Opal Adsorbs and Stabilizes RNA – A Hierarchy of Prebiotic Silica Minerals

Abstract

A widely held ‘RNA first’ model proposes that RNA gave organic matter on Earth its first access to Darwinism. Such a proposal, which requires a mechanism to generate RNA from a prebiotic ‘soup’, must also manage the intrinsic instability of any RNA so formed. Here, we show that silicon dioxide (silica, SiO₂), in the form of synthetic opal, adsorbs and stabilizes RNA from aqueous solution. The extent of adsorption on fully amorphous silica is less, as is the extent of adsorption on the surface of crystalline quartz. We show that the RNA adsorbed on opal is considerably more stable than the same RNA molecule free in aqueous solution at pH 9.5. This provides a mechanism by which any RNA formed in a prebiotic environment could have been concentrated and stabilized so that it could have later participated in the first Darwinian biology.

Key words

prebiotic chemistry - RNA - silica - opal - amorphous silica

• References and Notes

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• 16 Experimental Section The 83 nt long single-stranded RNA molecule use in these experiments had the following sequence: 5′-CGCUGUACGCAACACAAGGCUUAUGGUGUAUCCUCCUGGAUC ACGUGUGGU ACGUACUGUCCGAUUAUUUCUAAUCGGGAUAC-3′ The amplifications were achieved with the following primers, forward and reverse: 40NLib-Rev: 5′-GTATCCCGATTAGAAATAATCGG-3′ 40NLib-Fwd: 5′-TGCTAATACGACTCACTATAGGGCGCTGTACGCAACAC-3′ RNA Preparation The 83 nt single-stranded PNA molecules were prepared by T7 in vitro transcription from a synthetic DNA template that had the corresponding sequence placed after a T7 RNA polymerase promoter. The RNA was purified by PAGE and EtOH precipitated. For ³²P-5′-labeling, 10 pmol RNA were dephosphorylated with calf intestinal phosphatase (CIP, New England Biolabs) and radioactive phosphate added back with T4 polynuclotide kinase (PNK, New England Biolabs). RNA was finally purified with Qiagen nucleotide removal kit. Final ³²P-labeled RNA concentration was ca. 0.1 μM. Synthesis of SiO₂ Minerals The samples were made with a modified Stöber process,¹⁷ by which spheres of amorphous silica approximately 300 nm across are formed by precipitation. The heavily hydrated spheres have a large amount of hydroxyl (OH) groups, and can be fabricated by compression into rod-like structures (sample C) with average pores of 96 Å, and 129.7 m²/g specific surface area as measured by nitrogen BET with a NOVA-2200 instrument (Quantachrome). To make natural-like opal (samples A and B), spheres are centrifuged, re-dispersed in ultrapure water, and allowed to settle for up to 3 months. This allows them to form a packed crystal structure, which gives the Bragg diffraction typical in opals. The sample is dried and heat-treated over 1000 °C to give it cohesive strength. This material is ca. 26% porous with fully interconnected pores and average pore size of 780 Å.¹⁸ Before use, the materials were washed with H₂O₂ (30%), then MilliQ water, then absolute EtOH, and then air dried at r.t. Macroscopic surface areas were determined by microscopy, and the area of the defined faces determined by GeneSnap (Syngene). Adsorption on SiO₂ 5′-Labeled RNA (0.5 nM, 100 fmoles RNA total) in mQ-H₂O (200 μL, Millipore) was allowed to interact with each mineral piece by tumbling in 1.5 mL tubes for 45 min at r.t. After incubation, supernatants were transferred to clean 1.5 mL tubes; each piece was washed with 500 μL mQ-H₂O for 5′ (wash 1), or overnight at 4 °C (wash 2). Sample B was also subjected to a third wash with 100 mM aq NaF (pH 9.5) for 5 min, and a fourth wash in aq NaF for 2 d. After each, supernatants were transferred to new tubes. All fractions (including minerals) were read by Cherenkov countering. Fractions bound were calculated as cpm remaining in the pieces divided by total cpm. Reverse Transcription and PCR Amplification Reverse transcription reactions had a final volume of 160 μL to accommodate whole mineral pieces. 5′-Labeled primer 40NLib-Rev was pre annealed with RNA on the mineral surface in KCl and Tris-Cl. Samples were boiled in a water bath for 5 min, and then slowly cooled to 37 °C over ca. 45 min by turning off the hot plate. Tubes were then transferred to a constant 37 °C water bath, and dNTPs, M-MuLV buffer, and M-MuLV reverse transcriptase added to the reaction. Final reaction conditions were 50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 1.5 mM dNTPs mix (0.375 mM each), 75 nM reverse primer, and 2.5 U/μL M-MuLV reverse transcriptase (New England Biolabs). Samples were loaded along with a 5′-labeled 10 bp DNA step ladder (Promega) on 15% PAGE containing 7 M urea, and ran in 1X TBE. Gels were dried for 1 h at 80 °C, exposed overnight to phosphorimager screens, and screens scanned with a phosphorimager (Personal Molecular Imager, BioRad). PCR amplifications (100 μL) were carried out for 25 cycles on aliquots (30 μL each) of these RT samples. PCR reactions contained 40NLib-Rev reverse primer (2 μM), 40NLib-Fwd primer (2 μM), dNTPs (0.2 mM each), Taq polymerase (0.0375 U/μL, New England Biolabs), and 1X Taq buffer (New England Biolabs). PCR cycling was as follows: 5′ @ 95C, [30′′ @ 95 °C, 30′′ @ 58 °C, 45′′ @ 72 °C] × 25, 5′ @ 72 °C, ∞ @ 4 °C. Product mixtures were loaded on 1.8% agarose gel stained with EtBr, alongside with a 25 bp DNA step ladder (Promega). A negative control without template was also prepared and loaded on the gel.
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