ADAR activation by inducing a syn conformation at guanosine adjacent to an editing site

Abstract

ADARs (adenosine deaminases acting on RNA) can be directed to sites in the transcriptome by complementary guide strands allowing for the correction of disease-causing mutations at the RNA level. However, ADARs show bias against editing adenosines with a guanosine 5′ nearest neighbor (5′-GA sites), limiting the scope of this approach. Earlier studies suggested this effect arises from a clash in the RNA minor groove involving the 2-amino group of the guanosine adjacent to an editing site. Here we show that nucleosides capable of pairing with guanosine in a syn conformation enhance editing for 5′-GA sites. We describe the crystal structure of a fragment of human ADAR2 bound to RNA bearing a G:G pair adjacent to an editing site. The two guanosines form a Gsyn:Ganti pair solving the steric problem by flipping the 2-amino group of the guanosine adjacent to the editing site into the major groove. Also, duplexes with 2′-deoxyadenosine and 3-deaza-2′-deoxyadenosine displayed increased editing efficiency, suggesting the formation of a Gsyn:AH+anti pair. This was supported by X-ray crystallography of an ADAR complex with RNA bearing a G:3-deaza dA pair. This study shows how non-Watson–Crick pairing in duplex RNA can facilitate ADAR editing enabling the design of next generation guide strands for therapeutic RNA editing.

Dissecting the basis for differential substrate specificity of ADAR1 and ADAR2

“…Indeed, we found that an arRNA harboring a G-A mismatch yielded the highest editing level within a SMAD4-ORF target, followed by arRNA harboring a G-G mismatch, whereas the fully complementary arRNA yielded background levels of editing (Figure 5E-F) . These findings are in line with reports by 58,59 . Collectively, our findings establish how editing at target sites can be induced either by introducing mismatches at a relatively distant fixed offset via a mechanism impacting recognition through the RBDs, or in close vicinity to the target site via a mechanism likely impacting recognition through the deaminase domain.…”

Selective Inhibition of ADAR1 Using 8-Azanebularine-Modified RNA Duplexes

“…Previously, we used gel-based assays such as gel shift and endonuclease footprinting experiments to probe ADAR2-RNA interactions and give insight into potential substrates for X-ray crystallography screens. ,,, However, the use of these techniques for ADAR1 studies has been complicated by nonspecific binding from protein aggregates, especially for longer ADAR1 constructs (containing one or more dsRNA or Z-DNA/RNA binding domains). Estimation of relative binding affinities by means of IC 50 values in the in vitro deamination assay described here offers an alternative method for biochemical and biophysical investigations of ADAR1-RNA interactions.…”

“…Since the 8-azaN hydrate lacks a good leaving group, the 8-azaN reaction cannot proceed forward, hence preventing catalytic turnover. The use of dsRNAs containing 8-azaN at the reactive site allows for the mechanistic trapping of ADAR2-RNA complexes for binding and structural studies. ,− , However, whether this nucleoside analog alone or integrated into an RNA structure can likewise be used for ADAR1 investigations is still unexplored. In this study, we then sought to employ a panel of short RNA duplexes (≤16 bp) bearing 8-azaN to probe the ADAR1 catalytic domain substrate recognition.…”

Calculated pKa Values for a Series of Aza- and Deaza-Modified Nucleobases

“…Single-atom mutagenesis has been carried out with many nucleic acids to gain a better understanding of their structure–function relationships. 13 − 18 In contrast, the addition of a nitrogen atom to a purine nucleobase gives the azapurine class of modification. The addition of a nitrogen atom has led to unique pH-dependent fluorescent properties of the nucleobase while maintaining Watson–Crick (WC) base-pairing capabilities.…”

“…As such, the physical properties of the nucleobase can be altered at the single-atom level. Single-atom mutagenesis has been carried out with many nucleic acids to gain a better understanding of their structure–function relationships. − In contrast, the addition of a nitrogen atom to a purine nucleobase gives the azapurine class of modification. The addition of a nitrogen atom has led to unique pH-dependent fluorescent properties of the nucleobase while maintaining Watson–Crick (WC) base-pairing capabilities. , Azapyrimidines (occasionally referred to as triazine nucleosides) have demonstrated utility in probing immunosuppression and purine metabolism pathways. , Aza nucleosides also have reported anticancer, , antiviral, , and antibacterial capabilities.…”