Convergent Evolution of Adenosine Aptamers Spanning Bacterial, Human, and Random Sequences Revealed by Structure-Based Bioinformatics and Genomic SELEX

Abstract

SUMMARY Aptamers are structured macromolecules in vitro evolved to bind molecular targets, whereas in nature they form the ligand-binding domains of riboswitches. Adenosine aptamers of a single structural family were isolated several times from random pools but they have not been identified in genomic sequences. We used two unbiased methods, structure-based bioinformatics and human genome-based in vitro selection, to identify aptamers that form the same adenosine-binding structure in a bacterium, and several vertebrates, including humans. Two of the human aptamers map to introns of RAB3C and FGD3 genes. The RAB3C aptamer binds ATP with dissociation constants about ten times lower than physiological ATP concentration, while the minimal FGD3 aptamer binds ATP only co-transcriptionally.

Discovering Human RNA Aptamers by Structure-Based Bioinformatics and Genome-Based In Vitro Selection

“…More recently, modifications to the in vitro selection procedure have aimed to identify naturally occurring aptamers and other functional nucleic acids by using genome-derived DNA pools as templates for selections. In the case of adenosine, but not GTP, both synthetic and genomic DNA selections revealed a number of structurally conserved aptamer sequences (Burke & Gold, 1997;Curtis & Liu, 2013;Davis & Szostak, 2002;Sassanfar & Szostak, 1993;Vu et al, 2012). These adenosine-binding motifs are sequence-independent and represent a rare example of convergent molecular evolution spanning both genomic and synthetic sequence space.…”

“…2.1). Nuclear magnetic resonance and mutation studies have shown that these conserved nucleotides and flanking helices are required for the formation of a binding pocket to allow base stacking and hydrogen-bonding interactions with the ligand (Dieckmann, Butcher, Sassanfar, Szostak, & Feigon, 1997;Dieckmann, Suzuki, Nakamura, & Feigon, 1996;Jiang, Kumar, Jones, & Patel, 1996;Vu et al, 2012). Although the sequence compositions of the flanking helical motifs vary, the adenosine-binding loop is largely sequence conserved, and both of these properties are exploited with structure-based search algorithms.…”

“…In principle, the pool can sample the entire genome of the target organism at single-nucleotide resolution (in both directions, with respect to the engineered RNA polymerase promoter), independent of expression of individual genes, but lacks sequences corresponding to spliced and otherwise processed transcripts. Structural characterization of the (Burgstaller & Famulok, 1994;Sassanfar & Szostak, 1993;Vu et al, 2012). The bulged guanosine nucleotide necessary for ligand binding is in bold.…”

“…Binding buffer should facilitate binding of aptamers onto immobilized ligands. Elution buffer, in addition to being similar to its binding counterpart, contains elevated concentration of magnesium chloride to accommodate chelating by negatively-charged ligands (e.g., adenosine triphosphate; Vu et al, 2012), to maintain sufficient amount of Mg 2+ to allow RNA to fold into a stable tertiary structure. In addition, the concentration of the free ligand should mimic its physiological concentration within the cell or exceed the approximate concentration of the ligand on the beads.…”

“…4.1.1a Binding buffer: 140 mM KCl, 10 mM NaCl, 20 mM TrisÁHCl, 5 mM MgCl 2 . 4.1.1b Elution buffer: 140 mM KCl, 10 mM NaCl, 20 mM TrisÁHCl, 5 mM MgCl 2 , desired concentration of free ligand (in case of dior triphosphorylated ligand, such as ATP, Mg 2+ concentration needs to be increased by the ligand concentration to avoid changing the free Mg 2+ concentration in the solution; e.g., we supplemented 5 mM ATP with an additional 5 mM MgCl 2 ; Vu et al, 2012). 4.1.1c Harsh elution buffer: 8 M urea and 5 mM EDTA.…”