Poly(U) RNA-templated synthesis of AppA

Abstract

Simple nucleotide templating activities are of interest as potential primordial reactions. Here we describe the acceleration of 5 ′ -5 ′ AppA synthesis by 3 ′ -5 ′ poly(U) under normal solution conditions. This reaction is apparently templated via complementary U:A base-pairing, despite the involvement of two different RNA backbones, because poly(U), unlike other polymers, significantly stimulates AppA synthesis. These interactions occur in moderate (K + ) and (Mg 2+ ) and are temperature sensitive, being more efficient at 10°C than at 4°C, but absent at 20°C. The reaction is only slightly pH sensitive, despite potentially relevant substrate pK a 's. Kinetic data explicitly support production of AppA by interaction of stacked 2MeImpA and pA nucleotides paired with a single molecule of U template. At a lower rate, AppA can also be produced by a chemical reaction between 2MeImpA and pA, without participation of poly(U). Molecular modeling suggests that 5 ′ -5 ′ joining between stacked or concurrently paired A's can occur without major departures from normal U-A helical coordinates. So, coenzyme-like 5 ′ -5 ′ purine dinucleotides might be readily synthesized from 3 ′ -5 ′ RNAs with complementary sequences.

Non-Watson–Crick RNA synthesis suited to origin functions

“…Thus the chemical reaction is second order overall in the nucleotides, and the GppG-stimulated reaction is third order overall: first order in two A nucleotides plus first order in GppG. This resembles previous cross-templated reactions, which behave in a similar second-and third-order fashion overall (Puthenvedu et al 2015;Majerfeld et al 2016). …”

“…The lane marked "0" is an unincubated complete reaction, "inc" is the untemplated reaction after 19 h at 12°C, and "inc + GppG" implies the same incubation but with 10 mM GppG added. This is the chromatographic system previously used for detection of AppA (Puthenvedu et al 2015), and the identity of the product has been confirmed by HPLC (D Puthenvedu and M Yarus, in prep.). By determining the fraction of total radioactivity in the product spot, product concentration can be calculated from known total [pA].…”

“…AppG and AppA in Figure 2B are particularly notable; they contain two purines, but are inactive. Thus, GppG is most likely acting via two A:G base pairs, somewhat as does poly(U) ordering two A's via U:A pairs (Puthenvedu et al 2015; see Discussion).…”

“…These linear homopolymers encode coenzyme-like AppA or GppG, respectively, apparently by pyrimidine-purine base-pairing. For example: Puthenvedu et al 2015) pG Majerfeld et al 2016) Linear polymer templates can be envisioned as products of clay-mediated polymerization (Ferris et al 1996) of activated nucleotides. In this formulation, linear homopolymers from clay act as primitive "genes," and coenzyme-like ribodimers act as "gene products."…”

“…The single resolved product spot, ahead of substrate 5 ′ [ 32 P]A, is AppA, which appears spontaneously, but at an accelerated rate when GppG is added. Note that as for the substantially different cross-templating systems (Puthenvedu et al 2015;Majerfeld et al 2016) This chromatographic method can therefore be used to quantitate the velocity of AppA synthesis under varied conditions, as shown in Figure 2. For example, note that GppG is at a molecular concentration of 10 mM, and poly(U) is at a total nucleotide phosphate concentration of 10 mM.…”