Prebiotic Synthesis of Biomolecules Prakash C. Joshi (Ferris Group) New York Center for Astrobiology & Department of Chemistry and Chemical Biology Rensselaer Polytechnic Institute Troy, NY 12180, USA 1

Some milestones in prebiotic chemistry The Miller/Urey experiment AtmosphereComposition ReducingCH 4, NH 3, H 2 O, H 2 CO 2, N 2, H 2 O, H 2 CO 2, H 2, H 2 O NeutralCO 2, N 2, H 2 O OxidizingCO 2, N 2, H 2 O, O 2 Simulation of a primitive Earth environment CH 4 + NH 3 → HCN + 3H 2 CO + NH 3 → HCN + H 2 O

Biomolecules from HCN Background: ► John Oro 1,2 reported formation of amino acids and adenine by heating HCN (15 M) in strong ammonia. 5HCN → Adenine (DNA & RNA base) ► Clifford Matthews 3 suggested peptide synthesis from HCN in water. 1.Arch. Biochem. Biophys. 96, 1962, Arch. Biochem. Biophys. 94, 1961, Nature 215, 1967, Adenine

Biomolecules from HCN Our Approach: ►0.1 M HCN, pH 8, room temperature ►Three major class of biomolecules (amino acids, purine and pyrimidine) were isolated and characterized. Journal of Molecular Evolution 17, 1981, Polymer Preprints, American Chemical Society 20, 1979, Journal of Molecular Evolution 11, 1978, Origin of Life, Ed. H. Noda, Japan Sci. Soc. Press, 1978, pp Biosystems 9, 1977,

Prebiotic Peptides from HCN ? Result No peptide linkage were detected in HCN Oligomer by NMR, pyrolysis, biuret test and other chemical tests for CO-NH linkage. Ferris, Joshi et al., Structural studies on HCN oligomer, J. Mol. Evol. 17, 1981, Ferris, Science, 203, 1979,

Formation of Purine and Pyrimidine's From HCN

Photochemical Decarboxylation of Orotic Acid, Orotidine and Orotidine 5'-Phosphate Ferris & Joshi, Journal of Organic Chemistry 44, 1979, Ferris and Joshi, Science 201, 1978,

Prebiotic Synthesis of RNA Objective Synthesis of RNA from mononucleotides by clay mineral catalysis. Investigation of homochiral selection in clay mineral catalyzed RNA synthesis. The long range goal is to test for their catalytic activity.

“RNA World” 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*. Most theories of the origin of biological organization have suggested that for RNA, chain length in the range of monomers are needed to make a genetic system viable **. *Gilbert, W. Nature, 1986, 319, 618; Altman, C. et al., Cell, 1983, 35, 849; Cech, T.T. et al., Cell, 1981, 27, 487; Orgel, L, Science, 2000, 290, **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.

Model Reactions of Activated Nucleotides on Montmorillonite 11

Determination of Excellent, Good and Poor Catalysts by HPLC HPLC traces obtained from three representative types of montmorillonite. Volclay (Excellent), Little Rock (Good) and Chambers (Poor) Joshi et al. JACS,131, 2009,

Oligomers from D-ImpA and D-ImpU: Analytical tools Oligomers OD 260 time High Performance Liquid Chromatography (HPLC) n=17 n=33↓ Mass Spectral Analysis 32 P-labelled Products of polyU from MeadpU Separated by PAGE  Estimated maximum oligomer length around 55 nucleotides Gel Electrophoresis

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,

A Comparison of Oligomerization Between Activated Ribo- and deoxyribo-Nucleotides on Na + -Montmorillonite 15

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

17 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 18

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 (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,

Summary Montmorillonite clay minerals are not only an excellent catalyst for the synthesis of RNA but they also facilitate chiral and sequence selection.

Octonary Reaction D, L-ImpA, D, L-ImpU, D, L-ImpG and D, L- ImpC on Na + -Montmorillonite 22

128 Future Research 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

Small trans-aminoacylating RNA complexes* Small transamino- acylating RNA complexes. A. C3 RNA B. Intermediate trans complexes. C. Final GUGGC/GCCU complex. *Turk R M et al. PNAS, 2010, 107, (Laboratory of Michael Yarus)

Base pair recognition by a given intercalator is a necessary, but not sufficient, condition for achieving intercalation-mediated ligation. Horowitz E D et al. PNAS 2010;107: ©2010 by National Academy of Sciences

Model Protocell Capable of Copying DNA Jack W. Szostak The simple protocells consist of only genetic material surrounded by fatty acid membrane. This form of a cell is thought to have been able to grow, repilcate and evolve.