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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
1
“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
6
A U G C
NO
O
OHHO
P
O
-O
N
N
ImpNNa+
Na+-Montmorillonite
pH 6-9, 25oC,3 days
(pN)2 - (pN)n
N
NNH
N
NH2
NH
NH
O
O
NH
NNH
N
O
NH2
N
NH
NH2
O
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
1
2C 2L 3 4 5
6
78
910¯
11¯¯
OD260
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
12
BaseO
O
OHHO
P
O
-O
N
N
ImpA
Na+
Montmorillonite
pH 8, 25oC,3 days
(pA)2 - (pA)n
where base = Adenine
RNA Synthesis: Yes
where base = Adenine
BaseO
O
HHO
P
O
-O
N
N
ImpdA
Na+
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
A l
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
19
Examples of linear and cyclic dimer structures
N
NN
N
NH2
O
OHO
HO
N
NN
N
NH2
O
OHOH
OP
O
-O
3'
5'
3'
NH
O
ONO
OOH
HO
NH
O
ONO
OHOH
O
P
O
2'
5'
O-
N
NN
N
NH2
O
OHO
O
N
N N
N
NH2
O
OH O
O
P
O O-
P-O O
Unwanted
a b
a) D, D-3’,5’-ApA; b) D, D-2’,5’-UpU; c) D, D-c-3’,5’-ApA
c
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
Joshi & Aldersley (2013) J. Mol. Evolution: 76, 371-379.
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
Joshi & Aldersley (2013) J. Mol. Evolution: 76, 371-379.
23
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?
N
NN
N
NH2
O
OHOH
OPN
O-
O
N
NH
O
ONO
OHOH
OPN
O-
O
N
D-ImpU
D-ImpA
N
N N
N
NH2
O
OH OH
O P N
O-
O
N
HN
O
O NO
OH OH
O P N
O-
O
N
L-ImpU
L-ImpA
*** * *** *
*** * *** *
Homochiral Selection
Quaternary Reactions of D, L-ImpA and D, L-ImpU on Na+-Montmorillonite
24
O
OH OH
O NP
O-O
N
(pN)2 to (pN)11
(as determined by HPLC
where N = D/L-A, D/L-U)
N
NN
N
NH2
HN
N
O
O
O
OHOH
ONP
O O-
N O
OHOH
ONP
O O-
N
NH
N
O
O
N
N N
N
NH2
O
OH OH
O NP
O-O
N
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
26
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
27
N
NN
N
NH2
O
OHOH
OPO-
O
NH
N
N
O
NH2N
O
OHOH
OPO
O-
N
NH2
ONO
OHOH
OPO-
O
NH
O
ONO
OHOH
OPO-
O
N
N
N
N
NN
NN
N
N N
N
NH2
O
OH OH
OP
-O
O
HN
N
N
O
H2N NO
OH OH
O PO
-O
N
NH2
O N O
OH OH
O P-O
O
HN
O
O NO
OH OH
O P-O
O
N
N
N
N
NN
NN
Na+-Montmorillonite
pH 7, 25oC, 3 days
2x4n
(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, D-pU2'pGL, L-pU2'pG
D, L-pA2'pUL, D-pA2'pU
D, L-pA2'pGL, D-pA2'pG
D, L-pA2'pCL, D-pA2'pC
D, D-pU2'pGL, L-pU2'pG
D, L-pA3'pUL, D-pA3'pU
D, L-pA3'pGL, D-pA3'pG
D, L-pA3'pCL, D-pA3'pC
D, D-pU2'pGL, L-pU2'pG
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, D-pG2'pUL, L-pG2'pU
D, L-pU2'pAL, D-pU2'pA
D, L-pG2'pAL, D-pG2'pA
D, L-pC2'pAL, D-pC2'pA
D, D-pG2'pUL, L-pG2'pU
D, L-pU3'pAL, D-pU3'pA
D, L-pG3'pAL, D-pG3'pA
D, L-pC3'pAL, D-pC3'pA
D, D-pG2'pUL, L-pG2'pU
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-pG2'pCL, L-pG2'pC
D, D-pC3'pUL, L-pC3'pU
D, D-pC3'pGL, L-pC3'pG
D, L-pU2'pCL, D-pU2'pC
D, D-pG2'pCL, L-pG2'pC
D, L-pC2'pUL, D-pC2'pU
D, L-pC2'pGL, D-pC2'pG
D, L-pU3'pCL, D-pU3'pC
D, D-pG2'pCL, L-pG2'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
O
O
OH
123
45PP
Activation of the nucleotides?
Goal: Synthesis of a biopolymer as an enzyme and self replicator
Base + Sugar + H3PO4 = NucleotidenNucleotides = Functional RNA
Why RNA?
N
NN
N
NH2
O
OHOH
OPH
O-
O
AMP
N
NN
N
NH2
O
OHOH
OPN
O-
O
N
ImpA
Activation of Mononucleotides
NO
O
OHHO
P
O
-O
N
N
N N
H2N
H3C
where N= A, U, G, or C
MeadpN
NO
O
OHHO
P
O
-O
N
NImpN
+
Na+
41
Preparation of an Activated Nucleotide
Imidazole salt of5'-AMP
N
NN
N
NH2
O
OHOH
OPN
OH
O
NPh3P
(PyS)2 in DMF
N
NN
N
NH2
O
OHOH
OPHOOH
O N
HN
DMSO, DMF
Imidazole salt of5'-AMP
A Comparison of Oligomerization Between Activated Ribo- and deoxyribo-Nucleotides on Na+-Montmorillonite
43
where base = Adenine
BaseO
O
HHO
P
O
-O
N
N
ImpdA
Na+
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
OHHO
P
O
-O
NN
ImpA
Na+
Montmorillonite
pH 8, 25oC,3 days
(pA)2 - (pA)n
where base = Adenine
RNA Synthesis: Yes
LOWLOWHIGHHIGH
Poor
Excellent
Excellent
Poor
Excellent
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
