Impact of 3-deazapurine nucleobases on RNA properties

Abstract

Deazapurine nucleosides such as 3-deazaadenosine (c3A) are crucial for atomic mutagenesis studies of functional RNAs. They were the key for our current mechanistic understanding of ribosomal peptide bond formation and of phosphodiester cleavage in recently discovered small ribozymes, such as twister and pistol RNAs. Here, we present a comprehensive study on the impact of c3A and the thus far underinvestigated 3-deazaguanosine (c3G) on RNA properties. We found that these nucleosides can decrease thermodynamic stability of base pairing to a significant extent. The effects are much more pronounced for 3-deazapurine nucleosides compared to their constitutional isomers of 7-deazapurine nucleosides (c7G, c7A). We furthermore investigated base pair opening dynamics by solution NMR spectroscopy and revealed significantly enhanced imino proton exchange rates. Additionally, we solved the X-ray structure of a c3A-modified RNA and visualized the hydration pattern of the minor groove. Importantly, the characteristic water molecule that is hydrogen-bonded to the purine N3 atom and always observed in a natural double helix is lacking in the 3-deazapurine-modified counterpart. Both, the findings by NMR and X-ray crystallographic methods hence provide a rationale for the reduced pairing strength. Taken together, our comparative study is a first major step towards a comprehensive understanding of this important class of nucleoside modifications.

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

“…To understand base-pairing and catalytic properties of nucleobases in functional RNA, knowledge about their acid−base properties is crucial. 7,26 To quantify the acid−base properties of c 1 G, we conducted pH-dependent UV-spectroscopic titration experiments. Figure 3 shows an overlay of spectra for the c 1 G nucleobase that were used for pK a determinations.…”

“…Thus far, diverse deazanucleosides have been utilized for RNA atomic mutagenesis experiments; these are 3-deazacytidine (c 3 C), ,, 7-deazaadenosine (c 7 A), ,− ,, 3-deazaadenosine (c 3 A), , 1-deazaadenosine (c 1 A), ,,, and 7-deazaguanosine (c 7 G). , Furthermore, an efficient synthesis of 3-deazaguanosine (c 3 G) and the corresponding phosphoramidite has been reported recently and adds to the deazanucleoside tool box. , The missing piece, however, is 1-deazaguanosine (c 1 G), which is urgently needed for RNA atomic mutagenesis studies to probe the role of active site guanosines in catalysis of diverse ribozymes and for ligand recognition in the binding pockets of many riboswitches. In this work, we present a novel synthetic route toward c 1 G, the corresponding phosphoramidite and its incorporation into oligoribonucleotides by RNA solid-phase synthesis.…”

A High-Pressure, High-Temperature Flow Reactor Simulating the Hadean Earth Environment, with Application to the Pressure Dependence of the Cleavage of Avocado Viroid Hammerhead Ribozyme

“…A treatment for denaturing of ASBVd(−):HHR prior to HPLC analysis did not affect the retention time of ASBVd(−):HHR. According to the literature [ 21 , 22 ], running HPLC at high temperatures seems to be useful. However, this is not the case for the present system, so it was possible to run HPLC at 35 °C.…”

“…HPLC analyses were carried out using an LC10A HPLC system (Shimadzu, Kyoto, Japan) or an LC-2040 Plus (Shimadzu, Kyoto, Japan) on an anion-exchange column (diameter 2 mm and length 75 mm, DNA-NPR, TOSOH, Tokyo, Japan) at a flow rate of 0.15 mL/min and using a gradient of 0.375–0.975 M NaCl at pH 9.0 with 7.5 M urea and 0.02 M 2-amino-2-hydroxymethyl-1,3-propanediol (Tris) buffer, or using an Infinity II (Agilent, Santa Clara, CA, USA) on an anion-exchange column (diameter 4.6 mm and length 75 mm, DNA-NPR, TOSOH, Tokyo, Japan) at a flow rate of 0.75 mL/min and using a gradient of 0.375–0.975 M NaCl at pH 9.0 with 7.5 M urea and 0.02 M Tris buffer. The HPLC method was modified on the basis of our previous method for oligonucleotide analysis [ 20 ] with the addition of a large amount of urea for denaturation of RNA [ 21 , 22 ]. All these HPLC analyses with the same gradient buffer, but using different systems with different sizes of the anion-exchange columns were consistent.…”

Convenient Synthesis of 3‐Deazapurine Nucleosides (3‐Deazainosine, 3‐Deazaadenosine and 3‐Deazaguanosine) Using Inosine as a Starting Material

“…3‐Deazapurine nucleosides have been incorporated into oligonucleotides, and these nucleoside analogs have been used as important probes in studies on protein–nucleic acid interactions (Cosstick et al., 1990; Ono & Ueda, 1987; Saito‐Tarashima et al., 2016). Quite recently, oligonucleotides with 3‐deazaadenosine have been used in atomic mutagenesis studies on functional RNAs (Bereiter et al., 2021). Despite the utility of 3‐deazapurine nucleosides, they are usually prepared using a classical glycosylation method involving 3‐deazapurine bases and sugars.…”