Evolution of functional nucleic acids in the presence of nonheritable backbone heterogeneity

Abstract

Multiple lines of evidence support the hypothesis that the early evolution of life was dominated by RNA, which can both transfer information from generation to generation through replication directed by base-pairing, and carry out biochemical activities by folding into functional structures. To understand how life emerged from prebiotic chemistry we must therefore explain the steps that led to the emergence of the RNA world, and in particular, the synthesis of RNA. The generation of pools of highly pure ribonucleotides on the early Earth seems unlikely, but the presence of alternative nucleotides would support the assembly of nucleic acid polymers containing nonheritable backbone heterogeneity. We suggest that homogeneous monomers might not have been necessary if populations of heterogeneous nucleic acid molecules could evolve reproducible function. For such evolution to be possible, function would have to be maintained despite the repeated scrambling of backbone chemistry from generation to generation. We have tested this possibility in a simplified model system, by using a T7 RNA polymerase variant capable of transcribing nucleic acids that contain an approximately 11 mixture of deoxy- and ribonucleotides. We readily isolated nucleotide-binding aptamers by utilizing an in vitro selection process that shuffles the order of deoxy- and ribonucleotides in each round. We describe two such RNA/DNA mosaic nucleic acid aptamers that specifically bind ATP and GTP, respectively. We conclude that nonheritable variations in nucleic acid backbone structure may not have posed an insurmountable barrier to the emergence of functionality in early nucleic acids.

Fig. 1.

Sequence and sugar content of the MNA starting pool. (A) Schematic of DNA template including a 5′ T7 promoter and random regions flanked by constant primer binding sites. Random regions consist of 64 degenerate bases (Top) or a designed stem loop (5′ CTCGCGAACGAG 3′) flanked on both sides by 26 degenerate bases (Bottom). (B–E) Anion-exchange HPLC chromatograms; absorbance at 260 nm (y axis) vs. time, 0–30 min (x axis). Peak numbers denote negative charge at pH 8.0. (B) 2, 5′-AMP; 3, 5′-ADP; 4, 5′-ATP. Gray line depicts 0–2 M KCl gradient. (C) Chemically synthesized degenerate DNA oligomer standards with no terminal phosphates. Peak 2, trinucleotide; 3, tetranucleotide; 4, pentanucleotide; 5, hexanucleotide; 6, heptanucleotide. (D) Full-length MNA, denoted by asterisk (*). (E) MNA base digests generated from a 9∶1 (dNTP∶rNTP) substrate ratio transcription reaction. Peak 2, ribonucleotide 2^′/3^′ monophosphate (r); 3, dinucleotide (d-r); 4, trinucleotide (d-d-r); 5, tetranucleotide (d-d-d-r); 6, pentanucleotide (d-d-d-d-r); 7, hexanucleotide (d-d-d-d-d-r), where d denotes deoxyribose and r denotes ribose.

Fig. 2.

In vitro selection scheme and progress. (A) Transcribed, ³²P- labeled MNA consisting of approximately 50% deoxy- and ribonucleotides was incubated with a ligand-free precolumn for 1 h. Flow-through from this column was incubated with either ATP- or GTP-derivatized agarose for 30 min. After a wash regime, aptamers were specifically eluted by four 30-min incubations with free ligand, then reverse-transcribed (RT) and PCR-amplified to generate the next pool of template DNA. (B) Elution profile from rounds 1 (○) and 8 (●) of the ATP aptamer selection. (C) Percent of input MNA eluted by free ligand washes for each round of the ATP (white) and GTP (gray) selections. In the first round, the precolumn retained approximately 10% of MNA; in subsequent rounds approximately 50% of total counts were retained.

Fig. 3.

MNA ATP aptamer 74 column binding assays. (A) Sequence of the previously identified DNA ATP aptamer (Top) and the MNA ATP aptamer 74 (Bottom), (for PBS sequences see Fig. S4, legend). Common aptamer sequence motifs are shown in bold. (B) Elution profile. MNA (○, n = 6), DNA (▵, n = 3), or RNA (□, n = 3) was incubated with ATP-agarose (2 mM) for approximately 10 min. Fractions of one column volume were collected for a series of nonspecific and ATP washes (2 mM). Error bars represent SEM. (C) Specificity of the MNA and DNA ATP aptamer 74. Atomic positions (gray) recognized by the MNA or DNA ATP aptamer 74 were determined by the competitive elution of DNA or MNA bound to ATP-agarose with ATP analogues (2 mM). Total MNA or DNA (second value, when shown) eluted by each analogue wash followed by a subsequent ATP wash (internal control) is reported as 100%. The average percentage (of two independent experiments) of aptamer eluted by each analogue is shown; the range of values did not exceed 7%. In most cases, after all wash regimes, residual binding by the MNA to the derivatized agarose ranged from 15 to 40%.

Fig. 4.

MNA GTP aptamer 812 column binding assays. (A) DNA sequence of the MNA GTP aptamer 812 showing the G-rich motif (bold) and six complementary bases of the reverse PBS (underlined). (B) Elution profile. MNA (○, n = 6), DNA (▵, n = 3), or RNA (□, n = 3) was incubated with GTP-agarose (5 mM) for approximately 10 min. Fractions of one column volume were collected for a series of nonspecific and GTP washes (5 mM). (C) Specificity of the MNA GTP aptamer 812. Atomic positions (gray) recognized by the MNA GTP aptamer 812 were determined by the competitive elution of MNA bound to GTP-agarose with GTP analogues (5 mM). Total MNA eluted by each analogue wash and a subsequent GTP wash (internal control) is reported as 100%. The average percentage (of two independent experiments) of aptamer eluted by each analogue is shown; the range of values did not exceed 5%. In most cases, after all wash regimes, background binding by the MNA to the derivatized agarose ranged from 15 to 40% of MNA.