Template-directed synthesis of a genetic polymer in a model protocell

Abstract

Contemporary phospholipid-based cell membranes are formidable barriers to the uptake of polar and charged molecules ranging from metal ions to complex nutrients. Modern cells therefore require sophisticated protein channels and pumps to mediate the exchange of molecules with their environment. The strong barrier function of membranes has made it difficult to understand the origin of cellular life and has been thought to preclude a heterotrophic lifestyle for primitive cells. Although nucleotides can cross dimyristoyl phosphatidylcholine membranes through defects formed at the gel-to-liquid transition temperature, phospholipid membranes lack the dynamic properties required for membrane growth. Fatty acids and their corresponding alcohols and glycerol monoesters are attractive candidates for the components of protocell membranes because they are simple amphiphiles that form bilayer membrane vesicles that retain encapsulated oligonucleotides and are capable of growth and division. Here we show that such membranes allow the passage of charged molecules such as nucleotides, so that activated nucleotides added to the outside of a model protocell spontaneously cross the membrane and take part in efficient template copying in the protocell interior. The permeability properties of prebiotically plausible membranes suggest that primitive protocells could have acquired complex nutrients from their environment in the absence of any macromolecular transport machinery; that is, they could have been obligate heterotrophs.

Fig. 1

Conceptual model of a heterotrophic protocell. Growth of the protocell membrane results from the incorporation of environmentally supplied amphiphiles, while division may be driven by intrinsic or extrinsic physical forces. Externally supplied activated nucleotides permeate across the protocell membrane and act as substrates for the non-enzymatic copying of internal templates. Complete template replication followed by random segregation of the replicated genetic material leads to the formation of daughter protocells.

Fig. 2

Ribose permeability of fatty acid based membranes. Influence of (A) head group charge, (B) head group size, (C) membrane fluidity. (D) Comparison of decanoic acid based membranes with myristoleic acid based membranes. All binary lipid mixtures were 2:1 molar ratios of fatty acid:additive; a 4:1:1 ratio of DA:DOH:GMD was used. Ribose permeabilities are relative to that of MA membranes. MA, myristoleic acid; MA-OH, myristoleoyl alcohol; MP, myristoleoyl phosphate; GMM, glycerol monoester of myristoleate; PA, palmitoleic acid; GMPA, glycerol monoester of palmitoleate; OA, oleate; GMO, glycerol monoester of oleate; Sorb, sorbitan monooleate; LA, lauric acid; DA, decanoic acid; DOH, decanol; GMD, glycerol monoester of decanoic acid.

Fig. 3

Time courses of nucleotide permeation through fatty acid based membranes. (A) Nucleotide permeation across MA:GMM membranes. ■, AMP; □, AMP + 3 mM MgCl₂; •, ADP; ○, ADP + 3 mM MgCl₂, , ATP; △, ATP + 3 mM MgCl₂. (B) Permeation of AMP derivatives across MA:GMM membranes. •, adenosine-5′-monophosphate; ○, 2′-deoxyadenosine-5′-monophosphate; □, 2′-amino-2′,3′-dideoxyadenosine-5′-monophosphate; ◆, 2′-deoxyadenosine-5′-phosphorimidazolide. (C) Permeability of activated nucleotides across MA:GMM membranes. ■, adenosine-5′-phosphorimidazolide; •, 2′-amino-2′,3′-dideoxyadenosine-5′-phosphorimidazolide; ○, 3′-amino-3′-deoxyadenosine-5′-phosphorimidazolide. (D) Nucleotide permeation across DA:DOH:GMD and DA:DOH membranes. ■, AMP; □, dAMP; X, 2′-amino-2′,3′-dideoxyadenosine-5′-monophosphate; ◆ & , adenosine-5′-phosphorimidazolide; •, 2′-deoxyadenosine-5′-phosphorimidazolide; ○, 2′-amino-2′,3′-dideoxyadenosine-5′-phosphorimidazolide. All are for 4:1:1 DA:DOH:GMD membranes except for , which is 2:1 DA:DOH.

Fig. 4

Template-copying chemistry inside vesicles. Vesicles contained encapsulated primer-template complexes, and template-copying was initiated by the addition of activated monomer to the external solution. (A) Nonenzymatic dC₁₅-template copying in solution (lanes 1−6) and inside 2:1 MA: GMM vesicles (lanes 8−13) at 4 °C. (B) Template copying reaction in 4:1:1 DA:DOH:GMD vesicles at 25 °C. (C) Template copying reaction in 2:1 MA:farnesol vesicles at 4 °C. (D) Template copying reaction in POPC vesicles at 4 °C. (A-D) Arrow denotes full-length product. See methods for reaction conditions.