Nonenzymatic RNA Ligation in Water

We describe the nonenzymatic ligation of RNA oligomers in water. Dimers and tetramers are formed in a time-, pH-, and temperature-dependent reaction. Ligation efficiency depends on oligonucleotide length and sequence and is strongly enhanced by adenine-based nucleotide cofactors. Ligation of short RNA fragments could have liberated the prebiotic polymerization systems from the thermodynamically demanding task of reaching a (pre)genetically meaningful size by stepwise addition of one precursor monomer at the time. We describe the nonenzymatic ligation of RNA oligomers in water. Dimers and tetramers are formed in a time-, pH-, and temperature-dependent reaction. Ligation efficiency depends on oligonucleotide length and sequence and is strongly enhanced by adenine-based nucleotide cofactors. Ligation of short RNA fragments could have liberated the prebiotic polymerization systems from the thermodynamically demanding task of reaching a (pre)genetically meaningful size by stepwise addition of one precursor monomer at the time. The origin of informational polymers is not understood. Considering only extant nucleic acids and according to the simplest possible logic, the progression of events could have been: synthesis of nucleic bases, formation of nucleosides thereof, activation by phosphorylation, polymerization of short oligonucleotides, their elongation, and eventually their survival through replication and evolution. The bottom-up approach to the origin of (pre)genetic polymers requires a definition of the minimal information. A minimal complexity of two different nucleotides (1Rich A. Kasha M. Pullman B. Horizons in Biochemistry. Academic Press, New York1962: 103-126Google Scholar, 2Crick F.H.C. J. Mol. 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Biol. 1968; 38: 121-128Crossref PubMed Scopus (85) Google Scholar), a ribozyme composed of only these two different nucleotides was developed (9Reader J.S. Joyce G.F. Nature. 2002; 420: 841-844Crossref PubMed Scopus (77) Google Scholar). It was reasonably claimed that “without at least two different subunits, there is no information and thus no basis for Darwinian evolution” (9Reader J.S. Joyce G.F. Nature. 2002; 420: 841-844Crossref PubMed Scopus (77) Google Scholar). The “genetic” meaning of such a minimal molecular information is intrinsically limited. Evolution entails maintenance and at the same time the possibility of accepting variations of the macromolecular information. At some point in Darwinian evolution, nucleic polymers had to arise that were long enough to satisfy these divergent necessities. The nonenzymatic polymerization process has been studied for decades, the results essentially showing that polymers of several tens can be obtained (reviewed in Ref. 10Orgel L.E. Crit. Rev. Biochem. Mol. Biol. 2004; 39: 99-123Crossref PubMed Scopus (739) Google Scholar) from preactivated precursors. The prebiotic validity of a process requiring complex preactivations has been questioned (4Wächtershäuser G. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1134-1135Crossref PubMed Scopus (80) Google Scholar, 10Orgel L.E. Crit. Rev. Biochem. Mol. Biol. 2004; 39: 99-123Crossref PubMed Scopus (739) Google Scholar, 11Orgel L.E. Trends in Biochem. Sci. 1998; 23: 491-495Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). An additional difficulty is to be found in the standard state Gibbs free energy change (12van Holde K. Halvorson H.O. van Holde K.E. The Origins of Life and Evolution. Alan R. Liss, Inc., New York1980: 31Google Scholar), which essentially states that condensation reactions are extremely inefficient in water. Considering the crucial step of elongation, the limit to the formation of nucleic polymers endowed of (pre)genetic potential is established by the intrinsic instability of long polymeric forms. If the polymerization process is slow, an equilibrium between synthesis and degradation of the polymer would rapidly be reached, preventing the accumulation of sufficiently long, (pre)genetically meaningful information. Polymer elongation by successive condensation steps adding one monomer at the time is not a likely mechanism for the accumulation of (pre)genetic information. Focusing on the stability problem, a detailed analysis was performed of the relative stabilities of the key chemical bonds (3′-phosphoesteric, 5′-phosphoesteric, and β-glycosidic) in RNA (13Saladino R. Crestini C. Ciciriello F. Di Mauro E. Costanzo G. J. Biol. Chem. 2006; 281: 5790-5796Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) and in DNA (14Saladino 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 RNA, this analysis led to the conclusion that conditions exist in which the key bonds are more stable in the polymer than in the precursor monomer. In these conditions (water, temperature between 60 and 90 °C), the polymer is the fittest form (13Saladino R. Crestini C. Ciciriello F. Di Mauro E. Costanzo G. J. Biol. Chem. 2006; 281: 5790-5796Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 15Saladino R. Crestini C. Ciciriello F. Costanzo G. Di Mauro E. Helv. Chim. Acta. 2007; 4: 694-720Google Scholar). The presence of free phosphates (16Saladino R. Crestini C. Neri V. Ciciriello F. Costanzo G. Di Mauro E. ChemBioChem. 2006; 7: 1707-1714Crossref PubMed Scopus (53) Google Scholar) and a defined window of pH values (17Ciciriello F. Costanzo G. Pino S. Crestini C. Saladino R. Di Mauro E. Biochemistry. 2008; 47: 2732-2742Crossref PubMed Scopus (16) Google Scholar) further stabilize the polymer and widen the thermodynamic niche in which the accumulation of information is favored. If a condition is identified in which a ribozyme-like activity is exerted by a sequence-homogeneous oligonucleotide leading to sequence duplication, the basis would be established for the simplest replicatory system eventually able to incorporate mutations and evolve. We focus on the problem: if short RNA polymers form in a aqueous an intrinsic of RNA exist that their thus the and were from The degradation of RNA was in on and is an oligonucleotide the sequence F. A. E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The were from and provided in the standard was further by a was for at the temperature of the to be a time to and the The pH values on a pH are RNA and RNA of the oligonucleotide RNA were The oligonucleotide was on a the was by a the RNA, in pH was by the addition of of and of at and The was in in and at one was for point of of RNA The Ligation and ligation of the of RNA were in of at the of RNA was for A of was to a of 90 The was in and for A was by the addition of of of of pH of of the addition of the was The were the were of this and the were at the and conditions the were in of for at and on a analysis was performed and was by the difficulty of size for RNA the of the was by the of their in such in the of and nucleotides long and of their a and The of these is in the same for the from a complex of fragments of the and from from from of is a that from It both and and is at in pH and in was pH of at pH at from is from It the sequence of RNA to yield and for is at in pH in of nucleotides from at pH at was by in in the presence of an adenine-based nucleotide The is of oligonucleotide length and and the of formation of and from both a on the and from a a of RNA oligomers were in at 60 at pH The formation of only was The formation of requires the presence of a detailed in The formation of the in time that of the the relative in are in The of formation of the relative to that of the is in The of the reactions leading to the formation of the were at and 60 on to that for 60 in and The for the reactions leading to tetramers were not of the complexity of the for their formation The was the of that is formed at time of ligation reactions the of the for the of the ligation are and are by this to ligation to the of the to form the to the of two would the and in the For further the In the in was The of ligation the of is in A detailed of the activity is in analysis of ligation ligation were of of analysis of ligation reactions and have been to the ligation of in the conditions 60 pH which has been to The ligation are The relative yield of tetramers a of the oligonucleotide is in The of formation of tetramers is in formation the of at which the of yield is of the ligation The ligation were of of analysis of ligation reactions and have been to the ligation of in the conditions 60 pH which has been to The ligation are The relative yield of tetramers a of the oligonucleotide is in The of formation of tetramers is in formation the of at which the of yield is of the ligation in a oligomers a were studied in to the to two purine are a for the ligation events to The results that this is not the and that ligation for both of A efficiency was for the in and An additional for the of a at the is the stability in of the step to the In a of fragments by this the presence of the thus in the of In addition to the in the oligomers by the of the analysis of ligation system is the ligation in the condition that for which has been to formation is only in the presence of the and The to the ligation in the presence of and at the is the ligation in the condition that for which has been to formation is only in the presence of the and The to the ligation in the presence of and at the in a The mechanism of RNA is K. J. Chem. Chem. 23: Google Scholar, G. R. PubMed Scopus Google Scholar). The of the requires of the an G. R. PubMed Scopus Google Scholar) in two the and the the to a is a of and steps are by and The degradation a of a of the a G. R. PubMed Scopus Google Scholar), which is in a in the of The stability at temperature of oligonucleotide A and of in a of pH is in (17Ciciriello F. Costanzo G. Pino S. Crestini C. Saladino R. Di Mauro E. Biochemistry. 2008; 47: 2732-2742Crossref PubMed Scopus (16) Google Scholar). are stable between pH and Ligation and degradation of by the two reactions was from the that the of The half-life of the oligomers was the of the of this Ref. F. Costanzo G. Pino S. Crestini C. Saladino R. Di Mauro E. Biochemistry. 2008; 47: 2732-2742Crossref PubMed Scopus (16) Google Scholar). At 60 the half-life of the is The of ligation are The of the ligation was In addition to and were in the same conditions the analysis for and in ligation was by and and only of the reaction. was not at The relative values of are in for longer not the In the conditions 60 pH the and nucleotide are and no (13Saladino R. Crestini C. Ciciriello F. Di Mauro E. Costanzo G. J. Biol. Chem. 2006; 281: 5790-5796Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, G. Saladino R. Crestini C. Ciciriello F. Di Mauro E. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). the can be to the chemical the of molecular from degradation The formation of and tetramers not of and of entails a ligation by the of the two by two and a polymerization The two only if the and are in the and in the relative two conditions are only by the oligomers a the length of the in the of by from one in the of in If is not and the is the and not be in the were is to the of the that and is in more the results of the same analysis performed on and The of the is to that of the The formation of the at a is the J. Holde K.E. J. Mol. Biol. PubMed Scopus Google Scholar) that size is the for the formation of at pH The of the pH in the ligation mechanism is The ligation of the was in the and in the presence of and and and In the of the the ligation of the monomer to yield is and the is not formed at the that ligation in the of an molecular is in The in In this no ligation is in the of no is and the formation not of the molecular size than the monomer size are formed on the of form is that is the of leading to a the in the efficiency by a the formed in are of size In the the size of the formed is by the in a degradation obtained by of a that is not in the one an and nucleotide for which no was and is that of a a that would conclusion is by the that of RNA by the RNA was in in the conditions in the presence of of The were in and the was from the were by the results showing that at the RNA not of is to be and a to at In the of RNA is to be by and Scopus Google Scholar), describe a of the of of and of RNA formation of the are in the of RNA and in The can be by the obtained in In RNA oligomers in and The of efficiency is and the by at least of only the and in the presence of and the performed of In A a is in which the oligomers were and The two were in at the same in only the being at the only the of ligation at least one be in the on the of the two on the the ligation reactions of the in the formation of the and of the is in the two on the additional fragments are to the in the of ligation The are at the of the The of the of these values a of the the values a of the of the the ligation an The that form more than and for are more than longer both in and in The by of the in the of the is The between the is in the that the not one not ligation was performed on a of different The was of sequence in this only at least one could be The that a complex of ligation events The of the are in showing that the efficiency of the ligation events to the oligonucleotide is that the ligation the is that the the that the sequence not in the ligation events and that a complex of fragments is obtained In of RNA oligomers in and according to a mechanism that not of the that depends on the sequence The of is the stable form of at pH and temperature and at pH and The of at temperature and pH is a The equilibrium between the two is established by and size J. Holde K.E. J. Mol. Biol. PubMed Scopus Google Scholar, Scopus Google Scholar, A. J. Mol. Biol. PubMed Scopus Google Scholar, C. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). the ligation depends on the pH values by these between the and forms. A of was and and the ligation reactions were at and that the the Scopus Google Scholar, C. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) is the pH ligation at the lower temperature not at the one in and At these pH values at 60 in Ref. Scopus Google Scholar). We have that in and that no are exerted by the for not was to the pH variations the of water. The for a on the pH in this is formation of bonds in A by of by and J. Chem. Scopus Google Scholar, J. Chem. Scopus Google Scholar, R. R.A. Biochemistry. PubMed Scopus Google Scholar), has detailed the of bonds the that be in from the that the two through their their In ligation could between the and of two oligomers in through their phosphates and a in a the ligation could at the in the divergent In this the of a this and the from an The the and of adenine nucleotide and the of of the oligomers of the obtained in the step of the are not in the results obtained can be on the that in both the are in the reactions A and in the of the additional ligation steps on the same of ligation reaction. of these two possible reactions the formation of The to ligation between and the extant ligation reactions the only could between two in the In this the of the and would be by the between the two the temperature the energy for the The bonds thus formed in would yield a which is in the a size is for of The of this of events are the and the of the oligomers are In the of both only one in the of different length The of the of a at one of the two of the the formation of are at the formed For their formation of two is by of has been for DNA of both short M. G. R. 2007; PubMed Scopus Google Scholar) and long A. A. S. J. Chem. B. 2008; PubMed Scopus Google Scholar). A of has not been for RNA in 2: PubMed Scopus Google Scholar), there is no to a Ligation would thus between to in of is in by the of the The a mechanism the of the not be the could the of the the for DNA S. Biol. PubMed Scopus Google Scholar). information on the of formed have performed and is a and bonds from the in a of a of and tetramers of in the for at that the are to by the The the in the same conditions only in the bonds formed are bonds of the the possibility that bonds than bonds were in the ligation reaction. The of the bonds was by of an RNA that had ligation in the standard conditions 60 is a The was on the The of was and to be composed of The of which are at the of the that have The is at the of the and is the only that is not of a formed of the bonds of the be of the of the of The two one in the formed and one of the in the monomer is at the The of the the of relative to that of In would of The is thus that of the bonds formed in the ligation are of the We describe the ligation of in at is the least demanding molecular sequence RNA a from the in possible and is of potential prebiotic The was for An and and at pH the temperature by pH the pH the temperature to the reaction. is of the formation of a RNA, established in J. Holde K.E. J. Mol. Biol. PubMed Scopus Google Scholar, Scopus Google Scholar, A. J. Mol. Biol. PubMed Scopus Google Scholar, C. Acad. Sci. U. S. A. 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In the formation of is for the two ligation which efficiency depends on the limit between and of the mechanism the formation of and pH are The presence of and of strongly and The that are formed in their size is to the size of the formed in their presence that the of the nucleotides is in In this is that of was C. J. V. G. J. 2008; PubMed Scopus Google Scholar), not by The that are on adenine for their M. J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google and M. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The are in the that RNA can to The and by was the focus of and several of the the process leading to ligation are are 2: PubMed Scopus Google Scholar). The states were found to have lower free energy than the states for purine for no a one was obtained J. J. Chem. Scopus Google Scholar). The process has been to be and the was to be lower at J. J. Chem. 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The the through a The to be are by and are further by adenine The obtained for the and the is the one to the a by the that these and In this system the RNA has a to that of an the their and the thermodynamic of the system is that of the the at the limit of their further the Gibbs free energy We describe an that the of by and in Ref. Scopus Google Scholar) the ligation efficiency that is in the same The of ligation has potential in prebiotic It to the of the thermodynamically polymerization to a (pre)genetic meaningful It has the intrinsic potential of the of complex If the of different from in the the could not only to would widen the of in which ligation could and at ligation would be An Darwinian would thus be We have (17Ciciriello F. Costanzo G. Pino S. Crestini C. Saladino R. Di Mauro E. 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A. 2002; PubMed Scopus Google Scholar) to describe the of a RNA sequence from a genetic point of that the molecular The nonenzymatic ligation of oligonucleotide the formation of the same of and to prebiotic The process is on than is a The that the replicatory events were on is