Alexander ChetverinInstitute of Protein Research

of the Russian Academy of SciencesPushchino, Moscow Region

alexch@vega.protres.ru

MOLECULAR COLONIES:A plausible form

of compartmentalizationin the RNA world

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.

Oparin AI (1924) Origins of Life. Moscow, Moskovhii Rabochii. English translation by Ann Synge.

“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”.

Compartmentalization in liposomes

Szostak JW, Bartel DP, Luisi PL (2001) Nature 409, 387-390.

Growing and division (self-replication) of liposomes can be coupled to RNA replication

Oberholzer T, Wick R, Luisi PL, Biebricher CK (1995) Biochem. Biophys. Res. Commun. 207, 250-257.

However, replication of liposomes andself-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.

2 C13COOH1 C13COO-Glycerol

2 C13COOH1 C13COO-Glycerol

2 C13COOH1 C13COO-Glycerol

4 C9COOH1 C9COH1 C9COO-Glycerol

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100

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100

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10075

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Rem

aine

d in

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lipos

omes

, %

Time, hours

ATP (±Mg2+), AMP, ADP

ADP + Mg2+

AMP + Mg2+

A

ImpA (adenosine-5’-phosphorimidazolide)

AMP, dAMP

ImpA

3'-NH2-ImpA

AMP, dAMP

ImpA

3'-NH2-ImpA

Mansy SS, Schrum JP, Krishnamurthy M, Tobé S, Treco DA, Szostak JW (2008) Nature 454,122-125.

Lipid membrane is a strong barrier fornucleotide exchange with the surrounding solution

15-nt primer

30-nt product

Time, hours

Nonenzymic poly(С)-directed synthesis of poly(G) inside and outside liposomes

Mansy SS, Schrum JP, Krishnamurthy M, Tobé S, Treco DA, Szostak JW (2008) Nature 454,122-125.

Inside Outside

Substrate (5 mM 3'-NH2-ImpG) was added to the surrounding solution

Compartmentalization in molecular colonies

Qβ-replicase amplifies RNAs exponentially

In only 10 min, it produces up to 1010 copies of a single RQ RNA molecule(a cognate Qβ replicase template)

Haruna I, Spiegelman S (1965) Science 150, 884–886.

Qβ replicase synthesizes a variety of RQ RNAs(for Replicable by Qβ replicase) without the addition

of a template (“spontaneously”)

Eigen: RQ RNAs are generated de novo (self-originate)?

• Mixing of Qβ replicase with ATP, GTP, CTP and UTP.• Incubation for 1–3 hours at 37°C.• Product analysis by gel electrophoresis.

The gel is stained with ethidium bromide(stains polynucleotides, but not mononucleotides).

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.

223 nt

120 nt

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

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

Chetverin A, Chetverina H, Munishkin A (1991) J. Mol. Biol. 222, 3-9

Closed dish Open dish

Molecular colonies (nanocolonies, “polonies") provide for compartmentalization of biochemical reactions in

the absence of membranes

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

Molecular colonies form when template nanomolecules (DNA or RNA) are amplified in an immobilized medium whose polymer matrix possesses pores in the nanometer range

Selection of RNA molecules for the ability to replicate

Nonreplicable RQ RNA fragments

5' fragment3' fragment

Replicating RNA

Recombination

Membrane with NTPs

Agarose with Qβ replicase

Onefragment

Two fragments

Chetverin A, Chetverina H, Demidenko A, Ugarov V (1997) Cell 88, 503-513.

Reactingsequences

14 mm

Reaction well with a dry polyacrylamide gel

Gel swelled under cover slip

Gel secured the with adhesive foil

Growing DNA colonies by carrying outPolymerase Chain Reaction (PCR) in a

polyacrylamide gel

Obelin gene(756 bp)

GFP gene(1570 pb)

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106

108

107

0 30 100

Number of DNA molecules seededNumber of DNA

molecules in a spot

• Nearly all seeded molecules produce DNA colonies• Each colony contains up to 108 copies of a gene

Gene amplification in molecular colonies

Samatov TR, Chetverina HV, Chetverin AB (2005) Nucleic Acids Res. 33, e145.

Drying the PCR gelIncubation

(2 hours at 37°C)Soaking in a transcription cocktail

After PCR

Gene expression in molecular colonies: Transcription

After transcription

Samatov TR, Chetverina HV, Chetverin AB (2005) Nucleic Acids Res. 33, e145.

Signal intensity increases as a results of transcription

Protein synthesis was monitored byGFP (green fluorescent protein) fluorescence

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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.

Gene expression in molecular colonies:Transcription + Translation

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.

Relevance to the problem of the origin of life

Could life originate in a clay?

1940s: Clay minerals might play an important rolein 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.

Clay mineral Repeating layers Density, g/cm3

Specific surface area, m2/g

Ion exchange capacity, meq/g

KaoliniteKaolinite type:1 tetrahedral1 octahedral

2.60-2.68 8-20 0.03-0.15

MontmorilloniteSmectite type:2 tetrahedral1 octahedral

2.35-2.70 600-800 0.8-1.5

Properties of layered clays

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

Montmorillonite structure

OSi

HAl

Layer spacing 1-2 nm;increases several fold upon hydration

Williams LB, Canfield B, Voglesonger KM, Holloway JR (2005) Geology 33, 913–916.

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.

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.

Montmorillonite is a safe haven for organic molecules

Served as a “primordial womb” for the life?

Has been found in meteorites and on Mars.

Poulet F et al. (2005) Nature 438, 623–627.

Ferris JP, Ertem G, Agarwal VK (1989) Orig. Life Evol. Biosphere 19, 153−164.

Montmorillonite can concentrate nucleotidesfrom even very dilute solutions

• Within 24 hours at 25°C, рН 7, 50 mg (ca. 20 μL) of dispersed clay absorbs 10% (in the presence of Na+) to 90% (in the presence of Mg2+) 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.

Na+

Ca2+

Mg2+

Franchi M, Ferris P, Gallori E (2003) Orig. Life Evol. Biosph. 33, 1–16.

- double stranded RNA

Montmorillonite strongly bindsRNA or DNA,

especiallysingle stranded

and in the presence of

divalent cations

Ferris JP, Hill AR Jr, Liu R, Orgel LE (1996) Nature 381, 59-61.

Montmorillonite catalyzes synthesis of >40 nt-long oligo(A)

Substrate: 15 mM adenosine-5’-phosphorimidazolide, added daily

Primer: [32P]dA(pdA)8pA, adsorbed on montmorillonite

• 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.

2 4 6 8 14Reaction time, days (at25°C)

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

Reaction time, hours Huang W, Ferris JP (2006) J. Amer. Chem. Soc. 128, 8914-8919.

Incr

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00 n

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Time, min

Montmorillonite particles increase 100-foldthe rate of liposome formation…

Hanczyc MM, Fujikawa SM, Szostak JW (2003) Science, 302, 618-622

+ Montmorillonite

…and become incorporated inside the liposomes, along with the adsorbed RNA

Hanczyc MM, Fujikawa SM, Szostak JW (2003) Science 302, 618-622.

Fluorescently labeled RNA

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).

Hyd

roph

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face

Hyd

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surf

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Antimicrobial peptides are short (20 to 40 amino acid residues)amphipathic α-helical peptides able to form pores across lipid bilayers

Lipid membranes originated subsequent to peptides?

ConclusionsMolecular 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.

Financial support:

•Program “Molecular and Cell Biology”

•Russian Foundation for Basic Research

Principal collaborators:

H.V. ChetverinaT.R. Samatov