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Alexander Chetverin Institute of Protein Research of the Russian Academy of Sciences

Molecular colonies: A plausible form of compartmentalization in the RNA world. Alexander Chetverin Institute of Protein Research of the Russian Academy of Sciences Pushchino, Moscow Region alexch@vega.protres.ru.

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Alexander Chetverin Institute of Protein Research of the Russian Academy of Sciences

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  1. Molecular colonies: A plausible form of compartmentalization in the RNA world Alexander Chetverin Institute of Protein Research of the Russian Academy of Sciences Pushchino, Moscow Region alexch@vega.protres.ru

  2. Oparin was the first who realized that no evolution in the prebiotic world was possible without a competition and hence compartmentalization, some form of segregation of biomolecules from the environment. His 'coagulates' (bits of a gel) and then 'coacervates' (colloid vesicles) were the first models of primitive cells. “The moment when the gel was precipitated or the first coagulum formed, marked an extremely important stage in the process of the spontaneous generation of life. At this moment… the transformation of organic compounds into an organic body took place. Not only this, but at the same time the body became an individual… and set itself apart from the environment surrounding it”. “Only the most complicated and efficient could grow and develop, all the rest either ceased to develop or perished”. Oparin AI (1924) Origins of Life. Moscow, Moskovhii Rabochii. English translation by Ann Synge.

  3. Compartmentalization in liposomes

  4. Growing and division (self-replication) ofliposomes can be coupled to RNA replication Szostak JW, Bartel DP, Luisi PL (2001) Nature 409, 387-390. Oberholzer T, Wick R, Luisi PL, Biebricher CK (1995) Biochem. Biophys. Res. Commun. 207, 250-257.

  5. However, replication of liposomes and self-replication of ribozymes are hardly compatible Many ribozymes have optimal activity in the presence of high concentrations of divalent metal ions. In such conditions, vesicles composed of acidic phospholipids would aggregate, possibly interfering with growth and division. Szostak JW, Bartel DP, Luisi PL (2001) Nature 409, 387-390.

  6. Lipid membrane is a strong barrier for nucleotide exchange with the surrounding solution ATP (±Mg2+), AMP, ADP AMP, dAMP 100 100 ADP + Mg2+ 75 75 ImpA (adenosine-5’- phosphorimidazolide) AMP + Mg2+ 50 50 2 C13COOH 1 C13COO-Glycerol 2 C13COOH 1 C13COO-Glycerol A 25 25 Remained in the liposomes, % 100 AMP, dAMP 100 75 75 ImpA 50 ImpA 3'-NH2-ImpA 50 4 C9COOH 1 C9COH 1 C9COO-Glycerol 25 2 C13COOH 1 C13COO-Glycerol 3'-NH2-ImpA 25 Time, hours Mansy SS, Schrum JP, Krishnamurthy M, Tobé S, Treco DA, Szostak JW(2008) Nature454,122-125.

  7. Nonenzymic poly(С)-directed synthesis of poly(G) inside and outside liposomes Time, hours 30-ntproduct 15-ntprimer Substrate (5 mM3'-NH2-ImpG) was added to the surrounding solution Outside Inside Mansy SS, Schrum JP, Krishnamurthy M, Tobé S, Treco DA, Szostak JW (2008) Nature454,122-125.

  8. Compartmentalization in molecular colonies

  9. Qβ-replicase amplifies RNAs exponentially Haruna I, Spiegelman S(1965) Science 150, 884–886. In only 10 min, it produces up to1010copies of a single RQ RNA molecule (a cognate Qβ replicase template)

  10. Qβ replicase synthesizes a variety of RQ RNAs (for Replicable by Qβ replicase) without the addition of a template (“spontaneously”) • Mixing of Qβ replicase with ATP, GTP, CTP and UTP. • Incubation for 1–3 hoursat 37°C. • Product analysis by gel electrophoresis.The gel is stained with ethidium bromide(stains polynucleotides, but not mononucleotides). 223 nt 120 nt Eigen: RQ RNAs are generated de novo (self-originate)? Sumper M, Luce R (1975) Proc. Natl. Acad. Sci. USA 72, 162–166. Biebricher CK, Eigen M, Luce R (1981) J. Mol. Biol. 148, 369–390. Biebricher CK, Eigen M, Luce R (1986) Nature 321, 89–91.

  11. Louis Pasteur, 1860:No life could arise in a boiled meat broth unless solid particles heavier than air were allowed to enter Pasteur’s “swan” flask

  12. Detection of airborne RQ RNAs • Qβ replicase- and NTP-containing agarose is poured into 2 dishes • The dishes are incubated for 1 hour: one dish is closed, the other is open • The agarose is stained with ethidium bromide Open dish Closed dish Chetverin A, Chetverina H, Munishkin A (1991) J. Mol. Biol. 222, 3-9

  13. Molecular colonies (nanocolonies, “polonies")provide for compartmentalization of biochemical reactions in the absence of membranes Molecular colonies form when template nanomolecules (DNA or RNA) are amplified in an immobilized medium whose polymer matrix possesses pores in the nanometer range Each colony is made up of clustered copies of one parental molecule (a molecular clone) Chetverin A, Chetverina H, Munishkin A (1991) J. Mol. Biol. 222, 3-9

  14. Selection of RNA molecules for the ability to replicate Membrane with NTPs NonreplicableRQ RNA fragments 5' fragment 3' fragment Reacting sequences Agarose with Qβ replicase Recombination One fragment Two fragments Replicating RNA Chetverin A, Chetverina H, Demidenko A, Ugarov V (1997) Cell 88, 503-513.

  15. Growing DNA colonies by carrying out Polymerase Chain Reaction (PCR) in a polyacrylamide gel Reaction well with a dry polyacrylamide gel Gel swelled under cover slip Gel secured the with adhesive foil 14 mm

  16. Gene amplification in molecular colonies 109 Obelin gene (756 bp) 108 107 108 GFP gene (1570 pb) 107 106 0 30 100 Number of DNA molecules in a spot Number of DNA molecules seeded • Nearlyallseeded molecules produce DNA colonies • Each colony contains up to 108copies of a gene Samatov TR, Chetverina HV, Chetverin AB (2005) Nucleic Acids Res. 33, e145.

  17. Gene expression in molecular colonies: Transcription Incubation (2 hours at 37°C) Drying the PCR gel Soaking in a transcription cocktail Signal intensity increases as a results of transcription After PCR After transcription Samatov TR, Chetverina HV, Chetverin AB (2005) Nucleic Acids Res. 33, e145.

  18. Protein synthesis was monitored by GFP (green fluorescent protein) fluorescence 30 Number of seeded DNA molecules 100 1 2 Reaction time, hours Gene expression in molecular colonies:Transcription + Translation Since genes are co-localized with their expression product, they can be selected according to properties of the synthesized proteins Samatov TR, Chetverina HV, Chetverin AB (2005) Nucleic Acids Res. 33, e145.

  19. Relevance to the problem of the origin of life Molecular colonies can perform all essential functions of a living cell, including replication of the genetic material and its expression (transcription and translation), and can undergo evolution. Unlike liposomes, compartmentalization is achieved because of a relatively low rate of diffusion of macromolecules in a porous matrix as compared with low-molecular substances. RNA colonies similar to those generated in Qβ replicase-containing agarose might have served as a pre-cellular form of compartmentalization in the RNA world. Instead of agarose, RNA colonies might be formed in a wet clay or another porous minerals existed on the Earth.

  20. Could life originate in a clay?

  21. 1940s: Clay minerals might play an important role in the early chemical processes leading up to the origin of life Minerals could concentrate and catalytically transform organic molecules toward biofunctionality and confer on them structural properties such as chirality. Goldschmidt VM (1947) Geochemical Aspects of the Origin of Complex Organic Molecules on the Earth, as Precursors to Organic Life. Published: New Biology 1952, 12, 97-105. Bernal JD (1949) The physical basis of life. Proc. Phys. Soc. 62, 537-558.

  22. Properties of layered clays Yeremin NI (2004) Non-metallic minerals, 2nd ed. Publishing House of Moscow University.

  23. Montmorillonite structure O Si H Al Layer spacing 1-2 nm; increases several fold upon hydration

  24. Montmorillonite is a safe haven for organic molecules Not only it prevents decomposition of methanol under “black smokers” conditions (300 °C, 100 MPa ), but also promotes synthesis of diverse organic compounds that may be precursors to biomolecules. Williams LB, Canfield B, Voglesonger KM, Holloway JR (2005) Geology 33, 913–916. Provides a 3-fold protection of the adsorbed RNA from the UV light (judging by retention of a ribozyme activity). Biondi E, Branciamore S, Maurel MC, Gallori E (2007) BMC Evol. Biol. 7, S2. Has been found in meteorites and on Mars. Poulet F et al. (2005) Nature 438, 623–627. Served as a “primordial womb” for the life?

  25. Montmorillonite can concentrate nucleotides from even very dilute solutions • Within 24 hoursat 25°C, рН 7, 50 mg (ca. 20 μL) of dispersed clay absorbs 10% (in the presence ofNa+) to90% (in the presence ofMg2+) of AMP dissolved at a 15 μM concentration in 1 L (a 50,000-fold greater volume) of water. • Hence, the concentration of the nucleotide in the clay becomes up to 50,000-fold higher, i.e., ca. 750 mM. Ferris JP, Ertem G, Agarwal VK (1989) Orig. Life Evol. Biosphere 19, 153−164.

  26. Na+ - double stranded RNA Ca2+ Mg2+ Montmorillonite strongly binds RNA or DNA, especially single stranded and in the presence of divalent cations Franchi M, Ferris P, Gallori E (2003) Orig. Life Evol. Biosph. 33, 1–16.

  27. 2 4 6 8 14 Montmorillonite catalyzes synthesis of >40 nt-long oligo(A) Reaction time, days (at25°C) • In the absence of montmorillonite, the primer is mostly elongated by 2 nucleotides (maximally 10) due to a rapid hydrolysis of the activated substrate. • In the presence of montmorillonite, condensation is 100-1000 times faster than hydrolysis, whereas only 10 times faster in its absence. Substrate: 15 mM adenosine-5’- phosphorimidazolide, added daily Primer:[32P]dA(pdA)8pA, adsorbed on montmorillonite Ferris JP, Hill AR Jr, Liu R, Orgel LE (1996) Nature 381, 59-61.

  28. With 1-methyl adenine as activating group, 40 nt-long oligo(A) is formed on montmorillonite within 8 hours, without any primer or additional substrate portions The correct 3'-5' inter-nucleotide linkages are preferentially formed 2 8 24 48 72 Huang W, Ferris JP (2006) J. Amer. Chem. Soc. 128, 8914-8919. Reaction time, hours

  29. Montmorillonite particles increase 100-fold the rate of liposome formation… + Montmorillonite Increase in optical density at 400 nm Time, min Hanczyc MM, Fujikawa SM, Szostak JW(2003) Science, 302, 618-622

  30. …and become incorporated inside the liposomes, along with the adsorbed RNA Fluorescently labeled RNA Hanczyc MM, Fujikawa SM, Szostak JW(2003) Science 302, 618-622.

  31. Thus, montmorillonite is able to: • concentrate from the surrounding solution and stabilize activated ribonucleotides; • catalyze polymerization of the ribonucleotides; • provide for a certain chiral homogeneity of the synthesized polyribonucleotides (RNA); • immobilize polyribonucleotides (RNA), both templates and their copies; • bind single-stranded RNA more strongly than double stranded one (like a single strand-binding protein), which stabilizes the state in which replication is possible; • compartmentalize RNA in the absence of lipid membranes (provide for the formation of RNA colonies); • keep different RNA species together, thereby providing for the creation of mixed colonies and for their joint inheritance (by virtue of being adsorbed on the same clay microparticle); • form liposomes around RNA colonies (a new role at a later stage of evolution).

  32. Hydrophobic surface Hydrophilic surface Lipid membranes originated subsequent to peptides? Antimicrobial peptides are short (20 to 40 amino acid residues) amphipathic α-helical peptides able to form pores across lipid bilayers

  33. Conclusions Molecular colonies could become an experimental model for de novo creation of living cells. They are functional equivalents of cells, provide for assembling cellular components and checking which variations of the assembly ensure the full expression of the genome: • provide for compartmentalization of biological macromolecules, • are able to perform all biochemical reactions that make up the gene expression, • allow reaction components to be added or removed by simply soaking the gel (due to the absence of a membrane), • link the genotype and phenotype as necessary for natural selection, • are capable of evolution, i.e., the formation of new genetic material which can then be expressed. Molecular colonies growing in clay might have been a form of compartmentalization at an early step of evolution in the RNA world and during development of the protein world.

  34. Principal collaborators: H.V. Chetverina T.R. Samatov Financial support: • Program “Molecular and Cell Biology” • Russian Foundation for Basic Research

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