Origin of Informational Polymers

We have measured the stabilities of the bonds that are critical for determining the half-life of ribonucleotides and the β-glycosidic and 3′- and 5′-phosphoester bonds. Stabilities were measured under a wide range of temperatures and water/formamide ratios. The stability of phosphodiester bonds in oligoribonucleotides was determined in the same environments. The comparison of bond stabilities in the monomer versus the polymer forms of the ribo compounds revealed that physico-chemical conditions exist in which polymerization is thermodynamically favored. These conditions were compared with those determining a similar behavior for 2′-deoxyribonucleosides, deoxyribonucleotides, and deoxyribooligonucleotides and were shown to profoundly differ. The implications of these facts on the origin of informational polymers are discussed. We have measured the stabilities of the bonds that are critical for determining the half-life of ribonucleotides and the β-glycosidic and 3′- and 5′-phosphoester bonds. Stabilities were measured under a wide range of temperatures and water/formamide ratios. The stability of phosphodiester bonds in oligoribonucleotides was determined in the same environments. The comparison of bond stabilities in the monomer versus the polymer forms of the ribo compounds revealed that physico-chemical conditions exist in which polymerization is thermodynamically favored. These conditions were compared with those determining a similar behavior for 2′-deoxyribonucleosides, deoxyribonucleotides, and deoxyribooligonucleotides and were shown to profoundly differ. The implications of these facts on the origin of informational polymers are discussed. Minimal requirements for informational polymers to have originated under prebiotic conditions are a unitary chemical frame for the synthesis of nucleobases and a favorable thermodynamic energy balance for the polymerization of nucleotides to oligonucleotides. A Formamide-based Unitary Chemistry—To gather into a single reaction milieu all the precursors necessary for the assembly of the first informational polymers, a unitary chemical frame is needed. A series of observations suggest that formamide (NH2CHO) chemistry might have provided such a frame. To summarize the evidence: the one-carbon compound formamide is a highly versatile building block for the syntheses of all the necessary precursor nucleic bases. We have reported (Refs. 1Saladino R. Crestini C. Costanzo G. Negri R. Di Mauro E. Bioorg. Med. Chem. 2001; 9: 1249-1253Crossref PubMed Scopus (171) Google Scholar, 2Saladino R. Ciambecchini U. Crestini C. Costanzo G. Negri R. Di Mauro E. ChemBioChem. 2003; 4: 514-521Crossref PubMed Scopus (116) Google Scholar, 3Saladino R. Crestini C. Ciambecchini U. Ciciriello F. Costanzo G. Di Mauro E. ChemBioChem. 2004; 5: 1558-1566Crossref PubMed Scopus (77) Google Scholar, 4Saladino R. Crestini C. Neri V. Brucato J.R. Colangeli L. Ciciriello F. Di Mauro E. Costanzo G. ChemBioChem. 2005; 6: 1368-1374Crossref PubMed Scopus (61) Google Scholar and references therein) the synthesis of purine, N9-formylpurine, adenine, N9, N6-diformyladenine, hypoxanthine, cytosine, hydroxypyrimidine, 4(3H)-pyrimidinone, uracil, 5,6-dihydrouracil, thymine, 5-hydroxymethyluracil, AICA (5-aminoimidazole-4-carboxamide), FAICA (5-formylaminoimidazole-4-carboxamide), and urea. These compounds are obtained from formamide heated in the presence of simple inorganic catalysts such as silica, alumina, zeolites, CaCO3, TiO2, common clays, kaolin, montmorillonites, olivines, and cosmic dust analogues of olivines. In all cases and for each catalyst several compounds were simultaneously formed, as summarized in Ref. 4Saladino R. Crestini C. Neri V. Brucato J.R. Colangeli L. Ciciriello F. Di Mauro E. Costanzo G. ChemBioChem. 2005; 6: 1368-1374Crossref PubMed Scopus (61) Google Scholar. The richest yields were obtained in the presence of clays, TiO2, and cosmic dust analogues. Guanine is conspicuously absent, but a possible efficient substitute in base pairing might have been provided by hypoxanthine. Notably, formamide also yields purine acyclonucleosides through a formose-like condensation of N-formyl derivatives (2Saladino R. Ciambecchini U. Crestini C. Costanzo G. Negri R. Di Mauro E. ChemBioChem. 2003; 4: 514-521Crossref PubMed Scopus (116) Google Scholar), providing a solution to the long-standing problem of the lack of reactivity between nucleobases and ribose or 2′-deoxyribose. In addition, formamide is an efficient activator of nucleoside trans-phosphorylation (Refs. 5Schoffstall A.M. Origins Life. 1976; 7: 399-412Crossref PubMed Scopus (77) Google Scholar, 6Schoffstall A.M Barto R.J Ramos D.L. Origins Life. 1982; 12: 143-151Crossref PubMed Scopus (45) Google Scholar, 7Schoffstall A.M Mahone S.M. Origins Life Evol. Biosph. 1988; 18: 389-396Crossref PubMed Scopus (15) Google Scholar), 2E. Di Mauro and G. Costanzo, unpublished observations. further supporting its possible relevance in the prebiotic polymerization reactions that did lead to pre-genetic informational macromolecules. Formamide is easily formed by hydrolysis of HCN and is stable in liquid form over a wide range of temperatures (2.5-210 °C). This property favors its concentration from dilute aqueous solutions by moderate heating. These physico-chemical attributes and its presence along with that of H2O and HCN in planets, comets, asteroids, and in the interstellar space (8Millar T.J. Astrobiology: Future Perspectives.in: Ehrenfreund P. Irvine W.M. Owen T. Becker L. Blank J Brucato J.R. Colangeli L. Derenne S. Dutrey A. Despois D. Lazcano A. Robert F. Vol. 305. Kluwer Academic Publishers, Norwell, MA2004: 17-31Google Scholar) support its possible prebiotic role. A hypothetical, but plausible and simple, scenario could thus consist of the following: in the presence of a varied (perhaps complete) panel of nucleic acid precursors dissolved in a water/formamide solution at moderately high temperature and in the presence of a phosphate donor, formamide-based trans-phosphorylation reactions would lead to the formation of oligomers and kick start a molecular evolutionary process. We are not dealing here in any further detail with such a highly hypothetical and fragmentarily outlined synthetic process, our intention being limited to the indication of its chemical plausibility. The Thermodynamic Problem—In the origin of the informational polymers, their survival as macromolecules is even more problematic than the polymerization process itself. Do physico-chemical conditions exist, providing kinetic and/or thermodynamic advantage to the otherwise intrinsically more unstable polymeric form over the monomer? We focus here on RNA and describe a systematic comparison of the stability of the phosphoester bonds in the precursor monomers versus the stability of the same bonds when present in RNA, under a wide range of temperatures and water/formamide ratios. Such comparison was lacking and provides information directly relevant to the monomer versus polymer bias. The stability of the β-glycosidic bond in the same set of conditions was also determined. A parallel analysis was recently performed (9Saladino R. Crestini C. Busiello V. Ciciriello F. Costanzo G. Di Mauro E. J. Biol. Chem. 2005; 280: 35658-35669Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar) comparing the corresponding stability properties for 2′-deoxynucleosides, deoxynucleotides, and deoxyoligonucleotides. Thus, a direct comparison between relative bond stabilities in RNA and DNA is here for the first time provided under comparable conditions. Adenine, ribose, ribose 5′-monophosphate, adenosine, adenosine 5′-monophosphate (5′-AMP), and adenosine 3′-monophosphate (3′-AMP) were from Sigma Aldrich, analytical grade. The degradation of RNA in water, in formamide, and in formamide-containing aqueous solutions was studied on P1 RNA. This RNA has the sequence 5′-GGAAACGUAUCCUUUGGGAG-3′, was purchased from RNATEC, and was kindly provided by E. Caffarelli (10La Neve P. Altieri F. Fiori M.E. Scaloni A. Bozzoni I. Caffarelli E. J. Biol. Chem. 2003; 278: 13026-13032Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 11Gioia U. La Neve P. Dlakic M. Arseci M. Bozzoni I. Caffarelli E. J. Biol. Chem. 2005; 280: 18996-19002Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). HPLC Analysis—Samples were resuspended at a final concentration of 1 mg/ml in water or in the appropriate formamide reaction medium (usually in 0.5-1.0 ml). Temperatures and incubation times are indicated. 10-μl aliquots of the reaction mixtures were diluted to a final concentration of 50% formamide in a final volume of 20 μl and injected into a Supelcosil™ LC-18-S 5-μm HPLC column (Supelco) 15 cm × 4.6 mm. Elution was performed at a flow rate of 2 ml/min at room temperature with methanol:30 mm ammonium phosphate, pH 5.3 (2.5:97.5), UV 254 nm, pressure 1.5 atmosphere, on an HPLC Beckman System Gold instrument. Identification of the peaks was performed by comparison with real samples. RNA Preparation and Labeling; RNA 5′-Labeling—10 μmolP1RNA were labeled with [γ-32P]ATP using polynucleotide kinase (Roche Applied Science). The oligo was then purified on a 16% denaturing acrylamide (19:1 acrylamide/bisacrylamide) gel. After elution, the residual polyacrylamide was removed by a NuncTrap Probe purification column (Stratagene). 2 pmol (typically 30,000 cpm) RNA were processed for each sample. Ribooligonucleotide Degradation Protocols and Analyses—The 5′-labeled oligonucleotide was treated under the time, temperature, and solution conditions indicated where appropriate. To stop the reaction a solution of 5 × 10-4m (final concentration) of tetrasodium pyrophosphate (Sigma) dissolved in water, pH 7.5, was added to a final volume of 40 μl. The samples were vortexed for 1 min and then centrifuged at 13,000 rpm for 20 min. This procedure was performed twice. The supernatant was ethanol precipitated, resuspended in 5 μl of formamide buffer, heated for 2 min at 65 °C, and loaded on a 16% denaturing polyacrylamide gel (19:1 acrylamide/bisacrylamide). For these methods, see also Ref. 3Saladino R. Crestini C. Ciambecchini U. Ciciriello F. Costanzo G. Di Mauro E. ChemBioChem. 2004; 5: 1558-1566Crossref PubMed Scopus (77) Google Scholar. Half-lives of the Bonds in the Ribooligonucleotide—For oligonucleotides, the half-lives of the phosphoester bonds were determined from the rate of disappearance of the band representing the intact 20-mer molecule. Cleavage of RNA normally requires participation of the 2′-OH group as an internal nucleophile (12Morrow J.R. Aures K. Epstein D.J. Chem. Soc., Chem. Commun. 1995; 23: 2431-2432Crossref Google Scholar) by two “nucleophilic cleavage” events: the trans-esterification and hydrolysis reactions (Fig. 1). During the trans-esterification (pathway a), the 2′-OH nucleophile attacks on tetrahedral phosphorus (V) affording a 2′,3′-cyclic monophosphate I, which in turn can be hydrolyzed to a mixture of 3′- and 2′-phosphate monoester II and III, respectively (pathway b). Both steps can be catalyzed by a large range of chemical species, including protons, hydroxide, nitrogen derivatives, and metal ions. On the basis of this known mechanism two operative assumptions were made. 1) It was assumed that the cleavage of the 3′-phosphoester bond is largely more effective than the cleavage of the 5′-one and that for practical calculation purposes the 5′-phosphoester bond is not cleaved. In support, the cleavage of the 5′ terminally labeled RNA molecule only the cleavage of the the by 3′- and in the same of were This with DNA (9Saladino R. Crestini C. Busiello V. Ciciriello F. Costanzo G. Di Mauro E. J. Biol. Chem. 2005; 280: 35658-35669Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar), where under several conditions to the In the studied the of the phosphodiester is not sequence under the conditions This sequence (10La Neve P. Altieri F. Fiori M.E. Scaloni A. Bozzoni I. Caffarelli E. J. Biol. Chem. 2003; 278: 13026-13032Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 11Gioia U. La Neve P. Dlakic M. Arseci M. Bozzoni I. Caffarelli E. J. Biol. Chem. 2005; 280: 18996-19002Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) that conditions to be cleaved. the cleavage conditions this RNA is stable 2 of Ref. Neve P. Altieri F. Fiori M.E. Scaloni A. Bozzoni I. Caffarelli E. J. Biol. Chem. 2003; 278: 13026-13032Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). These conditions are (10La Neve P. Altieri F. Fiori M.E. Scaloni A. Bozzoni I. Caffarelli E. J. Biol. Chem. 2003; 278: 13026-13032Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 11Gioia U. La Neve P. Dlakic M. Arseci M. Bozzoni I. Caffarelli E. J. Biol. Chem. 2005; 280: 18996-19002Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) and are from the of the 5′-labeled P1 RNA as a of the formamide concentration reported on of each were treated for the indicated time at A panel is shown only for the the is shown for the analysis of the degradation of adenosine as a of the formamide concentration indicated in each nucleic The ribose is not in the HPLC analysis and is indicated in in the panel were with The half-life of the oligonucleotide was determined with procedure from of the disappearance of the intact 20-mer that molecule and the two assumptions reported sequence the half-life of a 3′-phosphoester bond in the is by the half-life of the oligonucleotide × the of 3′-phosphoester bonds in the The stability of the β-glycosidic bond of in water was determined in under several conditions of and Google Scholar). We describe here the of formamide on the stability of this were performed as a of formamide concentration in water and of In to its of the stability of this bond is a for the analysis of the stability of phosphoester bonds in nucleotides and in oligonucleotides. 2 the high pressure liquid high pressure liquid analysis of the degradation of adenosine as a of time and of the concentration of formamide in the aqueous medium to as indicated in each The panel on the provides the The the in this ribose to the base The cleavage of the bond yields ribose which is not under our and The same is in the of the Thus, the disappearance of adenosine is by the of For the nucleoside the process is In the HPLC peaks than those of adenosine and are Thus, cleavage than that of the β-glycosidic bond under this range of conditions. The half-life of this bond at in water is × min. of formamide first and then the stability of this 5 this by formamide on the stability of the β-glycosidic 5 also the of the same bond in and are from and see in the presence of a phosphate group in 3′- and in the the half-life of the β-glycosidic bond is in water and is by formamide The of the β-glycosidic bond in H2O is min for the and × min for the to formamide the high stability × of this bond is These are to temperature (Fig. and analysis of the degradation of 3′-monophosphate as the molecule is indicated in analysis of the degradation of 5′-monophosphate adenosine, as in 2 and The degradation of the were as a of temperature and formamide (Fig. the analysis shown is at °C). The disappearance of the in water is by the of the nucleoside thus being by the cleavage of the 3′-phosphoester In water the β-glycosidic bond is with a and the corresponding with a all the nucleoside is into and The presence of of formamide at the β-glycosidic bond the cleavage of the phosphoester The same analysis was performed on The of the analysis at are reported in The is similar to that of with the of the behavior in water these conditions the β-glycosidic bond is than the phosphoester bond for the the by formamide is and at The half-lives of the 3′- and 5′-phosphoester bonds as a of formamide and at temperatures are in the by water/formamide and temperature on the stability of the β-glycosidic and the phosphate bonds in the nucleoside and in its 3′- and for the β-glycosidic high stability in water 5 × for the cleavage by versus by (Fig. and by the presence of a phosphate in water (Fig. This is to is in where the phosphate group a on the β-glycosidic bond in water (Fig. in Ref. R. Crestini C. Busiello V. Ciciriello F. Costanzo G. Di Mauro E. J. Biol. Chem. 2005; 280: 35658-35669Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). For the phosphoester bonds (Fig. an for the for the 5′-phosphoester and a lack of by formamide, with the by formamide on the β-glycosidic The comparison of this set of properties with those in is under The of the in a 5′-labeled 20-mer under was treated at temperatures for the indicated times (Fig. in water or in the presence of or of the degradation The of degradation of the is from the disappearance of the molecule as a formamide concentration first and then RNA from the process. the of this of degradation performed at and and as for the appropriate range of temperatures reaction panel the half-lives of the 3′-phosphoester bond determined from similar to those reported in The half-lives of the and 3′-phosphoester determined from the HPLC in and are also The in that in water and at high formamide at and the 3′-phosphoester bond in the oligonucleotide is more stable than the corresponding in the the 5′-phosphoester in the oligo is stable that its cleavage could not be measured and The 5′-phosphoester bond in the is also more stable than the The the of 3′-phosphoester bond We have the stabilities of the bonds that are more relevant for the of the acid as a the β-glycosidic and the 3′- and 5′-phosphoester The stability of each bond varied on the molecular in which was In addition, of the bond stabilities were as a of the and chemical water and/or The of the presence of formamide were studied of the reported synthesis in its presence of a set of nucleic acid precursors and of its The hypothetical scenario for the synthesis of the pre-genetic nucleic polymers would thus consist of formamide-based syntheses in the presence of the appropriate of catalysts R. Crestini C. Costanzo G. Negri R. Di Mauro E. Bioorg. Med. Chem. 2001; 9: 1249-1253Crossref PubMed Scopus (171) Google Scholar, 2Saladino R. Ciambecchini U. Crestini C. Costanzo G. Negri R. Di Mauro E. ChemBioChem. 2003; 4: 514-521Crossref PubMed Scopus (116) Google Scholar, 3Saladino R. Crestini C. Ciambecchini U. Ciciriello F. Costanzo G. Di Mauro E. ChemBioChem. 2004; 5: 1558-1566Crossref PubMed Scopus (77) Google Scholar, 4Saladino R. Crestini C. Neri V. Brucato J.R. Colangeli L. Ciciriello F. Di Mauro E. Costanzo G. 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Chem. 2005; 280: 35658-35669Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar and of this β-glycosidic bond is by of formamide to and then at high concentration is This is for the for the and for the for the the presence of formamide the β-glycosidic bond from the concentration and by formamide is to the formation of an and highly bond the of nucleobases (9Saladino R. Crestini C. Busiello V. Ciciriello F. Costanzo G. Di Mauro E. J. Biol. Chem. 2005; 280: 35658-35669Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, PubMed Scopus Google Scholar, and Google Scholar). of the high of the the of the phosphate is also a relevant to be being more stable than were obtained in the of and phosphoester bonds are more stable in the than in the more for the of than for the In with a cleavage the formamide the stability of the phosphoester bonds in all cases P. M. J. Chem. Scopus Google Scholar). This is for (Fig. of Ref. R. Crestini C. Busiello V. Ciciriello F. 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DNA and are in Ref. A. ChemBioChem. 2005; 6: PubMed Scopus Google Scholar). a property of RNA at present and a support for RNA being an information than and are not in a or or the with the RNA the physico-chemical conditions in which informational polymers formed, and an A property of the polymeric that might information on the origin of informational polymers and on the problem is the of thermodynamic and to polymerization process and polymers this property with a This is and to in Ref. The Origins of Life and R. Scholar. from the that the condensation reactions are not thermodynamically in dilute aqueous solution or even at moderate water two possible solutions have been for their The Origins of Life and R. Scholar). The first is the mechanism by the of monomers by The is the from the by the water for the the that monomers a in prebiotic is highly on kinetic The in to the formation of for that of any of and in monomers and highly conditions. The of a is not thermodynamically syntheses of precursor monomers in an aqueous the reported the of a process and that the conditions for the syntheses of the first and for their are of and would have been These thermodynamic in the physico-chemical of water aqueous in in to and were The Origins of Life and R. Scholar) as reaction This is with the scenario into which our be with the that formamide have also been The reported in this on the stabilities of the relevant bonds versus and versus two thermodynamically which for DNA and and for DNA and °C, H2O and/or for RNA. of the provided the conditions that were for the process is not at the here the property of the RNA into an frame.