Nucleic Acid Catalysis under Potential Prebiotic Conditions

Abstract Catalysis by nucleic acids is indispensable for extant cellular life, and it is widely accepted that nucleic acid enzymes were crucial for the emergence of primitive life 3.5‐4 billion years ago. However, geochemical conditions on early Earth must have differed greatly from the constant internal milieus of today's cells. In order to explore plausible scenarios for early molecular evolution, it is therefore essential to understand how different physicochemical parameters, such as temperature, pH, and ionic composition, influence nucleic acid catalysis and to explore to what extent nucleic acid enzymes can adapt to non‐physiological conditions. In this article, we give an overview of the research on catalysis of nucleic acids, in particular catalytic RNAs (ribozymes) and DNAs (deoxyribozymes), under extreme and/or unusual conditions that may relate to prebiotic environments.


Introduction
The discoveryo ft he catalytic properties of nucleic acids by Cech and Altman in 1982-83 both redefined biological catalysis and provided compelling support for origin of life hypotheses centered around nucleic acid-based informations toragea nd catalysis, in particular the "RNA world" hypothesis first suggested by Alexander Rich, in which self-replicating RNA emerged prior to the evolutiono fD NA and proteins. [1][2][3] Despite the prevalence of the RNA World hypothesis and relatedc onjectures, such as different "pre-RNA" worlds [4] and mixed chimeric systemsincluding, for example, both RNA and DNA, [5] akey unanswered question is:u nder which environmental conditions did functional nucleic acids emerge and sustain themselves? Constraining the parameter space of ah abitable early Earth is crucial to understanding the emergence of life. One way of achieving this is to consider the sensitivity of nucleic acids to environmental conditions:i nw hat conditions can nucleic acids survive, and do conditions exist which can potentiate nucleic acid catalysis?E xploring conditions more exotic than dilute buffered solutions may yield answerst oi ntractable problems in origin of life and synthetic biology research. [6,7] Aw ide range of catalytic nucleic acids are known today.F or RNA (ribozymes), the most iconic example is the ribosome, [8] whose central role in peptide bond formation and thus protein synthesis designates it the most important ribozyme in modernb iochemistry,a nd the most obvious "smoking gun" of an early RNA world predating modern biochemistry.A nother ubiquitous ribozymet hat is essential in all free-living organisms is RNAseP,w hich processes the 5'-ends of precursor-tRNAs. [9,10] Other prominente xamples for ribozymes are small RNA-cleaving ribozymes such as the hammerhead (HH) ribozyme [11,12] (Figure 1A)a nd the hairpin (HP) ribozyme [13] (Figure 1B), which catalyze reversible self-cleavage to process the concatemeric products of rolling circle RNA replication into linear and circular RNA molecules. [14] Ar elatedf unction is carried out by self-splicing introns, [15,16] which catalyze their own excision from messenger,t ransfer,o rr ibosomal RNA via two sequential transesterification reactions of the phosphodiester backbone. In addition, in vitro selection experiments have revealed that the palette of RNA catalysis is far broader than these reactions and encompasses RNA ligation, [17,18] aminoacyl transfer,p orphyrin metalation [19] and CÀCb ond formation including the Diels-Alder reaction, [20] Michael addition, [21] aldol condensations [22] and others, [23] suggesting that an earlym etabolism might have been sustained by ribozymes.
While the main functiono fD NA in biology is the storageo f genetic information,alarge number of artificial DNAc atalysts have also been isolated by in vitro selection.T hese deoxyribozymes, or DNAzymes,c atalyze ar ange of bond forming reactions, including the Diels-Alder reaction, [24] Friedel-Crafts reactions, [25] RNA ligation (2'-5' and 3'-5'), [26,27] DNA ligation, [28] 5'phosphorylation, [29] adenylation, [30] RNA-nucleopeptide linkage [31] and porphyrin metalation. [32] The full range of DNA catalysis is reviewed in detail by Hollenstein, and an example of a RNA cleaving DNAzyme is shown in Figure 1D. [33] Finally,s ynthetic nucleic acids are also capable of catalysis. In particular, Ta ylor et al. selected artificial endonuclease and ligase enzymes from random pools of arabino nucleic acid (ANA), 2'-fluoroarabino nucleic acid (FANA), hexitol nucleic acid (HNA) andc yclohexene nucleic acid (CeNA). [34] While these studies convincingly demonstrate the broad catalytic potentialo fp olynucleotides, they leave open the question of whether some of these reactions couldh ave contributed to early biocatalysis, and whether they are compatible with the environmental conditions on early Earth.
Since the beginning of the Origin of Life field, great efforts have been made to determine,o ra tl east constrain, the conditions under which life originated. Definitive answers have been elusive, due to the extreme timescales under consideration and the combinedu ncertainties of when, where and how the first primitive forms of life emerged. The lack of fossil evidence of early life, the large number of possible geochemical environmentsa nd the difficulty in determining conditions on early Earth make this an almost intractable problem for origin of life researchers, amongstw hom there is little consensus on these questions. [35,36] In light of this, we and others have previously argued for af lexible approach to the problem, by performing experimentsu nder relaxed but plausible boundary conditions and using the resultst oi nform aboutp ossible plausible prebiotic environments. [37][38][39][40][41] The many studies that aim to constraint he global climate and conditions on early Earth allow some experimental boundaries to be set:A st oday,d ivalent magnesium and calcium were abundant in the oceans of early Earth.H istorical ocean solute composition is dependent on both pH and reducingp otential. Assuming an acidic ocean pH around 4G a, hydrogen   DNAzyme. The hammerhead (A) and hairpin( B) ribozymes catalyze the reversiblec leavage of the RNA substrate strand showniny ellow (black arrow indicates cleavage site). [42] The class Il igase (C) binds asubstrate strand (yellow) and catalyzes 3' OH nucleophilic attack on its own 5' triphosphate, leadingtop hosphodiester bondf ormation andreleaseo fi norganicp yrophosphate. [43] The 8-17 DNAzyme( D) is ametalloenzymecatalyzing RNA transesterification in the presenceo fd ivalent metali ons. [44] The substrate strandi sshown in yellow, with the ribonucleotide cleavage site marked in red.
Chem. Asian J. 2020, 15,214 -230 www.chemasianj.org 2019 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim sulfide presenti ns eawater would have created ar educing environmentrich in Fe 2 + ,but low in concentrations of free transition metal and group 12-16 ions due to the formation of insoluble sulfide compounds. [45,46] Early nucleic acid catalysis may have relied on Fe 2 + as ac ofactor,u ntil the advent of aerobic conditions caused the oxidation of Fe 2 + to Fe 3 + ,n ecessitating its replacement by Mg 2 + or other metal ions. [47] Oceanic pH, which is driven by atmospheric CO 2 concentrations, likely rose monotonically from pH 6.6 in the Hadean era to pH 7.9 by the Cambrian era. [48] However,o ther studies posit that oceanic pH in the late Hadean/ early Archean was as low as 3.5-5.4. [49,50] Further uncertainty is introduced if we consider that life may have emergedi nt he vicinity of ah ydrothermalv ents, where local pH may be either very low( pH 2-3) or very high (pH 9-11), depending on type, rather than in the bulk ocean. [51] Estimates of temperature are more variable, spanning climates ranging from frozent on ear boiling. Oxygen, iron and silicon isotope studies suggest temperatures of 70 8Cu pu ntil as late as 3.3 Ga, at heory additionally supported by evidence of al ow viscosityA rchean ocean. [52][53][54][55] However,e vidence of a temperate climatei sp rovided by geological carbonc ycle modelsa nd isotope evidence from cherts and sediments. [56][57][58] Studies of Archean glaciald eposits suggest the presence of ice caps or cold periodsd uring this time, [58] and somer esearchers argue that in the absence of extreme levels of greenhouse gases, ag lacialH adeanE arth is likely,a lbeit with intermittent periodsof" fire and brimstone" following major impacts. [59,60] Althought hese studies provide some useful constraints on the conditions at the Origin of Life, ab road range of conditions remain feasible. The exact microenvironment in which the first replicators emerged was likely more significant than the global conditions at the time. For example, 'warm little ponds' on land would be subjectt ot emperature, composition and concentrationf luctuations due to evaporation and condensation driven by day-night cycles, [61] eutectic phases in frozen environments lead to strong solute up-concentration and significant pH shifts, [62] and hydrothermal vents provide extreme temperature and pH gradients. [51] Any of these environments might provides helter from adversec onditions such as UV radiation, the surface intensity of whichw as several orders of magnitude higherthan today. [63] In this focus review,w ew ill explore the range of conditions under whichn ucleic acid catalysis is possible, highlighting how nucleic acids can adaptt oe xtreme conditions, and how these conditions can both support and potentiate function. In order to understand the emergence of life, we must understandt he environmental factorst hat would have acted upon the first functional nucleic acids, fore xample, in an RNA,p roto-RNA or mixed nucleic acid world scenario. In addition, many nucleic acid enzymes catalyze industrially relevant processes and, as such, challenging conditions may be required to increaser eaction rates, shift reactione quilibria or improve substrate or product solubility.I nb oth cases,r eaction conditions may deviate strongly from in vivo or typicalinv itro environments.
2. The role of metal ions in nucleic acid folding and catalysis

Folding of nucleic acids
The range of conditions in which catalytic nucleic acids are functional is largely determined by the mechanism by which nucleic acids can fold into catalytically active three-dimensional structures. Nucleic acid folding differs to that of proteins, which in many cases tend to fold via rapid, cooperative twostate thermodynamic transitions, with no detectable intermediate structures. [64] Nucleica cid chain compaction is driven by ion-mediated electrostatic interaction, conformational entropy, base pairing, base stacking, and noncanonical interactions. [65,66] Compared to proteins, the folding energy landscape of nucleic acids is convolutedd ue to the highn umber of competing, energetically similarf olding states, and nucleic acid molecules tend to adopt ar ange of conformations in solution. [67,68] The highly chargedp olyanionic backbone of nucleic acids usually prevents the irreversible aggregation of misfolded molecules. This means that, whilst activity may be lowered by adversee nvironmental conditions due to the presence of inactive or poorly active conformers, catalysis can occur under ab road range of environmental conditions. Consequently,c onditions that promote folding and the formation of active conformations are of particular interest, as they may directly improve the catalytic activity of nucleic acid enzymes.

Modes of metal ion-nucleic acid interaction
Ak ey variable determining nucleic acid foldinga nd activity is the presence of counterions, which helpt oo vercome the charge repulsion from the polyphosphate backboned uring compaction. For RNA, the most relevant cations under in vivo conditions are Mg 2 + and K + ,b oth of which interact with RNA predominantly through electrostatic forces. [69] In particular, Mg 2 + ions enable the formation of complex folds that allow nucleic acids to stabilizes pecific structures, recognize binding partnersa nd mediate catalytic processes. [70][71][72][73] Generally, interacting Mg 2 + can be divided into two populations ( Figure 2): diffusive ions, which surroundt he RNA as an ensemble of hydratedi ons that are non-specifically attracted to the negative chargeo ft he RNA, and am uch smaller group of partially desolvated ions, which bind to specific electronegative sites on the RNA itself. [74] Whilst these specific metal ion-RNAi nteractions mostly contributet ot he conformational specificity of an RNA structure (and thus in many cases to the active conformation of nucleic acid enzymes), diffusive ion-RNA interactions contributem ostt ot he thermodynamic stabilizationo ft he overall RNA fold. [75]

Impact of metal ions on nucleica cid catalysis
Given that magnesium is the seventh most abundant element in the Earth's crust, and that the Mg 2 + ion is the second most abundant cation (55 mm)i ns ea water after Na + ,i ti sc onceivable that similar Mg 2 + concentrationsw ere presenti na nA rche-an ocean, [76] or at varying levels in potential RNA world freshwater environments. However,m any other mono-, di-and polyvalent ions can also drive the folding of RNA (and other nucleic acids), including Mn 2 + ,C a 2 + ,F e 2 + ,S r 2 + ,B a 2 + ,N a + and polyamines. [66,77,78] The ion concentrationsr equired to achieve RNA folding vary betweent he differenti on types, as their charge density ande xcluded volume largely determine the strength of the coulombic RNA-ioni nteraction andt hus the overall compactness of the folded nucleic acid. [78] For example, the Tetrahymena group Ir ibozyme, which was derived from a self-splicing Tetrahymena preribosomal RNA and catalyzes ar eaction mimicking the first step of splicing, [79] requires micromolar concentrations of trivalent cations, millimolar concentrations of divalent ions but near-molar concentrationso fm onovalent ions for folding. [75] However, althought he Tetrahymena group Ir ibozyme folds into an ative-like state in the presence of various counterions, foldingo ft he catalytically active state requires site-specific binding of Mg 2 + or Mn 2 + . [75] All of the larger natural RNA enzymes,s uch as RNAseP [9,10] and the variouss elf-splicing introns, [15,16] depend on site-specific metal ion cofactors for chemical reactivity.L ikewise, the various artificial RNA ligase and polymerase ribozymes, which rely on nucleoside triphosphate activation chemistry,a re strict metalloenzymes with only poor tolerance towards metal ions other than Mg 2 + . [80] In view of this, it is quite surprising that moderni ntracellular conditions are somewhat challenging for nucleic acid folding and activity due to low free Mg 2 + concentrationso fa pproximately 1mm. [81] The need for higher levels of free Mg 2 + in vivo is alleviated by the presence of RNA chaperone proteins, which promote RNA folding and annealing. [69] The dependence on intracellular protein co-factors is well illustrated by RNAse P: at low ionic strength, the protein component of this complex is essential for activity in vivo and in vitro. [82,83] However,t he RNA itselfi sa ctive in vitro in the presence of 60 mm MgCl 2 . [2] The high divalention concentration required for RNA-only catalysis in vitro emphasizes that charge screening by either salt or the protein component is essential for folding and activity.N evertheless, optimal conditions are highly dependento nt he catalytic system in question.F or ex-ample,t he family of group II introns has ab road tolerance for Mg 2 + concentrations and near-optimal activity occurs between 0.1 to 100 mm in vitro. [84] Like ribozymes, DNAzymes use diffuse electrostatic and specific metal ion interactions for activity and folding. Notably,t he high stability, cost-effectivep roduction, and easy chemical modification of DNA has enabled the systematic selection of a large number of DNAzymes and aptamersc apable of selective metal ion detection. These DNAs can bind to and distinguish between an impressive range of species, includinga lkali metal ions, alkaline earth metal ions, transition metals,n oblem etals, post-transition metal ions and lanthanide and actinide ions for catalysis. [85] It should be mentioned that non-metallici ons can also support folding of nucleic acids into active conformations. For example,p olyamines can aid RNA folding;t he required MgCl 2 concentrationf or RNAsePR NA folding and activity is reduced from 60 mm to 10 mm in the presenceo f1m m spermidine. [2] However, enhancements in folding are dependento nt he characteristics of the polyamine counterion. Longerp olyaminesd estabilizef olded structures due to excluded volume effects, which can prevent ac omplete folding transition to the native state even under usually favorable folding conditions. [77] Lanthanides( Ln 3 + )a re also of interest, as their interactions with nucleic acids are very different from typical divalent metal ions due to their unusual coordination chemistry.I np articular, the absence of as trong ligand fielda llows for ahighdegree of structurald iversity in lanthanide complexes, as ligands alone dictate the symmetry and coordination of complexes. [86] As a result, lanthanides not only show ah igh affinity to the phosphate backboneo fn ucleic acids due to their high charge density (typically only mm concentrationsa re required for binding), but they can also directly interactw ith the nucleobase moieties. [87] Because of these unusual properties, the impact of lanthanideso nn ucleic acid catalysis is rather diverse:L n 3 + ions can accelerate as mall Pb 2 + -dependentr ibozymec alled the leadzyme, [88] yet they inhibit the hammerhead [89] and hairpin [90] ribozymes,a nd the RNA-cleaving 8-17 DNAzyme. [91] In addition, several strictly Ln 3 + -dependentR NA-cleaving DNAzymes were discovered by in vitro selection experiments, [92][93][94][95] suggesting that nucleic acide nzymes can directly harness the Lewis acid character of lanthanides for catalysis ( Figure 3). To the best of our knowledge,L n 3 + -specific ribozymes have not yet been described in literature, and at af irst glance rare earth metals have little relevance for origin of life scenarios due to their low aqueous solubility.H owever,l ow concentrationso f lanthanides are available,f or example, under hot acidic conditions in volcanic mudpots, and Ln 3 + ions are essential under these conditions for some acidophilic microbes that use methane as an energy source. [96] Thisr aises the possibility that prebiotic systems relyingo nn ucleic acid catalysis may have been able to harness lanthanides for certain reactions.

Metal ion inducedhydrolysis
While metal ionsa ssist nucleic acid foldinga nd catalysis in many cases, they are often also at hreat to the chemical integ- Figure 2. Schematic depicting dependence of RNA foldinga nd hydrolysis on divalentm etal ion concentration.U nder aqueousc onditions, divalent metal ions (in particular Mg 2 + and Mn 2 + )can enhance RNA folding by bothdiffuse binding and site-specific binding (highlighted in blue). In diffuse binding, hydrated Mg 2 + ions interact nonspecifically with the nucleic acid via longrange electrostaticinteractions. In site binding,d ehydrated or partially dehydrated Mg 2 + ions (highlighted in blue) interact specifically with anionicbinding sites, whicha re formed by the RNA fold to act as coordinatingl igands for the metal ion. At high M 2 + concentrations,metalion catalysis leads to increased RNA hydrolysis. rity of RNA ( Figure 2); [97] heavy metal ions such as Eu 3 + ,L a 3 + and Tb 3 + ,Pb 2 + ,and Zn 2 + catalyzerapid RNA cleavage in aqueous solutions. [97,98] Zn 2 + is only about 4% as active as Pb 2 + , and other metal ions such as Cd 2 + ,M n 2 + ,C u 2 + or Mg 2 + catalyze degradation one to two orders of magnitude slower than Zn 2 + . [99] However,a te levated temperatures and/orh igh ion concentrations, these seeminglyw eak catalysts (including Mg 2 + )c an reduce RNA half-lives down to minutes. [100] This means that environments with ah igh concentration of Mg 2 + and high temperatures, such as hydrothermalv ents, are unsuitable settings for RNA-based scenarios of molecular evolution. Likewise, free Ln 3 + ions are highly nucleolytic under basic conditions, as their ions form multinuclear complexesa nd cleave RNA nonspecifically at low mm concentrations with a rate acceleration as large as 10 8 -10 12 -fold. [101] DNA is much more resistantt owards metal ion-induced scission, and requires additional DNA-binding delivery agentsf or efficient cleavage under mild aqueous conditions. [102] An otable exception is the ability of Ce IV to accelerate DNA hydrolysis up to 10 11 -fold under neutral conditions,r educing the half-life of the phosphodiester linkagei nD NA from millions of years down to af ew hours. [101] Possible modes of metal ion-catalyzed nucleic acid hydrolysis include Lewis acid catalysis, Brønsted base catalysis, nucleophilic catalysis by metal-bound hydroxides and simple electrostatic stabilization of transition states by positively charged metal ions( Figure 3). The individual mechanisms of each metal ion class are still the subject of some debate and go beyond the focuso ft his review,b ut are discussed in excellent detail elsewhere. [101,[103][104] Facing the threat of degradationb ym etal ions, in particular in the case of RNA, it is interesting from aprebiotic perspective that an umber of nucleic acids are capable of efficient catalysis without divalent metal ions. In particular,s everal families of small nucleolytic ribozymes reversibly catalyze metal-independent and site-specific cleavage/ ligation of the RNA backbone, and can accelerate this reaction by approximately am illionfold using general acid base catalysis. [105] Similarly,p urely Na + -dependent DNAzymes were isolated by targeted in vitro selection. [106,107] Some of these (deoxy-)ribozymes will be discussedl ater in more detail, as they are compatible with aw ide range of conditions.

Prebiotic alternatives to Mg 2 + +
Of the various ions that can replaceM g 2 + during nucleic acid foldinga nd catalysis, Fe 2 + is of great prebiotic interest as it was likely to be highly abundant on Earth before the advent of photosynthesis. [31] Fe 2 + wass peculated to be present in microto low millimolar quantities during early Archean Earth. [31] Such concentrations are sufficient to replaceM g 2 + during RNA cleavage catalyzed by several DNAzymes. [109] As discussed in section 3, Fe 2 + was used during pH-dependent selection for RNA-cleaving ribozymes, where it enabled the discovery of novel catalytic motifs that are absent in typical selections using Mg 2 + . [110] Intriguingly,H siao et al. showedt hat substituting Mg 2 + with Fe 2 + in an anoxic environmente nabled various natural RNAs, such as tRNA or ribosomal RNA,t oc atalyze single-electron transfer reactions, which are typicallyl imited to cofactor-dependentprotein enzymes. [111] Thus, RNA might have catalyzed different electron transfer reactions, which are ap rerequisite for metabolic activity,before the rise of oxygen levels.
Zn 2 + has also been proposed as ak ey divalent transition metal ion inp rebiotic chemistry. [112] In this "Zinc World" hypothesis, porous and photoactive structures comprised of ZnS provided the substrate upon which CO 2 reduction and biomolecular polymerization occurred, driven by UV light. Indeed, Zn 2 + can substitute Mg 2 + as the only divalent metal ion during RNAsePc atalysis, but only in the presence of high concentrationso fa mmonium salts. [113] Zn 2 + was also shown to be strongly beneficial for DNA-catalyzedD NA cleavage. The artificial deoxyribozyme 10MD5isabimetallic metalloenzyme (analogous to many protein DNA endonucleases) that catalyzes the Mn 2 + /Zn 2 + -dependent DNA phosphodiester bond hydrolysis with at least a1 0 12 -fold rate enhancement. [114] In af ollow-up study,S ilverman and co-workersd emonstrated thato nly two base substitutions were necessary to alter 10MD5f rom hetero- Figure 3. Various modes of interaction between metal ions and RNA during RNA cleavage.T he reaction proceeds via at rigonalb ipyramidal transition state. The rate of reaction can be accelerated by Lewis acid stabilization of the leaving3 ' oxygen (A), facilitatingt he deprotonation of the attacking oxygen nucleophile (B), coordination of non-bridging oxygens (C) or coordination of an on-bridging oxygen in addition to the nucleophile (D), which promotes af avorablei n-line geometry for nucleophilic attack. The stabilizing metal ionand attacking base are shown in red andblue, respectively.Adapted from Forconie tal. and Frederiksenetal. [104,108] bimetallic to ap urely Zn 2 + -dependent monometallic DNAzyme. [115] Later,e ven faster ands maller deoxyribozymes which requireZ n 2 + alone forc atalysis were identified by in vitro selection. [116] In summary,t he availability of metal ions such as magnesium was mostl ikely not ac ritical factor for early nucleic acid enzymes( especiallyr ibozymes). However,i ti sp ossible that Fe 2 + ions in particular extended the catalytic properties of ribozymesu nder the anoxic conditions of the late Hadeana nd early Archean. Further research in this field could uncover new, unexpected catalytic nucleic acids that increaset he plausibility of an early metabolism mediated by nucleic acids.
3. The influence of pH on folding and catalysis 3

.1. Potential pH values in prebioticsettings
Another crucial physicochemical parameter for early nucleic acid catalysis and stability is pH. Estimates of environmental pH on early Earth are largely hypothetical (see introduction), but mostevidence suggests that oceanic pH was initially acidic (pH 6.6, [48] or lower [49,50] ).The theory that early molecular evolution originated at alkaline (pH 9-11) hydrothermalv ents, similar to the modern Lost City systems, has an umber of proponents, but is difficult to reconcile with an RNA-based origin due to the inherent labilityo fR NA to alkalineh ydrolysis, which occurs above pH 6a nd is strongly accelerated by higher temperatures and divalent metal ions ( Figure 4). [100,117] RNA is most stable at pH 4-5 with significant acid hydrolysis not occurring until below pH 2. Thus, more acidic vent types such as acidic volcanic lakes or comet ponds are credible early scenarios for RNA formation and catalysis. [51] DNA is less stable than RNA under acidic conditions due to increased depurination below pH 3, [118,119] but is more resistant to basic conditions as it does not possess the 2'-OH group required for base-catalyzed hydrolysis. AD NA-later scenario could therefore be in agreement with ag raduali ncrease of environmental pH over time. Indeed, high CO 2 levels in the Hadeane ra may have led to av ariety of acidic aqueous environments, [49] and the slow transition from acidic to slightly alkaline oceans could have driven the later emergence of the more stable DNA-baseds ystems. [48,120,121] 3.2. The impact of pH on nucleic acidcatalysis.
The direct effect of pH on catalysis is inherently dependento n the type and mechanism of the reaction. Catalysis by nucleic acids can occur via transition state stabilization (e.g. by hydrogen bondingo re lectrostatic stabilization), general acid and/or base catalysis (i.e. by enhancing the nucleophilicity of attacking groups by deprotonation or by stabilizing leaving groups by protonation), or by facilitatinga ctive conformationals tates such as the formation of an in-line transition state duringn ucleophilic attack. [122] For example, the reversible RNA cleavage reactionc atalyzed by smalln ucleolytic ribozymes, which is based on the nucleophilic attack of an O2' on an adjacent phosphorus atom, is in most ribozymes accelerated by general acid-base catalysis. [122] Here, two ionizableg roupss tabilize the developing negative and positive charges duringt he reaction by partial proton transfer in the trigonal bipyramidalp hosphorane transition state of the reaction( Figure 5). [122,123] Typically,o ptimal protont ransfer in enzymes requires functional groups with ap K a in the neutralr ange. [124] However,t he free form of the four canonical nucleobases have pK a values far from neutrality and are therefore suboptimal for general acidbase catalysis. [125] In somer ibozymes, the local molecular environment can cause ac onsiderable shift in the pK a of both general acid and base towards neutrality,asimilare ffect to that found in some proteins. [126][127][128] If both ionizable groupsa re sufficientlyp erturbed, the pH dependence of catalytic rates showsa"bell-shaped" pH rate profile, where the rates are maximal aroundp H7. [123,129] In other cases,s uch as for the hairpin (HP) ribozyme, the rates of RNA cleavage (and ligation) increaseu pt op H7,b ut plateau at higherv alues due to the high pK a of N1 in the catalytically activeguanosine base. [130] Generally,t he acid-basem echanism employed by smallr ibozymes makest hem robust towards changes in pH and enables significant cleavagea nd ligationa ctivity at pH > 6. However, the rate enhancement is limited by the small fractiono fr ibo- Figure 4. The impact of pH on RNA/DNA stability.A)Illustration of RNA and DNA stability in different pH ranges. At acidic pH < 2, RNA is prone to hydrolysis, whereas DNA is moresusceptible to depurination. At basic pH, the phosphodiester backbone of RNA hydrolyses rapidly,whereas DNA remains stable. B) Relative rate of RNA hydrolysis with respect to pH. Shown is an il-lustrativepH-rate profile for the cleavage of 3',5'-UpU at 90 8Cbased on the data reported by Jarvinen et al. [131] zymes that, on average, have the correct ionizations tate for general acid-base catalyzed cleavage (typically 1i n1 0 5 to 10 6 ribozymes for the HP ribozymea tn eutralp H [122] ). For the reverse ligation reaction the inverse ionization state is more favored, but the resulting rates are offset by al ow k cat due to the low reactivity of the neutral base moieties. [123] The phosphotransfer reactions of large metalloribozymes such as self-splicing introns, [132,133] RNAsePa nd artificial ligases that make use of triphosphate activation chemistry,s how a log-linear relationship between the rate of the chemical step and pH. [134] This is typical for ar eaction mechanism involving a pre-equilibrium loss of ap roton from ah ydroxylg roup before in-line nucleophilic attack. Likewise, most RNA-cleaving deoxyribozymes have al og-linear dependence of rate on pH with a slope near unity, [44,135] which is also consistentw ith the requirement for asingle deprotonation event duringt he reaction. pH levels also have an important effect on nucleic acid base pairing, ast he protonation state of nucleobases dictatest heir ability to form hydrogen bonds. In particular,a tl ow pH most nucleic acids are denatured( or at least destabilized) due to the protonation of G-C base pairs and resultant Hoogsteen base pair formation. [51] While this mechanism is detrimental forn ucleic acid folding, for example, of activer ibozymes, environmental pH cycles or gradients [136] mayh ave loweredD NA and RNA duplex melting temperatures,a nd therefore facilitated non-enzymatic and enzymatic copying reactions. [137] Furthermore, non-canonical AÀCa nd CÀCb ase pairs have been shown to occur under mildly acidic conditions, with AÀCb ase pairs at pH 5r eaching the stability of AÀUa nd GÀUb ase pairs under neutralc onditions. [138] Thus, differentp Hr egimes can enable the exploration of structuralm otifs and thus catalytic sequences that are otherwise inaccessible at neutral pH.

In vitro selection of nucleic acids catalysts undern on-physiological pH conditions
Indeed, in vitro selection experiments have shown that nucleic acids can be readily evolved towards improvedc atalysis at lower pH where the chemicals tability of the RNA backbonei ss trongly increased. For example, ad en ovo selection of self-cleaving ribozymesa tl ow pH resulted in av ariant that showed pH-dependentk inetics with an optimum of around pH 4. [139] Another study by Popović et al. investigated the effects of both pH and divalent cations on the isolation of self-cleavingR NA in iterative in vitro selectione xperiments from random libraries. [110] Depending on pH, and whether Mg 2 + or Fe 2 + was included as the divalent metal ion during selection, different sequences and secondary structurem otifs were isolated. Neutral pH in the presence of Fe 2 + led to the selection of hammerhead (HH)-like motifs, whilst at pH 5av ariety of previously unknown motifs were discovereda nd the abundance of HH motifs dropped to less than 0.1 %. Thus, both pH and substitutions between Fe 2 + and Mg 2 + strongly influence the relative fitnesso fd ifferent motifs.
Short RNA-cleaving DNAzymes have also been evolved to functioni nt rans at lowp H. The reaction proceeds optimally at pH of 4-4.5 in the absence of Mg 2 + ,d emonstrating that low pH can facilitate the Mg 2 + -free cleavageo fR NA by aD NAzyme. [140] Moreover,o ft he 20 clones sampled after selection, 14 did not share extensive sequence similarities, suggesting that the catalysis of the cleavager eaction atl ow pH has different or relaxed sequence requirements.
Ligation reactions represent an important catalytic function, for example, for nucleic acid self-replication. [141] Consequently, RNA ligasesh ave also been evolved to function at acidic pH. For example, random mutagenesis of ad erivative of the triphosphate-dependent class IR NA ligase ribozyme ( Figure 1C), followed by four rounds of evolution of the randomized pool under acidic pH, allowed for the selection of clones that function optimally at pH 4insteadofatneutral conditions. [142] Additional mutagenesis of the selected ribozyme furthere nhanced the rate of ligation by 8000-fold. [143] Kühne and Joyce implemented ac ontinuousi nv itro evolution strategy to progressively decrease or increaset he optimal pH of the class Il igase ribozyme, beginningw ith an optimal pH of 8.5. [144] The result was two highly active class Ir ibozyme variants with only very few mutationst hat shiftedt he optimal pH to either pH 5.8 or 9.8.
Early peptides ynthesis and even translation may have also occurred over ab road pH range. The peptidyltransfer reaction that takes place at the heart of the ribosome does not involve acid-base catalysis and so is relatively pH-insensitive. [145] Ac onsiderable decrease in peptideb ond formation is observed only at pH < 6.5 due to inactivation of the attacking amino group of the A-site aa-tRNA by protonation. [146,147] Notably,the activation of amino acids by aminoacetylation, ak ey step in protein biosynthesis, can also be catalyzedb yR NA under acidic condi- tions:K umar et al. reported the selection of ac alcium-dependent ribozyme capable of activating amino acids in this manner,w ith an optimal of pH 4.0-4.5. [148]

Heat tolerance of nucleic acid catalysis
Te mperature is af urther criticalp arameter in nucleic acid catalysis and stability ( Figure 6). As for proteins, reactionr ates increase with increasingt emperature, until the point at which activity falls due to denaturation. In the absence of magnesium, the duplex melting temperature (T m )o fnucleic acids is sufficiently low to reduce the catalytic potential at even slightly elevated temperatures. In addition, the faster reaction kinetics at elevated temperaturesa re offset by the increasing rate of phosphodiester hydrolysis, especially in the presence of divalent metal cations such as magnesium as discussed above, which prevents sustained catalysis.

Prebiotic temperatures and thermophilicRNAs
Te mperature estimations of the early Earth are am atter of debate. Severall ines of evidence exist that support ah ot climate duringt he Archean eon, 4t o2 .5 billion years ago, by which point the Earth's crust is thought to have cooled sufficiently to allow for the dawn of life. Based on oxygen and silicon isotope analyses in sedimentary rocks, [52,54,149] turbidity current deposits that suggest ap ossible low viscosity ancient ocean, [55] and the progressively decreasing thermostabilities of resurrected ancestral proteins, [150] Archean surface seawater temperatures have been interpreted to range between 60 8C and 80 8C. In contrast,t emperatures below 40 8Ca tt he surface have also been proposed based on evidencei ncluding deuterium andp hosphate isotope analyses, [56,57] and Archean glacial deposits suggest the presence of ice caps. [58] Indeed, more recent 3D climate-carbon models by Charnay et al. predict globalm ean temperatures between around 8 8C( 281 K) and 30 8C( 303 K) 3.8 billion years ago, suggestingt hat cold and even frozen environments may have been present on early Earth. [151] Hydrothermal vent temperatures are highly variable, with gradients from the hot interior (> 350 8C) to much colder seawater (or surrounding surface freshwater). [51] This precludes the occurrence of biochemical processes on or near to the surface of the vent, particularly given that the function of typical mesophilic nucleic acid enzymes is lost above % 70 8C, but conditions in the immediate surroundings may have been rather more amenable.
Despite the temperature sensitivity of RNA, living systems have adapted to survive at extreme temperatures. Comparison of homologous ribozymes in mesophilic and thermophilic organismsr eveals how sequence adaptations can lead to higher temperature stability. As tudy on RNase Ph omologs in mesophilic and thermophilic bacteria by Pan et al. observed that folding was more cooperative for thermophilic RNA, and the folding pathway proceeded via ad ifferent set of intermediate structures despite the high similarity of the final states. [152] Further work revealed that the thermophilic homologp ossesses severalm utationst hat increasei ts stabilityb yi ncreasing GC content ande liminating non-canonical base pairs. [153] In addition, insertions in diverse motifs throughout the thermophilic homolog structureincrease tertiary interactions and folding cooperativity while creating am ore densely packed core.

In vitro selection of thermophilicnucleic acid enzymes
Severalr eports focusingo nh eat adaptation of nucleic acid enzymes to highert emperatures have been published. Guo et al. used directed evolution to select for thermally stable variants of the Tetrahymena ribozyme. [154] Af amily of temperature stable variants were identified, which were slower than the originalribozymeb ut had 10.5 8Ch igher meltingt emperatures. Whilstt he consensus sequence of this family contained nine point mutations, only one served to strengthen the helical secondary structure. The remaining 8m utations increased tertiary interactions between adjacent motifs, thusi mproving the packing of the ribozyme structure and presumably favoring active conformations.
Saksmerpromee tal. discovered highly thermostablev ariants of the HH ribozyme. [155] Through in vitro selection,t wo groups of minimal HH ribozymes were isolated that exhibitedt rans catalytic activity at elevated temperatures due to strongt ertiary interactions between terminal loopsa nd internal bulges that strengthen ribozyme folding and ribozyme-substrate binding. High thermals tabilitym ay also be achieved without dedicated selection experiments: Vazquez-Tello et al. discovered that the SMa1H Hr ibozymef ound in the human parasite Schistosoma mansoni HH ribozyme is mosta ctive at % 70 8Ci n vitro without additional sequence optimizations. [156] Moreover, the same ribozyme can also be successfully cloned and expressed in the thermophile Thermus thermophilus where it catalyzes efficient cis-and trans-cleavage of mRNA in vivo at temperatures up to 80 8C. In this case, temperature modulates the rate limiting steps of the reaction:a t3 78C, catalysis is lim- Figure 6. Stability of (deoxy-)ribozymes with increasing temperature. In aqueouse nvironments, low and moderate temperatures support folding of typical secondaryand tertiary DNA and RNAstructures. Higher temperatures generally support the reversible melting and the resulting formation of unfolded single-strandedn ucleic acids. However,the individual melting points and pathways are strongly dependent on the overall number and strength of tertiary and secondary interactions, as well as the concentration of counter-ions.Generally,h ybridization of RNA is stronger than that of DNA. High temperatures also increase the rate of spontaneous and irreversible RNA backbonehydrolysis,which is typicallyn ot the case for DNA. ited by substrate dissociation, whereas at high temperature RNA degradation, ribozyme-substrate association, and secondary structure denaturation limit activity.
DNAzymes capable of high temperature catalysis have also been obtained by in vitro selection. Nelsone tal. selected a range of Zn 2 + -dependent RNA-cleaving DNAzymes with activity at 90 8C. [157] The selected sequences share little sequence similarityw ith other metal dependent DNAzymes, and only slightly enhancec leavage above background levels.I nterestingly,n os econdary structural features are predicted in the selected sequences at 90 8C, implying that the DNAzyme is capable of binding Zn 2 + and maintaining catalysis with minimal secondary structure.
These studies demonstrate that the catalysis of nucleic acids can be retained at elevated temperatures. Te mperature adaptation in ribozymes is generally achieved through additional RNA-RNAi nteractions stabilizing both the catalytically active conformation and RNA-substrate interactions, allowing activity to be sustained up to 80 8C. These adaptive mechanisms may generallya lso decrease the M 2 + dependency of nucleic acid folding and catalysis, which, in the case of RNA, helps to reduce degradation.M ore work investigating the stabilization of more primitive, short ribozyme systems is required to examine the range of temperaturest hat permitt he emergence or even self-replication of functional RNAsa ti ncreased temperatures. DNA is more resistant to degradation than RNA,s os elected DNAzymes can operate at up to 90 8Cb yr elying on metal cofactor binding rathert han the maintenance of aw ellfolded actives ite. It is as yet unknownw hether such systems are limited to simple reactions such as substrate cleavage.

Pressure as am odulator of nucleic acid catalysis
In addition to temperature and pH, hydrostatic pressure is also ap otentially importante nvironmental factor when considering oceanic or subterranean origins of life. High-pressure conditions are typically defined as 10 MPa or greater,c orresponding to aw ater depth of 1000 mo rm ore. 88 %o ft he volume of moderno ceans may be considered high pressure, with an average pressure of 38 MPa and am aximum on the abyssal plane of 110MPa. [158] Thus,a ny model of abiogenesis that includes deep-sea vents must account for hydrostatic pressure, which often has profound effects on biological systems by changingt he balance of intermolecular interactions. Longrange interactions such as Vand er Waals forces and salt bridges become weaker under compression, and shorter interactions such as hydrogen bonds are favored. Underp ressure, systemss hift towards low volumes tates in accordance with Le Chatelier's principle. In proteins, dissociation and unfolding is associated with al arge negative volume change (À30 to À110mLmol À1 ), whilst the DNA double helix dissociation hasa positive DV of 1-5 mL mol À1 . [159][160][161] This leads to dissociation and unfolding of protein systemsa sh ydrophobic surfaces become hydrated,b ut nucleic acid structures that are dependent on hydrogen bonding are stabilized. The double helical forms of DNA and RNA are typicallys tabilized by pressure, with ac oncomitant increasei nm elting temperature andn o major structuralc hanges other than slight structurald istortion due to compression of hydrogen bondingi nteractions. [162,163] The stabilizing effect is dependent on solutioni onics trength and T m ,w ith duplexest hat melt below 50 8Cb eing destabilized by pressure and those melt above 50 8Cb eing stabilized. [159] Certain non-canonical nucleic acids tructures, such as the DNA Gq uadruplex, exhibit negative DVsa nd melt under pressure. [161] RNA structures are also remarkably stable under high hydrostatic pressure:f ew structuralc hanges are observedi nt RNA Phe up to 1GPa. [164] Some RNA structures,s uch duplexes consisting of A-U base pairs, are slightly destabilized by pressure, and more critically the formationo ft ertiary interactions and docked conformationsr equired for ribozymec atalysis mayb e disfavored due to positive activation volumes. [165,166] Indeed, the observed rate of cleavage (k obs )a nd overall equilibrium constant of HP ribozyme self-cleavage decreases with increasing pressure. [166,167] However,d espite the overall retardation of the reaction, the actual self-cleavage step is accelerated by hydrostatic pressure and the decrease in rate is attributed to the positive activation volume of docking between catalytic loops. [168] The overall yields of RNA strand cleavage by certain hairpin (HH) ribozymes are improved by high hydrostatic pressure, whichc an even potentiate catalysis in the absence of the Mg 2 + typically requiredf or cleavage under ambient pressure. [169,170] Whilstt he hammerhead (HH) ribozyme also has a positivea ctivation volume associated with at ransitiont oa n active conformation (although significantly smaller than for HP ribozyme), no observable DV is associated with the cleavage reactionitself. [171] Molecular dynamics simulations have demonstratedt hat enhanced hydrogen bonding interactions in the core of the HP andH Hr ibozymes are responsible for an enhancement in the rate of cleavage under hydrostatic pressure. [172] The effect of hydrostatic pressure appearst oe xtend to deoxyribozymec atalysis:t he 10-23 DNAzyme was shown to be activeu nder pressure in the absence of magnesium, albeit with reduced overall yield. [169] These studies demonstrate that hydrostatic pressure can promote nucleic folding and compensate for al ack of magnesium in certain nucleic acid catalysts. The increasei nm elting temperature associated with pressurization could permit increasedr eactiont emperatures for weakly folding systems, and be used to avoid Mg 2 + -catalyzed degradation of RNA.W hen considering undersea environments, the resistance of nucleic acid to pressure-induced denaturationl ends support to an ucleic acid-based origin of life, especially when considering the drasticeffect of such conditions on protein folding.

Activity enhancement by freezing, evaporation and presence of organic solvents
Apart from the typical physicochemical parameters such as pressure, ionic conditions, pH and temperature described above,m ore exotic environmental conditions can strongly influence nucleic acid catalysis. An otable example is the extraordinary effect of dehydrating conditions on ribozyme and deox-

Freezingand dehydration induced ribozyme catalysis
The discovery that freezing or evaporation can enhanceo r even trigger ribozyme catalysis was serendipitous. The first reports of (undesired) HH ribozyme activitya ts ub-zero temperatures came from investigations of the autocatalyticp rocessing of dimeric tobacco ringspot virus satellite RNA (STobRV RNA) by Prody et al. [12] The authors reportedd ifficulties during longterm storageo fdimeric STobRV RNA duet os elf-processing into monomers during one week of storageo ft he RNA at À20 8Ca saprecipitate in 67 %e thanol. Similar observations of "unwelcome" RNA cleavage in hairpin ribozyme/yeast-mRNA constructs during repeated freezinga nd thawing were later also reported by Donahue and Fedor. [173] The first systematic investigationo ft his effect was carriedo ut in 1998 by Kazakov et al.,w ho reported efficient freezing-induced self-ligation of the hairpin (HP) ribozyme even in absence of divalentm etal ions such as Mg 2 + ,which are usually indispensable for catalysis in low-salt conditions. [174] Kazakov and his co-workersl ater expanded their work, and showedt hat alcohol-inducedd ehydration and simple evaporation also induced M 2 + -independent RNA ligation by HP ribozymes in both trans and in cis, while disfavoring the reverse cleavage reaction. [175][176][177] While divalent metal ions werei rrelevantf or the freezing-induced ligation, monovalenti ons had as trong impact on ligationy ields. In particular,s odium salts of acetate-phosphate-borate mixtures, EDTA, and acetate/LiCl led to increased ligation yields.
Af irst conjecture as to why monovalents alts are important for HP ribozyme catalysis under frozen conditions is provided by previouss tudies, which have shown that the absence of M 2 + can be compensated by high concentrations (> 1.5 m)o f monovalentc ations. [178] As already discussed above,s everalo f the small nucleolytic ribozymes such as the HP,H Ha nd VS ribozymesa re not obligate metalloenzymes (i.e. metal ions are not involved directly in catalysis) but rely on nucleotide-mediated generala cid base catalysis. M 2 + ions in dilute aqueous solution are still vital for tertiary RNA folding and stabilization of the active conformation. [179,180] The high concentrations of monovalentc ations required to substitute for divalent metal ions are readily availablei nt he aqueous phase of water-ice mixturesa tt emperatures above the eutectic point, in which the crystallization of nearly pure water crystals highly concentrates the remaining aqueous phase (Figure7). [181] The activation of the HP ribozyme by the high salt concentration in eutectic brine does not at first seem to explain the alcohol-induced activation of catalysis, since the typical alcohol concentrations used to trigger ribozyme catalysis are not sufficient to co-concentrate or precipitate monovalent counterions. [176,182] However, high concentrations of organic molecules such as primary alcohols or polyethers decrease the dielectric constanto ft he solvent, thereby strengthening cation-RNA interactions. [183,184] Thus, M 2 + -independentr ibozyme catalysis in presenceo fp rimary alcohols or poly(ethlyene glycol)( PEG) might, as in freezing, be at least partially due to the enhanced RNA-Na + interactions that can compensatef or the missing divalent metal ions. [176] Indeed, even under normal (aqueous) concentrations, ethanol at concentrations above 30 %s ignificantly increases the Mg 2 + -dependent activity of ribozymes and mitigates the effects of destabilizing mutations,a lthough higherl evels of ethanol in the presence of Mg 2 + diminishes this activity,p resumably due to RNA aggregation. [185][186][187] In addition to enhancing ion-ioni nteractions, dehydration induced by high levels of ethanol or PEG could also support ribozyme activity by promoting the formation of A-form helices (and therefore the catalytic loop structures of ribozymes defined by adjacent helical segments). [186,188] Kazakov et al. also reported that HP ribozyme-catalyzed ligation during evaporation is considerably improved by the presence of PEG, which had no impact on ligation under aqueous or frozen conditions or ethanol-induced ligation. The authors concluded that PEG might decrease the rate of evaporation, thereby extending the windows of partial dehydration where the water activity is still sufficient to allow hairpin ribozyme The left paneli llustrates adiluteaqueous system in an unfrozenstate. The right panels howsapartially frozen aqueous solution (e.g. ab inary NaCl-water systemc ontainingRNA)above the eutectic point. Solutes in the mother liquor( darkblue) are concentrated as al arge fraction of almost pure H 2 Oi s sequestered in the ice crystals (light blue).T his concentrationeffectl eads to ad ecreasedf reezing point of the mother liquora nd crystalg rowth stops when the equilibrium between the ice phase and the liquid phaseh as been reached.B )Illustratedv ariation in rate (dashed line) and ligation efficiency (solid line)oft he HP ribozyme( excess substrate concentration) in ap artially frozen, dilutebuffer solution (25 mm NaCl, 1mm Tris·HCl pH 7.5). [175] Both ligationr ate and yields are optimal between À4 8Ca nd À12 8C. At lower temperatures, the low thermal energy available in the system makes it difficult to surmount the activationbarrier for the reaction.A tt emperatures approaching 0 8C, meltingo ft he ice inactivatesh airpin ribozymec atalysis in absence of Mg 2 + .
catalysis. [189] The notion that at least some minimal hydration is required for HP ribozyme catalysis is also in agreement with the reports by Seyhan and Burge, who found that low but non-zerol evels of water activity are required for HP and HH ribozyme catalysis in dry RNA films. Intriguingly,h ydrated RNA films support cis and trans catalysis over ab road range of temperatures between À70 8Ca nd 37 8C( and probably above), which has potential implications for RNA catalysis under prebiotic conditions. [190] The formationo fa ctive ribozyme conformationsi nt he absence of divalent metal ions can be induced by conditions that promote electrostatic shielding and RNA compaction, such as partial dehydration,u p-concentration of monovalent cations, or reduced dielectric constant.F urthermore, the effective increase in RNA concentration during freezing facilitates RNA-RNA association, even from very stable monomeric structures, [191] and hasb een shown to induce the stretching and alignment of single stranded DNA,w hich in turn enables its adsorption onto av ariety of surfaces. [192] Freezing favors ligation in reversible transesterification reactions, even from highly fragmented ribozymes. [175,193,194] Freezing can enableh ighly thermodynamically disfavored reactions, such as ligationo fm onomeric 2',3 '-cyclicn ucleoside monophosphates to af ree 5' end of RNA. [195] While this reversal of exonucleolytic cleavage has an equilibrium constanto f % 2.2 m À1 under aqueous conditions (at 0 8C [196] ), it can be decreased % 20-foldb yf reezing to À9 8Ci nt he presence of 25 mm NaCl and 10 mm MgCl 2 ,e nablingq uantitative non-canonical 3'-5' nucleotidyltransferofR NA.
Both HP and HH ribozyme ligationy ields strongly benefit from repeated freeze-thaw (FT) cycling. [175,193,194] This effect can even be used to enablet he in trans assembly of long structured RNAs, such as the % 200 nt RNA polymerase ribozymes, from fragments between 20-30 nt. [193] The beneficial effects of FT cycles are likely the result of reducing the propensity of small ribozymes to form inactive or poorly active ribozymesubstrate complexes that attenuate bulk catalysis. Repeated freezing andt hawing leads to periodic disruption and re-formation of both active and unproductivec omplexes (in the absence or at low levels of M 2 + )t hereby providing unproductive complexes a"secondc hance" at catalysis.
Attwater et al. demonstrated the beneficial effectso fa frozen environment on strictly M 2 + -dependentr ibozymes such as the R18 RNA polymerase,w hichc atalyzes templated primer extension using nucleoside triphosphates. [197,198] The cold environmentc onsiderably extends the lifetime of the polymerase, whilst the concentratingp owero ff reezing above the eutectic temperature enables RNA polymerase activity even at extremely low (unfrozen) starting concentrationso fR NA,N TPs and Mg 2 + salts. The authors also investigated the impacts of different negative counter-ions to Mg 2 + ,a nd found that they markedly influence activity,p resumably duet ot heir influence on the eutectic freezing point, which dictates the concentrating effect of the eutectic brine. The ice microstructure has been shown to provide aq uasi-cellular compartmentalization enabling robustp henotype-genotype linkage, which is one of the key requirements for DarwinianE volution. [198] Indeed, this in-ice compartmentalization was later used by Attwater et al. to isolate ac old-adapted RNA polymerase ribozyme with considerably increased activity compared to ribozymes selected at ambient temperatures. [199] Recently,A ttwater et al. were also able to evolve an ice-adapted RNA trinucleotide polymerase ribozymet hat is able to copy itso wn 170 nt catalytic subunit via the ligationo fi ts almoste xclusively triplet-synthesized fragments. [200] 6.2. Freezingand dehydration induced deoxyribozyme catalysis Zhou et al reported the isolation of the DNAzyme EtNa ( Figure 8) from ar andom DNA library, which is specifically adaptedt oc atalyzeR NA cleavage in concentrated organic solvents containing only monovalentN a + . [201] EtNa showsarate enhancement of up to 1000-fold in 54 %e thanolc ompared to water in presence of 4mm NaCl, and is completely independent from divalent metal ions. The EtNa RNA cleavage rate can be directly used as ab iosensor for the precise measurement of alcohol levelsi ns pirits such as whisky or vodka. Interestingly, EtNa activity drastically decreases at ethanol concentrations beyond7 2% (v/v) ethanol, where the B-form helix of DNA is converted into the A-form that (in contrast to ribozymes) seems to be incompatible witht he formationo ft he active DNAzyme conformation. Given that EtNa shows cooperative binding of anda ctivation by Ca 2 + (in contrastt oM g 2 + ) [202] it can also be used as an ultrasensitive biosensor capable of de-tectingC a 2 + levels down to 1.4 mm Ca 2 + . [203] Eutectic freezing can also activate EtNa, while other DNAzymes that depend on divalent or trivalent metals are inhibited under these conditions. [204] This again highlights the interchangeability of freezing, organic dehydration or evaporation to achieve activation of metal-independent nucleic acid catalysts.

The potential of wet-dry cycles
The remarkable ability of dehydration to potentiate ribozyme function suggests that such conditions may have been important to the emergence of replicating RNA. Wet-dry cycles, per- Figure 8. Secondary structure of the EtNa DNAzyme. [201] The substrate strand is shown in yellow,with the ribonucleotide marked in red. The cleavage site is markedbya na rrow. haps driven by day-night cycles or geothermala ctivity on early Earth, have been proposed asp ossible drivers of the emergence of function. Viscous environments formed by water evaporation facilitate non-enzymatic RNA replication cycles slowing reannealing and therebyc ircumventing strand inhibition. [205] This effect was used by He et al. to form aH Hr ibozyme by the enzymaticl igation of short fragments, which was functional followingd ilution in water. [206] Wet-dry cycles can also be produced by the application of thermalg radients at an air-water interface (Figure 9). The resulting environment up-concentrates av ariety of components including RNA precursors and oligonucleotides, enabling a compelling variety of prebiotically important processes including precursor crystallization and phosphorylation. [207] Furthermore, the same environments ubstantially improves ribozyme catalysis ande ncapsulation within lipid vesicles.T he improved ribozyme catalysis is primarily the result of local high magnesium and RNA concentrationsa tt he air-water interface, but dehydration may also be significant.

Ultraviolet light
Exposure to UV radiation presents ac hallenge to the survival of prebiotic nucleic acids,a nd is often raised as am ajor problem in any RNA worlds cenario due to the elevated levels of surfaceU Vr adiation compared to the presentd ay. [208,209] Absorptiono fu ltraviolet photons by nucleobase aromaticr ings leads to an excited and highly reactivee lectronic state, which can give rise to chemical lesions such as adeninec ycloaddition to Ao rTin DNA, [210] as well as the formation of cyclobutane pyrimidine dimers in both DNA and RNA (Figure 9). [211] The effect of UV damage on nucleic acids has been investigated extensively (reviewed by Wurtmann and Wolin), [211] and UV-induced RNA-RNAc rosslinking is now an established methodf or characterizing tertiary or quaternary RNA structure. [212] Despite its deleterious effect of nucleic acids, ultraviolet radiation has been observed to promote prebiotic chemical reactions that yield ribonucleotides [213][214][215] and aminoa cids, [216] and has been proposed as ap ossible energy source to drive prebiotic chemistry on early Earth. [217] As such, UV radiation could provide an important link betweenp rebiotic chemistry and emergence of an RNA World, but only if radiation levelsr equired to drive such prebiotic reactions can be reconciled with nucleic acid stability under irradiation.K ey questionsa re:T o what degree can nucleic acid enzymes sustainp hotodamage and retain function?I si tp ossible for nucleic acid enzymes to adapt to strong UV environments?
Despite the well-documented exploration of UV-induced nucleic acidd amage, relativelyf ew insights are availabler egarding the role of UV exposure on functionalR NA (or other nucleic acid) enzymes. This may be in part due to ac omplex interplay between UV radiation and other factors influencing RNA catalysis, such as the presenceo fm etal ions. When exposed to UV radiation, tobacco mosaicv irus (TMV) RNA accumulates lesions in the form of uridine hydrates and pyrimidine dimers. However,i nt he presence of magnesium the rate of accumulation was approximately one-third than that in water,i mplying that folded RNA is more resistant to UV radiation damaget han the unfoldedr andom coil. [218] The influence of structure and conformation on nucleic acid UV sensitivity was further demonstrated by Kundu et al.,w ho reported an unexpected discrepancy between the UV sensitivities of dTdT dinucleotides in either RNA or DNA hairpins. [219] dTdT dinucleotides embedded in DNA hairpins, which typically adopt aB -form double strand,w ere susceptible to the formation of photolesions, whilst those in A-form RNA hairpins were protected from damage. The authors also demonstrated that the photosensitivity of the dTdT dinucleotides is modulated by sequence context, with the accumulation of dTdT lesionsr educed by neighbouring dA nucleotides, and almostc ompletely inhibited by neighbouringd Gn ucleotides. [219] It is fascinating that nucleic acids can gain UV resistance simply by adoptinga more compacth elical conformation, and the sequence dependence of UV photosensitivity suggestst hat adaptation of nucleic acids to strong UV environments could be possible. Despite this, it must be noted that the effect of UV exposure on functional RNA in vivo typically decreases function. [220][221][222] Recently,S aha and Chen monitored the function, folding, and kinetics of RNA aptamers that bind conditionally fluorescent ligandsi nv itro following UV induced photodamage. [223] One aptamer,S pinach2, retaineds ignificantl evels of fluorescence after UV exposure comparedt ot he malachite green aptamer.T his may be because al arge portion of the Spinach aptamer's binding site is comprised of ap hotostable G-quad- Figure 9. Schematic of ah eated rock pore. Thermal gradients at an airwater interface can result in an environmentwhich up-concentrates av ariety of components including ribozymes and ions. [207] The improved ribozyme catalysis is most likely the result of local high magnesiuma nd RNA concentrationsatt he interface. However,d irect dehydrationo ft he RNA at the temporallyd ried interfaceo nt he warm side (red) may also contribute to activity. Depending on the geometry of the system,e vaporated waterc ondenses at the cold side. The forming water droplets can fall back into the mother solution and wash off the dried components. This can lead to microscopic wetdry cycles.
ruplex. Single-stranded binding regions were found to be more UV sensitive,c onfirming that duplex formation is protective against UV radiation, [223,224] and that UV sensitivity significantly depends on folding and conformation. [219] While UV irradiation has been generally demonstrated to have ad etrimental on functional nucleic acids, some examples of UV-dependent nucleic acid catalysts have been reported. Chinnapen and Sen reportedt he in vitro selection of aD NAzyme with photolyase activity,U V1C, from ap ool of random sequences. [225] UV1C is capable of repairingd TdT dimers caused by UV exposure, and requires UV light to functioni na manner similart oe xtant protein photolyase enzymes ( Figure 10). The authors later demonstrated that aG -quadruplex neart he substrate bindings ite functions as both an antenna to absorb UV photonsa nd as an electron source for the repair reaction. [226] Intriguingly,aserotonin cofactor dependent photolyaseD NAzyme was later selected, which is able to repair both thymine and uracil dimers on ribose and deoxyribose backbones. [227] The discovery that nucleic acidsc an both harnessU Vr adiation and use this energy to repair photodamage is important, as it provides am echanism for early replicating systems to surviveh eavy UV irradiation on Early Earth. In the absence of such am echanism, early replicators would have to depend on environmental protectionf rom UV radiation, such as the protectivee ffect of montmorillonite clay particles, [228] or shielding by oceanicU Vabsorbers. [208]

Conclusion and perspectives
The activities of both ribozymes and deoxyribozymes are compatible with ab road range of potentially prebiotic conditions. Despite being less versatile and powerful than protein-based catalysis, nucleic acid catalysts are capable of escaping irreversible aggregation, while also tolerating or even benefiting from much harsher conditions such as freezing, drying or dehydration.M oreover,n ucleic acid catalysts often require only modest changes in their sequences to adapt to novel challenging conditions such as harsher pH values or higher temperatures, and can often tolerateo ra dapt to ab road range of different metal ion cofactors. These combined features make them ideal candidates fore arly biocatalysis, which presumably emerged andr emained functional outside the sheltereda nd constantm ilieu of the moderncell.
Despitet he large bodyo fr esearch, furthere xplorations of nucleic acid enzymesu nder prebioticc onditions may yield yet more unforeseen properties relevant for abiogenesis, andw arrant further investigation. For example, selection experiments under prebiotically plausible conditions beyonda queouss olutions in am odern oxygen-rich atmosphere could reveal further unexpected catalytic properties of ribozymes.I na ddition to the factors discussed in this review,o ther environmental factors such as mineral surfaces, [228,229,230] pH gradients, [231] high viscosities [206] or combination of variousd ifferent environments may furthere nhance the functional repertoire of early nucleic acids. For example, the clay montmorillonite inhibits HP ribozyme catalysis, but surfacea dsorption to this mineral offers protection against UV degradation. [228] Clay can also enhance recombination ribozymes and favor ligation by preferentially adsorbing longer RNA strands. [230] Furthermore, it is possible that heterogeneous complexes such as RNA/peptide complexeso rm ixed RNA/DNA (or alternative preRNA/preDNA) systems werei mportantf orerunners to modernb iochemistry,a nd allowed the catalysis of biochemical or replicative processes that "pure"R NA or DNA systemsa re presently incapable of. [5] Finally,i tr emains essential to further expand far-from-equilibrium scenarios to explore differents tages of molecular evolution (including nucleic acid catalysis) experimentally under heterogeneous conditions, such as the continuous provision of chemicalfuel and/or pH, temperature, or salinity cycles.