochiralit

yMonomer Dimer Trimer Tetramer Pentamer

Observed 50% 64% 76% 93% 97%

Calculated 50% 50% 25% 12.5% 6.25%

Ratio1 : 1 1 : 1.28 1 : 3.04 1 : 7.44 1 : 15.5

Joshi, Aldersley & Ferris (2013) Advances in Space Research, 51, 772-779.Joshi, Aldersley & Ferris (2011) Biochemical & Biophysical Res. Commun. 413, 594-598.Joshi, Aldersley & Ferris (2011) Orig. Life Evol. Biosph., 41, 213-236.Joshi, Pitsch & Ferris (2007) Orig. Life Evol. Biosph., 37: 3-26.Joshi et al. (2011) Orig. Life Evol. Biosph., 41, 575-579.

Constructing “RNA World” From Racemic Mixture of A, U, G and C on Na+-montmorillonite

H2N NO

Na+-Montmorillonite

pH 7, 25oC, 3 days

(n = chain length)

Search for catalytic RNA

Combinatorial ChemistryD,L-ImpA + D,L-ImpU + D,L-ImpG + D,L-ImpC on Clay

(128128 Possible Dimers)

D, D-pA2'pAL, L-pA2'pA

D, D-pU2'pUL, L-pU2'pU

D, D-pG2'pGL, L-pG2'pG

D, D-pC2'pCL, L-pC2'pC

D, D-pA3'pAL, L-pA3'pA

D, D-pU3'pUL, L-pU3'pU

D, D-pG3'pGL, L-pG3'pG

D, D-pC3'pCL, L-pC3'pC

D, L-pA2'pAL, D-pA2'pA

D, L-pU2'pUL, D-pU2'pU

D, L-pG2'pGL, D-pG2'pG

D, L-pC2'pCL, D-pC2'pC

D, L-pA3'pAL, D-pA3'pA

D, L-pU3'pUL, D-pU3'pU

D, L-pG3'pGL, D-pG3'pG

D, L-pC3'pCL, D-pC3'pC

D, D-pA2'pUL, L-pA2'pU

D, D-pA2'pGL, L-pA2'pG

D, D-pA2'pCL, L-pA2'pC

D, D-pU2'pGL, L-pU2'pG

D, D-pA3'pUL, L-pA3'pU

D, D-pA3'pGL, L-pA3'pG

D, D-pA3'pCL, L-pA3'pC

D, L-pA2'pUL, D-pA2'pU

D, L-pA2'pGL, D-pA2'pG

D, L-pA2'pCL, D-pA2'pC

D, L-pA3'pUL, D-pA3'pU

D, L-pA3'pGL, D-pA3'pG

D, L-pA3'pCL, D-pA3'pC

D, D-pU2'pAL, L-pU2'pA

D, D-pG2'pAL, L-pG2'pA

D, D-pC2'pAL, L-pC2'pA

D, D-pG2'pUL, L-pG2'pU

D, D-pU3'pAL, L-pU3'pA

D, D-pG3'pAL, L-pG3'pA

D, D-pC3'pAL, L-pC3'pA

D, L-pU2'pAL, D-pU2'pA

D, L-pG2'pAL, D-pG2'pA

D, L-pC2'pAL, D-pC2'pA

D, L-pU3'pAL, D-pU3'pA

D, L-pG3'pAL, D-pG3'pA

D, L-pC3'pAL, D-pC3'pA

D, D-pU2'pCL, L-pU2'pC

D, D-pG2'pCL, L-pG2'pC

D, D-pC2'pUL, L-pC2'pU

D, D-pC2'pGL, L-pC2'pG

D, D-pU3'pCL, L-pU3'pC

D, D-pC3'pUL, L-pC3'pU

D, D-pC3'pGL, L-pC3'pG

D, L-pU2'pCL, D-pU2'pC

D, L-pC2'pUL, D-pC2'pU

D, L-pC2'pGL, D-pC2'pG

D, L-pU3'pCL, D-pU3'pC

D, L-pC3'pUL, D-pC3'pU

D, L-pC3'pGL, D-pC3'pG

Summary

► Montmorillonite clay minerals are not only an excellent catalyst for the synthesis of RNA but they also facilitate chiral selection. ► Prebiotic RNA synthesis must have been a simple process requiring only saline clay minerals for the reaction to progress.

Autocatalysis Over Multiple Usages of Clay

Catalytic behavior was expected to decline with usage as occurs with oligomer synthesis.

This is not the case with the majority of dimers syntheses. The difference between the two curves is caused by

autocatalysis

Terahertz Spectroscopic Evaluation of Prebiotic RNA Synthesis in the Catalytic Interlayer of Montmorillonite

Wilke & Joshi*

How are activated nucleotides adsorb in the clay interlayer?

Why do purines adsorb more strongly on clay than pyrimidine's?

Understanding the cyclization reactions of activated nucleotides?

Influence of hydrophilic/hydrophobic interactions?

Understanding of the mechanism of chiral selection?

Application in exploration of biosignatures in explanatory systems.

*Applied Clay Science (2013) in Press

Building a Basic Living CellJack W. Szostak

Jack Szostak and his colleagues at Harvard Medical School are seeking to understand the origin of life through a series of experiments intended to build a basic living cell from scratch. Using a simple experiment, they now demonstrate that one of the key steps—creating a simple growing cell by tucking self-reproducing molecules into a membrane—may be startlingly simple.

Sweet Answer to the Origins of Life

John Sutherland

“We have discovered a way to generate the biomolecules needed to synthesize RNA from the simple molecules that were abundant on earth nearly

four billion years ago. Ironically, the feedstock molecule is HCN – a molecule that is acutely toxic to us.”

Powner & Sutherland (2011) Phil. Trans. R. Soc. B, 366, 2870-2877

The End

Basic Prebiotic Chemistry

Walter Gilbert

►Gilbert proposed the RNA world hypothesis for the origins of life based on a concept first proposed by Carl Woese in 1967*

*Gilbert W. (1986) “Origins of life: The RNA world”, Nature 319, 618.

Carl Woese

Sidney Altman’s Nobel Prize Winning Research

►Discovered RNase P, a ribonucleoprotein consisting of both a structural RNA molecule and a protein.

►He observed that the RNA component, in isolation, was sufficient to observe the catalytic activity of the enzyme.

RNase P

RNA fragment

Thomas Cech’s Nobel Prize Winning Research

►Studied the splicing of RNA in a unicellular organism, Tetrahymena thermophila.► He discovered that an unprocessed RNA molecule could splice itself to give RNA enzymes or ribozymes

RNase P

RNA fragment

Tetrahymena

From simple to complex compounds

Activation of the nucleotides?

Goal: Synthesis of a biopolymer as an enzyme and self replicator

Base + Sugar + H3PO4 = NucleotidenNucleotides = Functional RNA

Why RNA?

Activation of Mononucleotides

where N= A, U, G, or C

MeadpN

Preparation of an Activated Nucleotide

Imidazole salt of5'-AMP

(PyS)2 in DMF

OPHOOH

DMSO, DMF

Imidazole salt of5'-AMP

A Comparison of Oligomerization Between Activated Ribo- and deoxyribo-Nucleotides on Na+-Montmorillonite

With or without

Montmorillonite

pH 8, 25oC,3 days

(pdA)2

DNA Synthesis: NO

pKa values barely differ but higher rates in the presence of a vicinal hydroxyl may be best explained by interactions through hydrogen bonding between the 2’ or 3’-OH and a phosphoryl oxygen (Aastroem et al., J. Am. Chem. Soc., 2004, 126, 14710-14711).

•2’-OH: pKa = 12.14 (Velikyan et al., J. Am. Chem. Soc. 2001, 123, 2893-2894).

BaseOO

Montmorillonite

pH 8, 25oC,3 days

(pA)2 - (pA)n

RNA Synthesis: Yes

LOWLOWHIGHHIGH

Excellent

Sample I II II III IV V VI VII VIII IX X

# Cyclic Linear

C-bed 14.1 58.4 12.2 5.80 3.00 1.70 1.04 0.50 0.21 0.07 Trace

F-bed 92.9 2.30 0.07 0.02 Trace

A-bed 22.2 48.0 14.3 5.80 3.00 1.40 0.74 0.23 0.05 0.01 Trace

Catalytic Activity of Belle Fourche Clay Collected from Different Beds

Prebiotic RNA Synthesis by Montmorillonite Catalysis: Significance of Mineral Salts Prakash C. Joshi New York Center for Astrobiology & Department of Chemistry.

rna structures

functional rna

ac3 rna

chain length of oligomers

montmorillonite catalysis

maximumoligomer length

na montmorillonite

biopolymer formation

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