RNA dimer synthesis using montmorillonite as a catalyst: The role of surface layer charge

Applied Clay Science 83–84 (2013) 77–82

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Research paper

RNA dimer synthesis using montmorillonite as a catalyst: The role of surface layer charge Michael F. Aldersley ⁎, Prakash C. Joshi Department of Chemistry and Chemical Biology and the New York Center for Astrobiology, Rensselaer Polytechnic Institute, Troy, NY 12180, USA

a r t i c l e

i n f o

Article history: Received 9 July 2012 Received in revised form 31 May 2013 Accepted 2 August 2013 Available online 11 September 2013 Keywords: Montmorillonite Nucleoside Nucleotide Catalysis RNA Origin of life

a b s t r a c t The oligomerisation of activated nucleotides to form RNA is catalyzed by montmorillonite. However, the mechanism of this process and its use as a model for similar prebiotic chemistry is still under investigation. To date, amongst the more than two hundred clay minerals investigated as catalysts, only a few are excellent. We turned our attention to the less efficient catalysts to see how they performed in the synthesis of nucleotide dimers in which a nucleoside and an activated nucleotide produced only linear products of the form MpN. We have found that representative clay minerals, Otay and Chambers specifically, although poor at catalyzing the direct oligomerization of activated nucleotides, are able to promote these dimer syntheses. Sixteen reactions and thirty two products have been studied and found to vary in their outcome. The yields of 2′–5′ and 3′–5′-products were always less than those obtained with the excellent catalyst Volclay®. The results of this research provide a basis for further understanding of the physical processes in the mechanism of this catalysis and suggest that more clay minerals than hitherto expected may have the ability to catalyze RNA synthesis at the dimer level. These results have an important bearing upon the RNA world scenario for the origin of life. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Despite there currently being no definitive prebiotic route to RNA, it has been possible to generate long RNA oligomers by the montmorillonite-catalyzed reaction of activated monomers (Fig. 1) (Ferris, 2006). These reactions never occur with the naturally occurring minerals: we have reported the need to remove the interlayer metal cations and replace them with alkali metal and some alkaline earth cations before catalytic activity is observed (Aldersley at al., 2011; Joshi et al., 2009). This and other evidence (Aldersley et al., 2011) point strongly to the chemistry of the synthesis of RNA occurring within the interlayer spaces. Even so, not all montmorillonites are equally catalytic. We have introduced a classification scheme for the many types of clay tested (over 200) for their ability to catalyze RNA synthesis. The extent of catalytic activity is simply measured by the length of the oligomers formed from ImpA in a 3-day reaction as determined by HPLC: generating 7-mers or greater is designated as “excellent”, 4–7mers as “good” and less than 4-mers as “poor” (Joshi et al., 2009). Since the longer oligomers must necessarily start as dimers, we decided

Abbreviations: A, Adenosine; U, Uridine; G, Guanosine; C, Cytidine; AMP, Adenosine 5′-monophosphate; UMP, Uridine 5′-monophosphate; M, generic nucleoside; MpN, generic dimeric nucleotide; NMP, generic nucleoside 5′-monophosphate; NppN, generic pyrophosphate; ImpN, nucleoside 5′-phosphorimidazolide; RNA, ribonucleic acid; Huc, half unit cell. ⁎ Corresponding author. Tel.: +1 518 276 4080; fax: +1 518 276 4887. E-mail address: [email protected] (M.F. Aldersley). 0169-1317/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.clay.2013.08.009

to explore their synthesis (Joshi et al., 2012). Additionally, we sought to simplify the process of adsorption onto the clay minerals by exploiting the lack of charge on neutral nucleosides. Activated nucleotides would then be “quenched” by having the nucleoside present in excess. If the zwitterion of the activated nucleotide is the reacting species, then the overall combination of nucleoside plus activated nucleotide zwitterion is neutral. If the anion of the activated nucleotide is involved (Fig. 2), then the combination of reactants is negatively charged. This suggested that the reaction may, or may not, be independent of the charge upon the layer surface! Furthermore, the montmorillonites to be tested were from the “poor” catalyst group. Their catalysis of the oligomerization of activated nucleotides had already been investigated (Joshi et al., 2009). Our findings and those of others (Joyce and Orgel, 2006) have already led to the general conclusion that the RNA world could have been initiated by the mineral and metal ion catalyzed reactions on the early Earth and there has already been the identification of a relatively short length of RNA having catalytic properties (Turk et al., 2010). Thus, short RNA synthesis studies are important in understanding how functional RNA may be synthesized under pre-biotic conditions. However, these studies remain as merely a model since it is not known currently whether montmorillonites or nucleotides were present on the early Earth. The recent identification of phyllosilicates, including montmorillonite, on Mars (Bishop et al., 2008) raises the possibility that all the processes described may have taken place there too; even if the Martian clay minerals were “poor” catalysts, they may have possessed an ability to catalyze the synthesis of short RNA species, as we are proposing may have occurred on the early Earth. The synthesis of

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M.F. Aldersley, P.C. Joshi / Applied Clay Science 83–84 (2013) 77–82

these small nucleotides, as well as long oligomers, and the mechanism by which montmorillonite catalyzes the formation of the RNA remain central to our investigations: thus, • The investigation of “poor catalysts” for dimer synthesis may provide results that suggest a more widespread availability of short nucleotides in the prebiotic environment than hitherto. This is a requirement of the RNA world hypothesis (Coveney et al., 2012). • The clay catalyst provides both acidic and basic contributions to the catalytic process (Fig. 2) (Aldersley at al., 2011).

the corresponding NMP formed by hydrolysis. Standard nucleoside solutions (20 mM) were prepared in sodium chloride (0.2 M) and magnesium chloride (0.075 M). Reactions were also tried with a higher concentration of the nucleoside (up to 200 mM) but there was no appreciable improvement in the yields of dimers from these reactions. It is most important to recognize the contrast between these methods which produce only linear dimers of the general formula MpN in contrast with the oligomerization of ImpN in which cyclic dimers are a major product together with dimers pNpN, and longer oligomers.

2. Experimental

2.5. High performance liquid chromatography (HPLC)

2.1. General

HPLC analysis was performed using a Hitachi L-7000 HPLC system, operating at 260 nm with a diode array detector. The analytical separation of individual dimers was performed on a reverse phase Alltima C-18, 5 μ (4.6 mm × 250 mm) column (Alltech, Grace Davison) using a gradient system of 0.2% formic acid and 30% acetonitrile in water with 0.2% formic acid (1%/min). Dimer products MpN could readily be identified by their relatively long retention times compared with dimers, pNpN, and trimers, pNpNpN, from direct oligomerisation reactions of ImpN. In our work, for a given set of dimers MpN, the 2′–5′ isomer always elutes faster than the 3′–5′ isomer irrespective of the type of reverse phase column employed and the components used in the gradient systems. That the 2′–5′-linked dimers were eluted first in a reverse phase HPLC column is an empirically derived result based on every RNaseT2 enzymatic hydrolysis reaction studied: the 2′–5′-linked dimer is unaffected whereas the 3′–5′-linked product is hydrolyzed to 3′-Mp and the nucleoside N (Joshi et al., 2012).

The general methodology has already been reported (Joshi et al., 2009). Metal salts and nucleosides (Sigma) were of the highest analytical quality available. 2.2. Preparation of montmorillonite at pH 7 The montmorillonites used here are derived from Volclay®, Otay and Chambers types: the first was donated by the American Colloid Company. The other two were H-23 bentonite (Chambers, AZ) and H-24 bentonite (Otay, CA), both obtained from Ward's Natural Science Establishment, Inc., Rochester, NY, via the Department of Earth and Atmospheric Sciences at Rensselaer Polytechnic Institute. For simplicity, they are referred to throughout this work as Volclay, Otay and Chambers. Detailed technical and analytical information, both before and after refining, is available upon request. From these raw materials, H+-montmorillonite samples were prepared as described previously (Joshi et al., 2009). To provide montmorillonite samples at pH 7, 1 g of H+-montmorillonite was dispersed in 100 mL of water and was titrated with 0.02 M aqueous sodium hydroxide. At pH 7, the titration was terminated, the sample was centrifuged, the supernatant discarded and the montmorillonite was freeze-dried. 2.3. Preparation of activated nucleotides (ImpN) The phosphorimidazolides of the 5′-nucleotides (Fig. 1) were prepared as described previously (Prabahar et al., 1994). The purities of all of the ImpN as determined by reverse phase HPLC were greater than 99.5%. 2.4. Montmorillonite-catalyzed reaction of a nucleoside and an activated nucleotide

2.6. Isotherm determinations: Binding of activated monomers to montmorillonite (Joshi et al., JACS, 2009) Stock solutions of ImpN (150 mM) were prepared in a magnesium chloride (0.075 M) and sodium chloride (0.2 M) mixture at 4 °C. The solution was filtered through a 0.2-μm-nylon syringe filter (Alltech Associates, Inc., Deerfield, IL) and diluted to the desired molarity (10 mM–140 mM) in the same reagent. For adsorption isotherm measurements, the binding of ImpN (200 μL) to the montmorillonite (10 mg) was determined after incubating the solution at 4 °C for 60 min. After the 60 min incubation, the sample was centrifuged at 13,200 rpm (10 min) and the supernatant was analyzed by HPLC on an Alltima C-18 column. No oligomer formation or hydrolysis was ever detected in these binding studies under these conditions. The stock solution that had not been mixed with montmorillonite was also analyzed by HPLC and the difference in the peak areas was

These experiments at pH 7 and their analysis were similar to that already reported (Joshi et al., 2009, 2012). Experiments and controls were carried out with separate montmorillonite samples (Volclay, Otay and Chambers) that had been titrated to pH 7. Each combination of nucleoside M and an activated nucleotide ImpN over 3 days provides two products, the 2′–5′-linked and the 3′–′5′-linked MpN together with

Fig. 1. Anion of nucleotide-5′-phosphorimidazolide (ImpN, base = Adenine, Uracil, Guanine, Cytosine).

Fig. 2. Phosphodiester bond formation from a nucleoside and an activated nucleotide on montmorillonite with general base/acid catalysis. Metal cations have been omitted for clarity.

M.F. Aldersley, P.C. Joshi / Applied Clay Science 83–84 (2013) 77–82

79

Table 1 Yields of MpN with Volclay, Otay, Chambers and no montmorillonite. For a description of Q, see Section 3.2 n/d = not detected.

Target MpN

Nucleoside

D,D-ApA D,D-ApU D,D-ApC D,D-ApG D,D-UpA D,D-UpU D,D-UpC D,D-UpG D,D-CpA D,D-CpU D,D-CpC D,D-CpG D,D-GpA D,D-GpU D,D-GpC D,D-GpG

D-Adenosine D-Adenosine D-Adenosine D-Adenosine D-Uridine D-Uridine D-Uridine D-Uridine D-Cytidine D-Cytidine D-Cytidine D-Cytidine D-Guanosine D-Guanosine D-Guanosine D-Guanosine

Volclay

Otay

Chambers

Q

0.27

0.43

0.45

ImpN

%

D-ImpA D-ImpU D-ImpC D-ImpG D-ImpA D-ImpU D-ImpC D-ImpG D-ImpA D-ImpU D-ImpC D-ImpG D-ImpA D-ImpU D-ImpC D-ImpG

%

No clay

%

%

2′–5′

3′–5′

2′–5′

3′–5′

2′–5′

3′–5′

2′–5′

3′–5′

32 30 51 4.8 34 46 53 8.3 40 46 46 6.1 19 38 36 5.0

32 16 22 6.1 27 21 22 15 29 15 22 8.0 13 18 18 4.8

3.5 17 3.6 0.87 17 27 1.2 1.0 4.6 0.12 14 0.74 0.23 0.89 1.1 n/d

0.8 2.6 1.4 0.58 2.6 9.4 0.58 0.89 0.42 0.03 3.0 0.10 0.07 0.16 n/d n/d

4.0 12 3.8 1.3 12 25 3.0 0.27 7.3 0.49 16 0.77 0.73 1.5 0.86 n/d

2.4 2.9 0.7 0.81 2.9 13 1.8 0.25 0.31 0.02 3.9 n/d 0.34 0.09 n/d n/d

n/d 4.5 1.1 n/d 1.6 10 0.86 n/d 2.0 0.67 2.0 n/d 0.60 0.64 0.15 0.68

n/d 0.4 0.27 n/d 0.2 2.4 0.62 n/d 2.0 0.41 0.88 n/d 0.42 0.26 0.28 0.27

used to measure the extent of binding. The sets of data were analyzed using a Langmuir isotherm model although other isotherms have been used for comparison purposes. The equilibrium under analysis is that between the activated nucleotide in aqueous solution and the same nucleotide species adsorbed onto the clay surface: viz. Let:

This molar quantity equates to N0/Mr unit cells where N0 is the Avogadro's number. In a typical titration, let V be the volume (in L) required of a solution of molarity M of sodium hydroxide to titrate the sample to pH 7.

1. KL be the Langmuir equilibrium isotherm constant. 2. a be the amount of activated nucleotide adsorbed on the 10 mg of clay, equivalent to a measure of [ImpN (clay)], i.e. the quantity of activated nucleotide adsorbed upon the clay. 3. as be the saturation level of nucleotide adsorbed on the 10 mg sample of clay, equivalent to a measure of [ImpN (clay)]max. 4. c be the concentration of activated nucleotide in solution, [ImpN (aq)].

Number of sodium ions added at the end point ¼ V  M  N0

Then at equilibrium, K L ¼ a=cðas −aÞ: This expression can be rearranged to give:

Moles of sodium ions added at the end point ¼ V  M moles

Charge on the sodium ions added ¼ V  M  N0  e: Charge added from the sodium ions balances the charge on the clay mineral sample: generally the value of e is ignored and put equal to 1. So; charge per unit cell ¼ V  M  N0 =ðN0 =Mr Þ ¼ V  M  Mr So; Q; the charge per huc ¼ 0:5  V  M  Mr : For example, for V = 0.025 L (25 mL) and M = 0.05 mol/L, we can estimate the value of Q:

a=c ¼ K L as −K L a

Q ¼ 0:5  0:025  0:05  549

whence a plot of a/c versus a gives a line of slope-KL and intercept KL as, from which the value of as can be obtained. A further rearrangement of the expression yields:

Q ¼ 0:34:

a ¼ cK L as =ð1 þ cK L Þ:

3. Results and discussion 3.1. General results and trends

This enables a theoretical curve of the adsorption to be drawn for each data set for which KL and as are now known. When necessary, numerical methods were used to minimize the residuals existing between the theoretical curve and the curve obtained by analysis of the experimental dataset. The final correlation between the calculated points and the experimental data was always better than 0.95.

The results of the synthesis of 2′–5′ and 3′–5′ isomers of the sixteen nucleotides, MpN (where M, N = A, U, G, C) upon Otay, Chambers and Volclay are shown in Table 1. The yields of the corresponding dimers in Table 2 Values of KL and as from binding studies at 4 °C (Joshi et al., 2009).

2.7. From titration data to layer charge An estimate of the charge, Q, per huc can be determined by using titration data. Consider 1 g of the acid clay. This equals 1/Mr moles of unit cells of the clay where Mr is the molar mass of the unit cell (~549, http:// webmineral.com/data/Montmorillonite.shtml).

KL as KL as KL as

ImpA ImpA ImpU ImpU ImpC ImpC

Otay

Chambers

Volclay

Units

101 7.91 × 10−07 36 1.39 × 10−07 142 1.71 × 10−06

87 1.19 × 10−06 54 1.67 × 10−07 121 2.08 × 10−06

137 4.02 × 10−06 50 1.18 × 10−06 73 2.51 × 10−06

M-1 μmol/10 mg clay M−1 μmol/10 mg clay M−1 μmol/10 mg clay

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M.F. Aldersley, P.C. Joshi / Applied Clay Science 83–84 (2013) 77–82

control experiments from which the montmorillonite was absent are also shown in Table 1. In all cases, the remainder was an NMP, formed by hydrolysis of the related ImpN together with traces of NppN (b1%). As more protons locally become available from the hydrolysis product NMP, they can neutralize anionic species within the clay. These new acidic centers can assist in protonating the imidazolide so rendering imidazole a better leaving group (Aldersley et al., 2011). Clearly, more hydrolysis then occurs with these poor catalysts than with the excellent catalyst Volclay and the changes discussed above are consistent with the formation of traces of NppN since the local environment is made marginally more acidic (Aldersley et al., 2011). At pH 7 and 4°C, the activated nucleotides generally do not bind so extensively to the “poor” catalysts as they do to Volclay (Table 2) (Joshi et al., 2009) so that the material remaining in solution can also hydrolyze rather than react with the nucleoside on the montmorillonite. It is also clear that in some instances, the poor catalysts are inhibiting dimer formation and/or encouraging hydrolysis since the yields in the

absence of montmorillonite are higher than those with the poor catalyst (Table 1). Detectable dimer in some instances formed in the absence of the catalysts, most notably in the synthesis of UpU isomers. Thus, it is now clear that these poor catalysts, Otay and Chambers, do indeed catalyze dimer formation. The outcome of the reaction is different: a b c d e

for the sixteen reactions studied for the 2′–5′ and 3′–5′ products for each dimer synthesis for each of the two poor catalysts for Volclay compared with either of the poor catalysts in the absence of any clay mineral catalyst.

3.2. Effect of concentration of nucleoside Yields of dimers were virtually unchanged when different concentrations of the nucleosides were used, or when the clay mineral samples were shaken for prolonged periods (up to 4 h) with nucleoside before

Yields and Surface Layer Charge

Yields and Surface Layer Charge 80

Percentage Yield

80

a

70 60

60

2'5'ApA

50

10 0 0

0.1

0.2

0.3

0.4

0.5

Percentage Yield

80

0

0.1

0.2

0.3

0.4

0.5

80

c

70 60

60

2'5'CpA

50

2'5'GpC

30

2'5'CpG

20

2'5'GpU

40

2'5'CpC

30

2'5'GpA

50

2'5'CpU

40

d

70

2'5'GpG

20

10

10

0

0 0

0.1

0.2

0.3

0.4

0.5

80

Percentage Yield

2'5'UpG

20

10 0

0

0.1

0.2

0.3

0.4

0.5

80

e

70 60

60

3'5'ApA

50

3'5'UpC

30

3'5'ApG

20

3'5'UpU

40

3'5'ApC

30

3'5'UpA

50

3'5'ApU

40

f

70

3'5'UpG

20

10

10

0

0 0

0.1

0.2

0.3

0.4

0.5

80

Percentage Yield

2'5'UpC

30

2'5'ApG

20

2'5'UpU

40

2'5'ApC

30

2'5'UpA

50

2'5'ApU

40

b

70

0

0.1

0.2

0.3

0.4

0.5

80

g

70 60

60

3'5'CpA

50

3'5'GpC

30

3'5'CpG

20

3'5'GpU

40

3'5'CpC

30

3'5'GpA

50

3'5'CpU

40

h

70

3'5'GpG

20

10

10

0

0 0

0.1

0.2

0.3

Surface Layer Charge

0.4

0.5

0

0.1

0.2

0.3

0.4

0.5

Surface Layer Charge

Fig. 3. Dimer yields using data from Table 1. ◊ extrapolation to clay of smaller interlayer charge. a–d are the plots for 2′–5′ products from A, U, G, and C respectively with ImpN (N = A, U, G, C). e–h are the plots for 3′–5′ products from A, U, G, and C respectively with ImpN (N = A, U, G, C).

M.F. Aldersley, P.C. Joshi / Applied Clay Science 83–84 (2013) 77–82

81

addition of the activated nucleotide. The yields seem to be independent of the nucleoside and activated nucleoside concentrations so far studied when the ratios are approximately 2:1.

160

120

Q v KL ImpA

3.3. Mechanism for dimer formation

100

Q v KL ImpU

KL

140

The mechanism for the formation of a ribonucleotide dimer, MpN, from a nucleoside, M, and an activated nucleotide, ImpN, is very similar to that for dimer formation directly from ImpN (Aldersley et al., 2011). The clay must supply a proton to facilitate the imidazole acting as a leaving group and the presence of anionic sites on the clay surface can act a general bases (Jencks, 1972a, 1972b) to enhance the nucleophilic character of the nucleoside \OH groups (Fig. 2). By considering the volume of sodium hydroxide needed to bring 1 g of each of the acidic montmorillonite samples to pH 7, it is of course possible to estimate the charge per unit cell, and the equivalent surface layer charge, Q, of the montmorillonites (at pH 7) used in this work (Table 4). We have already shown that the enhanced charge on a clay surface can prevent activated nucleotide anions approaching one another closely enough to react (Aldersley et al., 2011). Such a problem is obviated here since one of the reactants is neutral. However, it is still clear from this work that a diminished yield of products has accompanied the increased surface layer charge on the Otay and Chamber clays compared with Volclay (a Wyoming type clay) (Table 4). All of the montmorillonites that are “poor” catalysts in our work (over 150!) need further evaluation in terms of their ability to catalyze dimer synthesis in the light of these latest findings. Conversely, a clay mineral with a smaller surface layer charge, let's arbitrarily say 0.18, that is smaller than that of Volclay (the exemplar 0.18 is shown as a dotted vertical line in Fig. 3a–h) may be expected to give even higher yields of dimers since it is anticipated that the yields and catalytic efficiencies will parallel the huc charge as they do in this small pilot study. Trend lines for this concept using the sets of dimers synthesized and studied here are shown in Fig. 3a–h. The search for a montmorillonite with just such a charge may be rewarding in the green synthesis of oligonucleotides (Joshi et al., 2012). Even larger surface layer charges than those present in Otay and Chambers would clearly produce vanishingly small yields of all dimers. Additionally, the new concept of fundamental particle charge (FPC) (Pratikakis et al., 2010) may prove to be a better tool for the comparison of clays in their ability to promote dimer synthesis as it has already been shown to be useful in a further understanding of the swelling behavior and viscosity of smectites. It will additionally assist in understanding the hydrophobicity of the montmorillonite/smectite surfaces and their tendency for reactions with hydrophobic molecules as well as contributing to an understanding of the process of delamination during formation of organophilic smectites. FPC is currently both novel and a topic of on-going research and collaboration.

Q v KL ImpC

80 60 40 20 0 0

0.1

0.2

0.3

0.4

0.5

Surface Layer charge, Q Fig. 4. Surface layer charge and Langmuir KL values: using data from Table 1.

addressed using mass spectrometric data suggesting G-quadruplex species that cannot readily be adsorbed into the montmorillonite interlayer spaces (Joshi et al., 2012). Reactions involving guanosine work well, notwithstanding its propensity to form gels (Yu et al., 2008), and this is further evidence that it is the adsorption of the activated nucleotide that is critical in these syntheses. 3.5. Purine–Purine, Purine–Pyrimidine and Pyrimidine–Pyrimidine combinations The possible purine–pyrimidine combinations of the associated nucleosides, even when experiments involving ImpG are included and demonstrate selectivity differences (Table 3) for the synthesis of the dimers MpN, where in Table 3, Nucleoside 1 = M and Nucleoside 2 = N. This has also been observed in the co-oligomerization of activated nucleotides (Miyakawa and Ferris, 2003). That the pyrimidine-base nucleosides are so poorly bound to the montmorillonite yet provide the best average yields of dimers may suggest that they rapidly adsorb and desorb compared with their purine counterparts. Conversely, the slightly lower average yields whenever a purine is involved (excluding that derived from ImpG) possibly reflect the stronger binding of purine species. Thus, a very subtle and complex interaction involving the structures of the nucleosides, the structures of their activated nucleotide counterparts and the charge on the layer surface is taking place throughout this study. 3.6. Cyclic nucleotide formation The dimer synthesis presented here has the advantage of simplicity over the oligomerization of the activated nucleotides, ImpN. For example, the oligomerization reaction provides cyclic nucleotides,

3.4. Reactions involving ImpG 1.00E-05

Table 3 Nucleoside base combinations encountered in this study: average yields derived from Table 1. Volclay

Otay

Chambers

No clay

%

%

%

%

Nucleoside 1 Nucleoside 2 2′–5′ 3′–5′ 2′–5′ 3′–5′ 2′–5′ 3′–5′ 2′–5′ 3′–5′ Purine Purine Purine Pyrimidine Pyrimidine Pyrimidine

Purine Purine ≠ G Pyrimidine Purine Purine ≠ G Pyrimidine

15 26 39 22 37 48

14 23 19 20 28 20

1.5 1.8 5.5 5.7 11 10

0.5 0.4 1.4 1.0 1.5 3.3

2.0 2.4 4.5 5.1 9.7 11

1.2 1.4 1.2 1.2 1.6 4.6

0.6 0.6 1.6 1.8 1.8 3.5

0.3 0.4 0.3 1.1 1.1 1.1

as µmole/10mg clay

Experiments involving ImpG (Tables 1 and 3) give lower yields in general than the other experiments. This issue has been extensively

Q v as ImpA Q v as ImpU

1.00E-06

Q v as ImpC

1.00E-07

0

0.1

0.2

0.3

0.4

0.5

Surface Layer charge, Q Fig. 5. Surface layer charge and maximum surface coverage: using data from Table 1.

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Table 4 Types of selected montmorillonites and their surface layer charge (Joshi et al., 2009). Clay type

Surface charge range, Q

Mean

Q, this work at pH 7

Volclay Otay Chambers

0.66–0.84 0.84–0.97 0.85–0.98

0.76 0.91 0.94

0.27 0.43 0.45

(pN)2, in relatively high yield. The cyclic compounds bind very strongly to the catalyst so that catalytic activity is curtailed and indeed, an extraction is needed to completely remove the cyclic products for analysis (Joshi et al., 2009). No analytical evidence for the formation of cyclic dimers has so far been found in the initial reaction of a nucleoside and an activated nucleotide. Indeed, there is no additional rational pathway for the formation of cyclic dimers with this particular chemistry. The cyclic dimer formation reaction is totally suppressed under these conditions. 3.7. Isotherm studies and their trend Langmuir isotherm studies for ImpA, ImpU and ImpC at pH 7 and 4 °C have been summarized in Table 2: an analogous study with ImpG is confounded by the formation of a thixotropic gel. The trend lines in the Figs. 4 and 5 between the KL and as values together with the corresponding surface layer charge serve to show the sensitivity of the adsorption to the structure of the activated nucleotide as well as the surface layer charges on the clays. We have extrapolated these isotherm values onto an as yet unknown clay sample with the lower surface layer charge of 0.18, Figs. 4 and 5, as with the dimer yields. The upward trend of the lines for KL from ImpU and ImpA is not matched by the behavior of the trend line for ImpC. Furthermore, these isotherm parameters are sensitive to pH for all the activated nucleotides in this study (Aldersley et al., 2011). Overall then, it can be predicted that this as yet unknown clay would catalyze oligomer formation from each of the ImpN (N = A, U, C) based upon the Langmuir isotherm extrapolations and additionally, catalyze more effectively the synthesis of dimers MpN (M, N = A, U, G, C) based upon the yield extrapolations. 4. Conclusions Having questioned the ability of certain montmorillonites to catalyze nucleotide dimer synthesis, the results presented show that these “poor” catalysts, Otay and Chambers, do indeed catalyze the synthesis of RNA dimers albeit with differing degrees of efficiency in the sixteen reactions and thirty two products reported. We now have a proof of concept that the poor catalysts can assist in the synthesis of shorter nucleotides, which, in turn, leads inevitably to the possibility that a larger quantity of shorter oligonucleotides could have been formed in a vast number of prebiotic locations. In contrast, a much smaller quantity of longer oligonucleotides would be formed in very few places. This follows simply and directly from our empirical finding of there being relatively few excellent catalysts compared with the very many poor catalysts. Consequently, the development of an RNA world scenario arising from these short oligomers is demonstrably more plausible, even though we are using only a model system. This alternative model exploits the otherwise non- or poor catalysts (Joshi

et al., 2009, Table 4) involving adsorption of a nucleoside onto the montmorillonite followed by addition of an activated nucleotide to the mixture. In this way, the nucleoside quenches the activated nucleotide and simple dimer MpN result (Joshi et al., 2012). This dimer synthesis completely eliminates cyclic dimers which cannot elongate further (Joshi et al., 2013), a vital consideration if larger quantities of longer oligomers are required. We are now in a position to use the same dimer synthesis reactions, incorporating as they do typical purine residues and typical pyrimidine residues, as probes of montmorillonite surfaces to determine even more specifically how the reactions are occurring. We intend future work on the “poor” catalyst collection that will extend our knowledge on the adsorption of nucleosides and activated nucleotides upon montmorillonite with the link to the charges upon the montmorillonite surfaces by way of this dimer synthesis approach. In view of the trends displayed in this work, a corollary suggests that a montmorillonite sample with a lesser surface layer charge than Volclay may be a superior catalyst in many if not all of these syntheses. Acknowledgments This research was supported by NASA Astrobiology Institute grant NNA09DA80A. References Aldersley, Michael F., Joshi, Prakash C., Price, Jonathan D., Ferris, James P., 2011. The role of montmorillonite in its catalysis of RNA synthesis. Appl. Clay Sci. 54 (1), 1–14. Bishop, Janice L., Dobrea, Eldar Z. Noe, McKeown, Nancy K., Parente, Mario, Ehlmann, Bethany L., Michalski, Joseph R., Milliken, Ralph E., Poulet, Francois, Swayze, Gregg A., Mustard, John F., Murchie, Scott L., Bibring, JeanPierre, 2008. Phyllosilicate diversity and past aqueous activity revealed at Mawrth Vallis, Mars. Science 321, 830–833 (Washington, DC, U.S.). Coveney, Peter V., Swadling, Jacob B., Wattis, Jonathan A.D., Greenwell, H. Christopher, 2012. Theory, modelling and simulation in origins of life studies. Chem. Soc. Rev. 41, 5430–5446. Ferris, James P., 2006. Montmorillonite-catalyzed formation of RNA oligomers: the possible role of catalysis in the origins of life. Phil. Trans. R. Soc. B 361, 1777–1786. Jencks, William P., 1972a. Requirements for general acid-base catalysis of complex reactions. J. Am. Chem. Soc. 94, 4731–4732. Jencks, William P., 1972b. General acid-base catalysis of complex reactions in water. Chem. Rev. 72, 705–718 (Washington DC, U.S.). Joshi, Prakash C., Aldersley, Michael F., Delano, John W., Ferris, James P., 2009. Mechanism of montmorillonite catalysis in the formation of RNA oligomers. J. Am. Chem. Soc. 131 (37), 13369–13374. Joshi, Prakash C., Aldersley, Michael F., Zagorevskii, Dmitri V., Ferris, James P., 2012. A nucleotide dimer synthesis without protecting groups using montmorillonite as catalyst. Nucleosides Nucleotides Nucleic Acids 31, 1–32. Joshi, Prakash C., Aldersley, Michael F., Ferris, James P., 2013. Progress in demonstrating homochiral selection in prebiotic RNA synthesis. Adv. Space Res. 51, 772–779. Joyce, Gerald F., Orgel, Leslie E., 2006. Progress towards understanding the origin of the RNA world, Cold Spring Harbor Monograph Series (2006), 43 (RNA World), 3rd edition. 23–56. Miyakawa, Shin, Ferris, James P., 2003. Sequence- and regioselectivity in the montmorillonite-catalyzed synthesis of RNA. J. Am. Chem. Soc. 125, 8202–8208. Prabahar, K. Joseph, Cole Timothy, D., Ferris, James P., 1994. Effect of phosphate activating group on oligonucleotide formation on montmorillonite: the regioselective formation of 3′,5′-linked oligoadenylates. J. Am. Chem. Soc. 116, 10914–10920. Pratikakis, A., Christidis, George E., Villieras, F., Michot, L., 2010. Fundamental particle charge and its significance. SEA-CSSJ-CMS Trilateral Meeting on Clays. Seville, Spain, 8-10 June 2010. Book of Abstracts. 154–155. Turk, Rebecca M., Chumachenko, Nataliya V., Yarus, Michael, 2010. Multiple translational products from a five-nucleotide ribozyme. Proc. Natl. Acad. Sci. U. S. A. 107, 4585–4589. Yu, Yuehua, Nakamura, Darren, DeBoyace, Kevin, Neisius, Adam W., McGown, Linda B., 2008. Tunable thermoassociation of binary guanosine gels. J. Phys. Chem. B 112 (4), 1130–1134.