1-Deazaguanosine-Modified RNA: The Missing Piece for Functional RNA Atomic Mutagenesis

Abstract

Atomic mutagenesis is the key to advance our understanding of RNA recognition and RNA catalysis. To this end, deazanucleosides are utilized to evaluate the participation of specific atoms in these processes. One of the remaining challenges is access to RNA-containing 1-deazaguanosine (c 1 G). Here, we present the synthesis of this nucleoside and its phosphoramidite, allowing first time access to c 1 G-modified RNA. Thermodynamic analyses revealed the base pairing parameters for c 1 G-modified RNA. Furthermore, by NMR spectroscopy, a c 1 G-triggered switch of Watson-Crick into Hoogsteen pairing in HIV-2 TAR RNA was identified. Additionally, using X-ray structure analysis, a guanine−phosphate backbone interaction affecting RNA fold stability was characterized, and finally, the critical impact of an active-site guanine in twister ribozyme on the phosphodiester cleavage was revealed. Taken together, our study lays the synthetic basis for c 1 G-modified RNA and demonstrates the power of the completed deazanucleoside toolbox for RNA atomic mutagenesis needed to achieve in-depth understanding of RNA recognition and catalysis.

Scaling Catalytic Contributions of Small Self‐Cleaving Ribozymes

“…RNA atomic mutagenesis of the functionality that is responsible for guanosine acid-base catalysis (namely, the N1-H moiety of guanine) have been lacking until recently. [37] The present study provides scaling of catalytic contributions for the pistol ribozyme class with unprecedented precision. Although it is tempting to assume that in general γ-and δ-catalysis-jointly constituting concerted general acid-base catalysis-make the largest contribution to the catalytic rate enhancement, [8] for pistol ribozymes this is not the case.…”

“…To achieve our goals, synthetic hurdles had to be taken first; those concerned access to the phosphoramidites of 1‐deazaguanosine (c 1 G), 3‐deazaguanosine (c 3 G), and xanthosine (X) needed for RNA solid‐phase synthesis. Since only for c 3 G containing RNA synthetic procedures were published, [35, 36] we have developed novel protocols for c 1 G and X modified RNA [37, 38] . A further requirement for our undertaking was the set‐up of a reliable and robust real‐time assay for direct monitoring of pistol ribozyme cleavage.…”

“…To date, only one other ribozyme class has been scaled for catalytic contributions with high precision. This is twister where “general base knockout” using c 1 G atomic mutagenesis caused a 275‐fold reduction in cleavage rate [37] . The precatalytic crystal structure of twister shows a hydrogen bond (2.6 Å) between the N1 atom of the active site guanine and the nonbridging pro ‐R oxygen of the scissile phosphate, [63] and hence, implicates that this guanine (G48) plays a significant role in phosphorane transition state stabilization (β‐catalysis).…”

“…One reason for this is that experimental means, i.e. RNA atomic mutagenesis of the functionality that is responsible for guanosine acid‐base catalysis (namely, the N1‐H moiety of guanine) have been lacking until recently [37] …”

A new route for the synthesis of 1-deazaguanine and 1-deazahypoxanthine

“…Previously published routes toward these compounds focused on ring closure of the imidazole part of the purine system after the pyrimidine core had been functionalized, however, the drawback of this approach is the passage of rather hazardous/explosive intermediates. Here, we present a new tactic for the syntheses of 1-deazaguanine and 1-deazahypoxanthine stimulated by a recently published route of our research group for the corresponding nucleosides [16,17], employing the same key reaction, namely the copper-catalyzed coupling of an aryl iodide with benzyl alcohol. We build on a commercially available imidazopyridine derivative and conceived a protecting group strategy to enhance solubility and selectivity to orchestrate the installation of the exocyclic amino and hydroxy groups.…”