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Szostak Reviewed work(s): Source: Proceedings of the National Academy of Sciences of the United States of America, Vol. 105, No. 36 (Sep. 9, 2008), pp. 13351-13355 Published by: National Academy of Sciences Stable URL: http://www.jstor.org/stable/25464047 . Accessed: 16/03/2012 07:25
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but thispossibility has not been explored because of the assumption that such conditions would disrupt vesicle integrity and lead to the release of contents.pnas.S. Results Retention of DNA Oligonucleotides at High Temperatures. 1). examined fourpure fatty acids at pH 8. have facilitated membrane is consistent acids nucleic that suggesting nutrient uptake The transporters. The authors declare no conflictof interest. La Jolla. implying that strand separation could been mediated have fluctuations protocells by thermal without the loss of genetic material temperatures. Joyce. because this composi tion leads to vesicles that tolerate a wide range of salt and pH conditions. high temperatures to help.105 | no. including of thermal instability is acids aremuch higher than forphospholipids (4).org/cgi/doi/10. For example.harvard.0805086105 | September 9. higher temperatures (^10-20% released after 1 h at 60-80?C). Den ver. A second question is whether problematic in view of the presumed thermal DNA would seem likely oligonucleotides (3). Again. if a genetic polymer is copied inside membrane vesicles. and dilution of below this concentration such as leads to vesicle are dissolution.S. amphiphiles fatty alcohols. 8).S. longer high-temperature incubation times did result in significant leakage (^20% re leased after 10 h at 100?C).mgh. o and fatty-acid glycerol esters fatty alcohols (7). has seemed there might be environmental conditions that could more facilitate nucleotide efficient uptake.which is approximately the pH at which half of the fatty acid is ionized and half is protonated. such as At elevated nucleotides very rapidly. phy. these environmental sensitivities do However. The fraction of the oligonucleotide thatwas released from the vesicles during the high-temperature incubation was determined by a second round of size-exclusion chromatogra oligodeoxynucleotide (dAi0). membrane polymer encapsulated show that vesicles of simple Here.M.M. and certain vesicles fatty-acid-based PNAS narrow posed solely of fattyacids are only stable within a fairly pH range (4). 6). how could the strands of the double-stranded product be separated? The possibility of thermal strand separation. IA).2008 | vol. and approved are July8. as we show here. fTo ? 2008 byThe National Academy of Sciences of the USA effect of water the hydrophobic the through acyl chains u 8 x Q.CO 80208. whom correpondence should be addressed.with no detectable release of the encapsulated oligo nucleotide (Fig. These observations provide strong phosphorimidazolides support for theplausibility of the heterotrophic protocell model but immediatelybring to mind several additional questions. including the presence of up to 4 mM Mg2+ (5.M. polyphosphates.edu. CA.We tocell membranes reasonably are composed permeable of fatty acids to nucleoside and protocell that is useful for assessing the interactions related and found that pro molecules place and that efficient template directed copying reactions can take in the vesicle interior after the addition of external activated nucleotides (1). to the environment.W. 6). the critical aggregate concentrations for fatty are buried in themembrane (9).T) vesicles were completely stable to 90?C.2:1 MA/GMM vesicles remained completely stable to 100?C. To test whether this temperature stability is a general feature of vesicles composed of single-chain amphiphiles or is a unique characteristic of 2:1 MA/GMM vesicles.We also examined linoleic acid (CI8:2) vesicles. with linoleic acid vesicles being completely stable to 90?C but Author contributions:S. We ini range by early to re at high temperature while separated by denaturation being in primitive tained within vesicles. We testedvesicle stability encapsulating a fluorescein-labeled by 10-mer DNA with the spon by the alternation with low temperature strand temperature. as in PCR. vesicles com Mg2+ and Ca2+ very destabi www.5. Surprisingly. and J. Mansy* of model of Molecular W. The at temperatures oligonucleotides ranging strands of encapsulated double-stranded from 0?C DNA can be opens up ways inwhich thermal fluctuations could be used to advantage by primitive cells.designed research. However. E-mail:szostak@molbio. This C18:2 fattyacid behaved similarly to theC18:l oleic acid. S. and which corresponds to the pH of maximum hydrogen bond donor to an adjacent ionized carboxylate (4. fatty-acid thermostable and retain internal RNA glycerol esters are extremely and DNA 100?C. of University Denver. divalent cations out cles.Thermostability Sheref S. and then incu bating aliquots of the purified vesicles at differenttemperatures for 1 h. with increasing amounts released at > CC I UJ X u based on the instability of fatty-acid vesicles under several environmental conditions that do not affectphospholipid vesi vesicles Also. 36 | 13351-13355 . Szostak+ and Jack protocell membranes and Integrative Biology.M. 3). Palmitoleic acid (CI6:1) and oleic acid (C18.S. Furthermore. 2008) thermostable. S. complex charged cross fatty-acid-based membranes excursions may high temperature before the evolution of advanced of these membranes thermostability taneous replication of encapsulated of template-copying chemistry at separation and nutrient uptake molecules from the protocell.1073/pnas. This remarkably of environments that could be observation tolerated extends cells the and of a self-replicating earliest cells may have consisted genetic a self-replicating within vesicle. The assumption genetic materials. because the salts of fattyacids tend of solution (5. removing unencapsulated at high origin of life | RNA world | synthetic biology We have recentlydescribed a laboratorymodel of a simple compatibilityof the protocell components. 2008 (received for review May 26.W. 1) or change in vesicle size (measured by dynamic light scattering). although stability to high pH can be conferred by admixture with to crystallize lizing to fatty-acidvesicles. Massachusetts General Howard Hughes Medical Institute.W. but did show 30-35% leakage of entrapped DNA after 1 h at 100?C (Fig.Most important. performed research. and J. allowing replication or perhaps the utilization of more highly charged such substrates as nonactivated or short nucleoside nucleotides. Freelyavailable online through the PNAS open access option. wrote the article. MA 02114 Edited by Gerald F. we examined vesicles made with a variety of amphiphiles and amphiphilemixtures by We using the same oligodeoxynucleotide retentionassay (Fig.S.S. Thus. tially examined the thermal stability of 2:1 myristoleic acid (MA)/monomyristolein (GMM) vesicles. this lipid leads tomore fluidmembranes with increased permeability to nucleotides at low temperatures (1). | vesicle | prebiotic by size-exclusion chromatography (SEC). Pure myristoleic acid (CI4:1) vesicles released small amounts of after stability because every protonated carboxylate can act as a of instability fatty-acid-basedvesicles (2. entropic consistent with membrane release as stabilization 1 h at 50?C. The Scripps Research The Biology and Center for Computational Institute. we composed single-chain and such as fatty acids.with free and encapsulated DNA measured by f luorimetry. increasing chain length leads to increased thermal stabilityof the bilayer membrane.S. and S. not extend to temperature.analyzed data. Boston. Department Hospital. *Presentaddress: Department of Chemistry and Biochemistry. and J.
.-. decanoic amphiphiles. presumably of vesicles by weakening composed acyl chain we subjected MA/GMM (2:1) vesicles to 20 cycles of 2 min at 20?C and 2 min at 90?C. ID) because thesemolecules the stability of short-chain vesicles [decanoic acid (DA)/decanol (DOH)/glycerol monoester of decanoic acid (GMD)::4. . IE).T:1] to a similar thermocycling of 30 s at 25?C and 30 s at 90?C).> 20 40 I ~- iooi . After then previously reported (4) are only stable at >50 mM total amphi these vesicles were phile concentration. increasing chain length from 14 to 16 to 18 carbons). of the glycerol im acid As vesicles. These experiments show that amphi phile mixtures of the kind thatmay have been present on the early least earth could at high temperature. We used an oligonucleotide of these related compounds (Fig. 60-70?C entrapped alcohol (MA-OH). .. Vesicles of 2:1 capric 13352 | www. 2:1 MA/farnesol. but were not stable enough to survive filtration crystallized monocaprin. . O. acid) vesicles. we proved. we average group and acid with decanol capric these in exhibited significantly periods of high temperatures overnight at room temperature.2:1 MA/GMM. Identical resultswere MA.-. . As noted above. to measure unable to their ability temperatures. .0% of contents corresponding per cycle. the highly branched isoprenoid alcohol farnesol de creased We vesicle interactions (Fig. of fatty acids (13). MA/GMM during prolonged at (2:1) vesicles presumably ionized car oligonucleotide could be acid/monocaprin of retaining gel an thermostable. adding GMM orM-OH to MA) or acyl chain interactions (e.2:1 MA/MA-OH. 4:1:1 decanoic acid/decanol/monocaprin. could form we acids in a typical examined mixtures (11.-Ai-f60 80 3.-. ID). Thermostability of model protocell membranes.V ? x5 \ *k ^ ' " \\ \ -V-L - ^ E ~ 5ci-'- - ?r I --^^ ? ^"^-^^ \\ 40- 1 4-?" CN. 2- ? - ? g ? 20OH-. per molecule. to purify by we were elevated (C10) saturated amphiphiles (Fig.5. The leakage of a fluorescein-labeled was monitored as a function of time. aldehydes.oleic acid.-. myristoleyl reflect the large ratio of head group size to acyl chain length for To decrease the of ternary mixtures head size.2:1 decanoic acid/decanol.g. pH 8. at than longer-chain unsaturated plausible prebiotically as Pure capric acid (C10:0. corresponding glycerol cases the addition Thus.-. regime (20 cycles 10-20% of the labeled DNA contents did leak out over 20 cycles. Therefore. and no leakage of encapsulated was observed (Fig. but less effectively than creased thermostabilityand retained oligonucleotides for 1 h at and could tolerate shorter Interestingly. In contrast. We are then examined stable release of encapsulated DNA the effect on vesicle incubation can interact stability 100?C. chromatography This gradually may instability GMM. (A) Influence ofthe acyl chain on vesicle stability. . at 50-60?C. The to multiple of vesicles is an important stability thermocycles characteristic because it demonstrates that vesicles of composed no 20 cycles. and glycerol scenario lagoon" "drying of capric acid with some by modified Fischer-Tropsch are envi of surviving simple single-chain amphiphiles capable ronments such as hydrothermal vents and hot that are springs for sites of early evolution candidates (14). synthesis and too unstable dAi0 and retain therefore./\ 100-n--a-a?4L-* - - -* - t5 100-ji-- - - -i - -*-? - > 1w- 60- ?405 0 \ vd 2 \ \ 2020 \ V \ \ V \ - \ \ \ \ " 100 2 \\ 40" c \ 5 20\ > 60i80~ \ " " - \ " ^\\ \ 40 60 (?C) 80 100 V- OH-1-.?i?j? 0 20 temperature c - 5t?'-'-'-"r D I4 ^^\ 1 \v N. . exhibiting ^20% 100?C. periods We wondered observed whether temperature or whether to multiple thermocycles rapid and for brief temperature result in vesicle disruption. with their expected. be enhanced either by strengthening head group interactions (e. after 1 h at of a series of were acid/decanol leak encapsulated capric were capable somewhat more additives to pure MA. observed but Vesicles started to and as of 2:1 by microscopy oligonucleotide. examined are more stability. also evidenced by dialysis and microscopy.-. esters 12).0-)-1-1-1 100 0 time (h) temperature (?C) time (min) from vesicles of the indicated composition dA10 oligonucleotide Fig. .2 M sodium bicine. We observed. loss examined the response of nucleic acid was of prebiotic-model Type (FTT) chemistry is likely to yield a mixture of hydrocar bons. A. However. DNA in both monoester can lipid conferred stabilityat 100?C. 1. IA). I" 0 25 50 75 100 5 35_ ^^_ 10 ^^"\^ 20 30 0-|-. vesicle thermostability up to 100?C for 1min with no detectable loss of encapsulated oligonucleotide (Fig.0805086105 Mansy and Szostak FRET pair strategy to demonstrate thatvesicle thermostability .5-1. To encapsulate nucleic acids and retain these at stability extends in large changes answer this question. size-exclusion chromatography (10). alcohols.g. In this case. linoleic acid. IB).1073/pnas.-r?^?^=J???-L 40 60 80 temperature (?C) OH-. composed To determine whether the thermostability of vesicles of could be fatty acids longer-chain similarly of palmitoleic examined mixtures acid and oleic monoester derivatives. (B) Influence of the head group on C14:1 vesicle stability. -l? -x \N .pnas. palmitoleic acid. .. (D) Influence ofthe head group on C10:0 vesicle stability. . #. (E) Time-dependent leakage from 4:1:1 decanoic acid/decanol/monocaprin model prebiotic vesicles at 100?C Solution conditions were 0. 2:1 decanoic acid/decanol. explored monocaprin. to a loss of 0.org/cgi/doi/10. DNA Strand Separation within Vesicles. MA. and because of the ability of the glycerol headgroup to provide two hydrogen boxylates bond donors that with the of MA. which can provide one hydrogen bond donor stabilizes MA Consistent with this argument. and 2:1 oleic acid/monoolein as for 2:1 MA/GMM (C) Time-dependent given for 2:1 palmitoleic acid/monopalmitolein leakage from 2:1 MA/GMM vesicles at 100?C. and we quantified the loss of entrapped oligonucleotide.
When are stable to elevated realized that these vesicles tempera would be whether nucleotide tures. -Sk^-O?o X ^ O 60"U 2^ 20. incubating (1). ADP + MgCI2. 2:1 MA/GMM. that activated nucleoside whether high temperatureswould also increase thepermeability of unactivated are much less bears that permeable two negative monophosphates.as seen fordimyristoyl phosphatidylcholine membranes (15). toboth 2:1MA/GMM and 4:1:1 capric acid/decanol/monocaprin vesicles with a 1-min incubation at 90?C. ADP permeated more rapidly monophosphates crossed the membrane over a 24-h period > DC t s x u were lowed by SEC able to resolve retained and leaked nucleotides.NAD. <10% of nucle monophosphates dramatically increased. Permeability can nucleotides diffuse ously shown that activated spontaneously so that nucleo across the fatty-acid-based bilayer membranes. Nucleotide permeation appears to operate via a MgCI2. A. . AAAA. temperatures. In the absence ofMg2+. we were unable compositions to successfully are clearly reconsti phosphate observed nucleoside the permeability characteristics compatible of encapsulated nucleotides from 100-nmMA/GMM vesicles AMP. followed by cooling to 20?C for reannealing. V. (B) Influence of 5' phosphates on 2:1 MA/GMM vesicle permeability at 90?C #. Complementary 3' strand separation and reannealing 19-mer oligodeoxynucleotides and mixed were annealed with an 5 0 were labeled with a fluorophore and a quencher at their5' that respectively. . AAA. <10 min forAMP). ADP crossed themembrane more slowly thanAMP (^20 min for complete equilibration of ADP vs. GMP + MgCI2. we wondered permeability at higher enhanced significantly temperatures. these are for the however.Un der the same solution conditions but at 23?C. 4:1:1 decanoic acid/decanol/monocaprin. at Elevated We have previ Temperatures. CMP + MgCI2. most likelydue to the strong inhibitionof DNA polymerases by even low concentrations of fatty acids (17). tutePCR within fatty-acidvesicles. A. in the presence of Mg2+. ATP + MgCI2. 2). ADP.We further probed the influence of charge bymeasuring the permeation ofADP and ATP (Fig. O. <0. S. both vesicle to the increased fluorescence signal that DNA and similar The reannealing. . D.\\ 20 30 40 50 \ \ > \ \ \ \ ^ \\ ? \i 60 70 80 90 100 strand melting complete of thermocycling within vesicles. and subsequent cooling allowed C^-k W 100_? O X. to control reactions and measured in solution as size-exclusion to thus yielding an increase in fluorescence. equilibrating across 2:1 our previous of the permeability was significantly to determine sought Unactivated of NMPs nucleotides.A0. By encapsulating at high temperatures. #. single such as oligonucleotides. 2. PCR within phospholipid vesicles has been previously documented. we rapid permeation of activated Because to observe membranes with uridine-5'-phosphorimidazolide MA/GMM within low-temperature nonactivated slower than data nucleotide demonstrated 30 s at 90?C. (A) Nucleotide mono Fig.UMP. than activated charges of the more NMPs instead polar because one. consistent with the higher affinityof ADP forMg2+ (16) and consistent with previously observed trends at 23?C (1).CMP. reaching completion within 10min for and deoxyadenosine-5'-monophosphate (dAMP) (Fig. O.not inside vesicles). oside (1). 3B). A.GMP. v/> > X Q. ends. UMP + MgCI2. the experimental setup. AMP + MgCI2. The control reaction was ofthe same composition but not subjected to a thermocycle. AMP. Simple exponential prebiotic model membranes more than previously robust clearly allowing appreciated. 3. thanAMP (complete equilibration in<3 min). solution reaction (that is. (A) Schematic representation of Fig. ATP. ATP permeation was much slower (>1 h for completion) and could not be fit to a curve. O. High-temperature phosphate permeability of 2:1 MA/GMM vesicles at 90?C T. AMP + . such as elevated that facilitate conditions. reannealing temperature (?C) nucleotide permeability. (0 Oligomer permeability of 2:1 MA/GMM vesicles after a 1-h incubation at each indicated temperature. T\\ '? ' _' -^= X ' ' I \ \\X within and can be exploited forDNA vesicles. . \\\ 5 10 i_i_i_i_i-1-1-1-1 ?^@>-?-@^-^^ \ 15 20 25 time (min) excess of unlabeled oligonucleotide duplex of exactly the same sequence (Fig.\\ 10 " 'I 40.105 | no. o 00 U| material uptake of critical nutrientswithout the loss of larger entrapped Although the vesicle stability and nucleotide permeability of membrane with PCR. UMP. After ensure that only encapsulated compositions is diagnostic fluorescence and consistent a result Nucleotide gave rise of strand separation increase with was chromatography was monitored. radiolabeled fol nucleotides. GMP. 36 | 13353 Mansy and Szostak . C. control. (B) Increase in fluorescence arising from intravesicular DNA strand melting and annealing. 3/4). White bars represent unlabeled strands of DNA. Heating above theTm of the dsDNA (75?C) leads to strand random reannealing of labeled and unlabeled oligonucletides.^ == B i A ? I-'-'-'-'-'-1 w '-'</ c Intravesicular DNA strand melting. can enter vesicles to the outside and take part in tides added on the inside of reactions template-directed primer-extension the vesicle we concerted flipping of an amphiphile-solute complex as opposed to packing defects arising fromgel to liquid phase transitions.2008 | vol. nonactivated with release the We monophosphates we nucleotides (1). B. III a b I s 0 12 3 time (min) 4 5 6 B ? ? I 4K W Tt\ X. Black bars represent DNA molecules either modified with a fluorophore (D) or quencher (Q). This assay was applied separation. AMP. D. CMP. but because of the impermeabilityof the PNAS | September 9.
with UniverSol Vesicle Stability and Oligonucleotide Permeability. 20). were ob Strand Melting Assay. All other chemicals were obtained from Sigma-Aldrich. dinucleoside diphosphates (NppN) required 4 min for 50% leakage from the vesicles. For of cycle. and the glycerol monoesters of fatty acids were obtained from Nu Chek Prep. Fluorescence was measured with a SpectraMAX Gemi niEM fluorescence plate reader (Molecular Devices). rather than a single lipid species. and which show increased permeability (1) to sugars and nucleotides. For the encapsulation of molecules.0805086105 Mansy and Szostak ." inwhich replication of the genetic material and growth the vesicle compartment interrupted separation at low to moderate tem proceed that lead interludes by high-temperature and the influx of additional nucleotides.5. that would cycle.Perhaps more interestingthan the simple ability bility thatprimitivecells could take advantage of thermal cycling to facilitate both strand separation of replicated genomic tem plates and the rapid uptake of nutrientsduring high-temperature excursions. it isplausible thatvesicles assembled fromsuch prebiotic might have permeability had greater ion tolerance. in environments tuations. collected with a Gilson FC 203B fraction collector. tetramer would be vesicles. 23). but can also reasonably to diurnal locations variations. where could be while enantiomers replication. we measured of Oligonucleotides. one could imagine a primitive "cell which the running buffer contained the same amphiphile composition as the vesicles at a concentration above their critical aggregate concentration. was synthesized with a 5' tetrachlorofluorescein and 5' modification. The 10-mer had a covalently attached 5'-fluorescein. 90?C for 1min. Such strand sepa scintillation fluid (MP biomedicals) on a Beckman Coulter LS 6500 multipurpose scintillation counter. the external to generate mixtures and erated complex mixtures of lipids.2 M sodium bicine. self-assembled large consideration protocell and/or frequent of appropriate suggest that even structures could survive temperature environments fluc for strong inhibitionof furtherpolymerization (21).1 mM) were encap sulated. CT). formation Permeability the nucleotides rather had than delivered to be encapsulated during vesicle across the membrane (18). Radioactive nucleotides (0. but the length of the leakage of genetic polymers fromprimitive cells unless the cell were composed possibility of attractive fairly long-chain for an environment fatty acids. would lead to significant membranes Another subject primitive cells to large thermal fluctuations is provided by the possibility of convection cells near or within surface hot cyclingofDNA molecules between moderate and high temper atures. The permeation of mononucleo is that mixtures more desirable pure A thirdsignificantand surprisingoutcome of our experiments of amphiphiles characteristics seem to form membranes have with than membranes studies that Previous composed shown that tolerant of the membrane side diphosphates and triphosphates. MA/GMM (2:1) samples were incubated at on 20?C for 2 min. All vesicle preparations were extruded 11 times through 100-nm filters with an Avanti miniextruder (Avanti Polar pore-size polycarbonate Lipids). amphiphiles were resuspended in the presence of the encapsulant. subsurface as well of early Thus. genetic polymers. except for the Oligonucleotide General Hospital dimer. solution. Our currentwork shows that such mixtures form membranes with with enhanced branes. as the leads to the example. sequences were poly(A). for replication. protocell replication schemes that invoke the sequential templated ligation of oligonucleotide substrates are only feasible for dinucleotides and from used trinucleotides. respectively. stere Discussion The high thermal stability of themodel protocell membranes we have studied has significantimplications for theorigin of that cellular life. Cell shear forces. In the absence of Mg2+. vents. On the early or hydrothermal springs cells have been shown. Samples were then loaded on a gel filtration column. fatty alcohols. and the vesicles were purified by gel filtration (Sepharose 4B). addition of fattyalcohols and fatty-acidglycerol esters to pure presence of divalent cations (5). For vesicles composed of mixtures of amphiphiles. the intracellular Alternatively.1073/pnas. and 20?C for 2 min before measurement SpectraMAX GeminiEM fluorescence plate reader (Molecular Devices) with excitation and emission at 518 and 539 nm. Because prebiotic compared chemical pure fatty-acid reactions undoubtedly dramatically mem gen fatty acids lead to membranes are more to the single. Isotopes were either 14Cor 3H and were obtained from Moravek Biochemicals and Radiochemicals. Materials and Methods Materials.pnas. our results very primitive. Vesicle Preparation. convection for the rapid an cells might fluid such convection earth. suggested that short oligonucleotides might cross the at elevated temperatures.org/cgi/doi/10. Nucleotide Permeability. Rayleigh-Bernard to allow in the laboratory. Thus. GGCCCTAGGCCGGGCGCAT-3' was synthesized with a 3' black hole quencher-1 (BHQ-1) modification. Fluorescently labeled oligodeoxynucleotides 5'-ATGCGCCCGGCCTAGGGCC-3' tained from Integrated DNA Technologies. pH 8. Therefore. Diurnal triggered assortment are fluctuations temperature frequently as a means scenarios of cyclic concentration prebiotic or as a means orative drying followed by redissolution. Nucleotide permeability measurements were made in0. At the most with basic level. subject adjacent a to reservoir of replicating containing protocells population sufficient for strand separa brief periods of high temperature. the oils were mixed before dispersion in aqueous the origin of the first cells need not be limited to sheltered surface and include locales. and were either synthesized at the Massachusetts DNA core facility or the Yale University Keck facility (New Haven. with 50% loss after 37 min. Fatty acids. the retentionof a series of oligomers within 100-nm MA/GMM (2:1) vesicles at 90?C. earth. Fatty-acid vesicles were prepared by oil dispersion in buffered solutions as previously described (22. temperature subject as locations near heat sources vents such as hydrothermal cells to tolerate temperature fluctuations is the possi near-surface and hot springs. Tetranucleotides were completely retained after 1 h at 90?C (Fig. lost to the environment rapidly a of substrates a are taken up that the substrates assuming environment. Vesicles were incubated at different temperatures in a Bio-Rad DNA Engine Peltier thermal cycler.but not a 10-mer oligonu cleotide. perhaps lead to random cells. thermostability. leading to amplification by PCR (19. 3C). thermostability. chemical transformations fluctuations even on within temperature could the early also evaporated drive periodic by evap of driving materials. The dimer was 3H-labeled NAD (Moravek Biochemicals and Radiochemicals). Separation of entrapped and unencapsu lated material was by gel filtrationwith Sepharose 4B resin (Sigma-Aldrich) in peratures. and analyzed by scintillation counting diurnal ration and nutrient uptake in vesicles. This behavior is in striking contrast to the situationwith neous set of initialnucleotides is generally regarded as essential ochemical purity is important for RNA presence of contaminating incorrect to the synthesis of useful oligonucleotides. The assay was as described for nucleotide permeability except that oligonucleotides were used inplace of mononucleotides. than more model homogeneous laboratory the availability of a relatively For homoge generation of substrate oligonucleotides of length>4 pool trapped molecules smaller than entrapped at 90?C. enhanced tion and nutrient by long and separated uptake random interludes at lower temperatures. Ves icle size was measured by dynamic light scattering with a PDDLS/CoolBatch 90T (Precision Detectors). 13354 | www. Trimer and tetramer oligo nucleotides were 5'-labeled by using 32P-y-ATP and T4 polynucleotide kinase (New England Biolabs). amphiphiles. would by environmental of genetic molecules into daughter invoked in example. to strand division.membrane. whereas trinucleotides (pNpNpN) leaked out more slowly.
vents and associated gradient 14. reactions. AW. S. Biochemistry17:3759-3768.M. Ritter Simoneit BR (1999) Lipid synthesis G. Fresta Mangone A.W.P. J. J constantsof 16. Chen IA. Orig Life Evol Biosph 31:103-118. (2008) Template-directed synthesis a genetic polymer ina model protocell. Nature 409:387-390.Orgel LE (1987)The case foran ancestral genetic system involving simpleanalogues of the nucleotides. S.Proc Natl Acad Sci USA 80:160-164. Schrum. Deamer DW. BurnsMA (2002) PCR ina Rayleigh-Bernardconvection cell.growth.Walde P. This work was supported of 1. membranes. Chen. 7. Biol 2:677-682. 22. Monnard PA. acids and alcohols: Conditions forstability Biochim BiophysActa 1559:1-9. M. etal. LuisiPL (1995) Polymerasechain reaction in liposomes. J Am Chem Soc 127:13213-13219. Dworkin JP(2005) Chemistry CurrChem 259:1-27. Tobe.single-chainamphiphiles. RushdiAl. tor ofthe Howard Hughes Medical Institute. of 13. BioTechniques 9:308-309. hydroxamicacid as ligands. Ricardo.AlbrizioM. Israelachvili.S. A. Chem 18. Salehi-Ashtiani Szostak JW(2005) RNA catalysis in K. Deamer DW (1978) Liposomes from ionic. Szostak JW.Science 302:618-622. 5. of fatty acid vesicles. and Surface Forces (Academic. Wick R. Hanczyc.Nature 454:122-125. O 2 Mansy and Szostak PNAS | September 9. Science 298:793. acid vesicles. Am Chem Soc 116:11649-11654. Ugaz VM. Edmonds P. B. was supported in part by the National Institutes of Health Award F32 GM07450601. Astrobiology 2:139-152.JChem Eng Data 45:70-74. F. P. Top and physicsof primitive 2. Hargreaves WR. Dufton. polymerase proteinencapsulated within phospholipid vesicles. Q. 17. (1991) Intermolecular 10. Chen IA. Swaminathan B (1990) Effect ionic of and nonionic detergents on the Taq polymerase. KanavariotiA. LibchaberA (2003) Exponential DNA replicationby laminar convection. PhysRev Lett 91:158103. and ATP) and salicyl binaryand ternarycomplexeswith ribonucleotides (AMP. Braun D.Weyant RS.ProcNatl Acad Sci USA 84:4398-4402. > CC I UJ X u (/) u wr! > X a.Bruckner. model protocellvesicles. Deamer DW (1994) Production of RNA by a Mol Evol 39:555-559.A. LuisiPL (1994) Autopoietic self-reproduction M. I. M. HoffmanSE (1985) Submarine hydrothermal environments as sites for the origin and evolution of life.Bell. JoyceGF. Breaker RR. and M. and division. Fujikawa SM. Iwasa. Deamer DW (2002) Influence ionic inorganic and polymerizationprocesses related to early formsof life: solutes on self-assembly for Implications a prebiotic aqueous medium. JN 9. Deamer DW. Joyce Schwartz GF.ACKNOWLEDGMENTS.105 j no.Miller SL. Simoneit BR (2001) Lipid formationby aqueous Fischer-Tropsch-type syn thesis over a temperature range of 100 to 400 degrees C. We thank J. F. 21.S. KrishnanM. Hanczyc MM. KhalilMM (2000) Complexation equilibria and determinationof stability ADP. 23. Szostak JW (2003) Experimental models of primitative cellular compartments:Encapsulation. ChakrabartiAC. 8. of 6. Seelig. Baross JA. 36 | 13355 . A. isan Investiga by NASA Exobiology Program Grant EXB02-0031-0018. Haines TH (1983)Anionic lipid headgroups as a proton-conductingpathway along the surfaceof membranes: A hypothesis. BartelDP. Goddard NL. ditions by Fischer-Tropsch-type 12. Oberholzer T. Apel CL.Orig LifeEvol Biosph 29:153-166. McCollom TM. J under hydrothermalcon 11. Sam for helpful discussions. 3. Apel CL. T. Mautner MN (2002) Self-assembledvesiclesof monocarboxylic and forthe encapsulation of biopolymers. London). 19. J. R. J.2008 | vol. Luisi PL (2001) Synthesizing life.Apel CL. Seebeck. 15.Biophys of Szostak JW(2004)A kineticstudy the growthof fatty J87:988-998. 4. Zhu. 20.Orig Life Evol Biosph 15:327-345.Deamer DW (2005) The formation glycerol monodecanoate bydeydration/ the condensation reaction: Increasing chemical complexity amphiphileson the early of earth. Orig LifeEvol Biosph 35:323-332. J. Mansy SS.
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