Prebiotic RNA Synthesis by Montmorillonite Catalysis: Significance of Mineral Salts Prakash C. Joshi New York Center for Astrobiology & Department of Chemistry and Chemical Biology Rensselaer 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 Altman Carl 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 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 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,

Model Reactions of Activated Nucleotides on Montmorillonite 6

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 Reactive surface:800m 2 /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) 4 O 10 (OH) 2.nH 2 O Site of Reaction: Clay Inter-layers

Analysis of RNA Oligomers by HPLC 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 NaClO 4 at pH 8. ImpA with 1M NaCl and montmorillonite

Determination of Chain-length of RNA Oligomers MALDI Mass Spectral Analysis Autoradiography Autoradiography n=17 n=33↓ 32 P-labelled Products Separated by PAGE→  Estimated maximum oligomer length around 55 nucleotides

A Comparison of Oligomerization Between Activated Ribo- and deoxyribo-Nucleotides on Na + -Montmorillonite 12

Chain length of Oligomers of 15 mM ImpA formed at varying pH of Volclay Volclay (pH) III*II**IIIIVVVIVIIVIIIIXX Control Origins of Life and Evol. Biosph, 36: 2006,

Catalytic action of Montmorillonite from different sources Montmorillonite III*II**IIIIVVVIVIIVIIIIX Belle Fourche (SD) Little Rock (Arkansas) Chambers (Arizona) Otay (California) Volclay (Am. Coll. Corp) Joshi et al., J. Am. Chem. Soc. 131, 2009,

Chain length → III IIIIVVVIVIIVIIIIXX Clay ↓ CyclicLinear C-bed Trace F-bed Trace A-bed Trace Catalytic Activity of Belle Fourche Clay Collected from Different Beds

Optimum Al 41 – 48% Optimum Mg 4.8 – 6.2% Optimum Fe 5 – 6% Al-Fe-Mg ternary diagram showing a tight cluster of catalytic montmorillonites

Influence of Salt Concentration in RNA Oligomer Chain Length Joshi & Aldersley (2013) J. Mol. Evolution: 76,

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

19 Examples of linear and cyclic dimer structures Unwanted abab 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: 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*ReagentChain length** HydrophilicLiCl 4 12 (NH 2 ) 2 C=NH12 HydrophobicNa 2 SO 4 12 LiCl12 NoneLiBr12 CsCl10 *Kool & Breslow (1988) J. Am. Chem. Soc., 110, **Determined by HPLC analysis Joshi & Aldersley (2013) J. Mol. Evolution: 76,

Role of Mineral Anions and Cations on Montmorillonite-Catalyzed RNA Synthesis SaltOligomer length*SaltOligomer length* LiCl12LiI9 NaCl11NaI9 KCl7KI5 LiClO 4 12NaCl11 NaClO 4 9NaBr10 KClO 4 6NaI9 Li + > Na + > K + Cl - > B - > I - *Determined by HPLC analysis Joshi & Aldersley (2013) J. Mol. Evolution: 76,

23 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?

Homochiral Selection Quaternary Reactions of D, L-ImpA and D, L-ImpU on Na + -Montmorillonite 24

Dimers formed by the Quaternary reaction of D, L-ImpA + D, L-ImpU on Montmorillonite (1) UppU (2) D, D & L, L-c-A 3’ pU 3’ p (3) Uridine (4) D, L & L, D-c-A 3’ pA 3’ p (5) 3’, 5’-c-UMP (6) D, D & L, L-c-A 3’ pA 3’ p (7) D, D & L, L-U 2’ pU (8) Adenosine (9) D, L & L, D-U 2’ pU (10) D, D & L, L-U 3’ pU & D, D & L, L-A 2’ pU (11) D, L & L, D-U 3’ pU (12) D, L & L, D-A 2’ pU (13) D, D & L, L-A 2’ pA (14) D, D & L, L-A 3’ pU (15) D, L & L, D-A 3’ pU (16) D, L & L, D-A 2’ pA (17) D, D & L, L-A 3’ pA (18) D, L & L, D-A 3’ pA Ion exchange HPLCReverse-phase HPLC Fraction collection

Homochiral Selection Homochirality of oligomers in a quaternary reaction of D, L-ImpA with D, L-ImpU on Na + -montmorillonite Homochiralit y MonomerDimerTrimerTetramerPentamer Observed50%64%76%93%97% Calculated50% 25%12.5%6.25% Ratio 1 : 11 : : : : Joshi, Aldersley & Ferris (2013) Advances in Space Research, 51, Joshi, Aldersley & Ferris (2011) Biochemical & Biophysical Res. Commun. 413, Joshi, Aldersley & Ferris (2011) Orig. Life Evol. Biosph., 41, Joshi, Pitsch & Ferris (2007) Orig. Life Evol. Biosph., 37: Joshi et al. (2011) Orig. Life Evol. Biosph., 41,

Constructing “RNA World” From Racemic Mixture of A, U, G and C on Na + -montmorillonite 27 2x4 n (n = chain length) Search for catalytic RNA

128 Combinatorial Chemistry D,L-ImpA + D,L-ImpU + D,L-ImpG + D,L-ImpC on Clay (128 Possible Dimers) D, D-pA 2' pA L, L-pA 2' pA D, D-pU 2' pU L, L-pU 2' pU D, D-pG 2' pG L, L-pG 2' pG D, D-pC 2' pC L, L-pC 2' pC D, D-pA 3' pA L, L-pA 3' pA D, D-pU 3' pU L, L-pU 3' pU D, D-pG 3' pG L, L-pG 3' pG D, D-pC 3' pC L, L-pC 3' pC D, L-pA 2' pA L, D-pA 2' pA D, L-pU 2' pU L, D-pU 2' pU D, L-pG 2' pG L, D-pG 2' pG D, L-pC 2' pC L, D-pC 2' pC D, L-pA 3' pA L, D-pA 3' pA D, L-pU 3' pU L, D-pU 3' pU D, L-pG 3' pG L, D-pG 3' pG D, L-pC 3' pC L, D-pC 3' pC D, D-pA 2' pU L, L-pA 2' pU D, D-pA 2' pG L, L-pA 2' pG D, D-pA 2' pC L, L-pA 2' pC D, D-pU 2' pG L, L-pU 2' pG D, D-pA 3' pU L, L-pA 3' pU D, D-pA 3' pG L, L-pA 3' pG D, D-pA 3' pC L, L-pA 3' pC D, D-pU 2' pG L, L-pU 2' pG D, L-pA 2' pU L, D-pA 2' pU D, L-pA 2' pG L, D-pA 2' pG D, L-pA 2' pC L, D-pA 2' pC D, D-pU 2' pG L, L-pU 2' pG D, L-pA 3' pU L, D-pA 3' pU D, L-pA 3' pG L, D-pA 3' pG D, L-pA 3' pC L, D-pA 3' pC D, D-pU 2' pG L, L-pU 2' pG D, D-pU 2' pA L, L-pU 2' pA D, D-pG 2' pA L, L-pG 2' pA D, D-pC 2' pA L, L-pC 2' pA D, D-pG 2' pU L, L-pG 2' pU D, D-pU 3' pA L, L-pU 3' pA D, D-pG 3' pA L, L-pG 3' pA D, D-pC 3' pA L, L-pC 3' pA D, D-pG 2' pU L, L-pG 2' pU D, L-pU 2' pA L, D-pU 2' pA D, L-pG 2' pA L, D-pG 2' pA D, L-pC 2' pA L, D-pC 2' pA D, D-pG 2' pU L, L-pG 2' pU D, L-pU 3' pA L, D-pU 3' pA D, L-pG 3' pA L, D-pG 3' pA D, L-pC 3' pA L, D-pC 3' pA D, D-pG 2' pU L, L-pG 2' pU D, D-pU 2' pC L, L-pU 2' pC D, D-pG 2' pC L, L-pG 2' pC D, D-pC 2' pU L, L-pC 2' pU D, D-pC 2' pG L, L-pC 2' pG D, D-pU 3' pC L, L-pU 3' pC D, D-pG 2' pC L, L-pG 2' pC D, D-pC 3' pU L, L-pC 3' pU D, D-pC 3' pG L, L-pC 3' pG D, L-pU 2' pC L, D-pU 2' pC D, D-pG 2' pC L, L-pG 2' pC D, L-pC 2' pU L, D-pC 2' pU D, L-pC 2' pG L, D-pC 2' pG D, L-pU 3' pC L, D-pU 3' pC D, D-pG 2' pC L, L-pG 2' pC D, L-pC 3' pU L, D-pC 3' pU D, L-pC 3' pG L, D-pC 3' 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 Cell Jack 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,

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 PP Activation of the nucleotides? Goal: Synthesis of a biopolymer as an enzyme and self replicator Base + Sugar + H 3 PO 4 = Nucleotide nNucleotides = Functional RNA Why RNA?

Activation of Mononucleotides 41

Preparation of an Activated Nucleotide

A Comparison of Oligomerization Between Activated Ribo- and deoxyribo-Nucleotides on Na + -Montmorillonite 43 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, ). 2’-OH: pK a = (Velikyan et al., J. Am. Chem. Soc. 2001, 123, ).

LOW HIGH Poor Excellent Poor Excellent

SampleIII IIIIVVVIVIIVIIIIXX #CyclicLinear C-bed Trace F-bed Trace A-bed Trace Catalytic Activity of Belle Fourche Clay Collected from Different Beds