Rational Design of RNA Editing Guide Strands: Cytidine Analogs at the Orphan Position

Abstract

Adenosine Deaminases Acting on RNA (ADARs) convert adenosine to inosine in double stranded RNA. Human ADARs can be directed to predetermined target sites in the transcriptome by complementary guide strands, allowing for the correction of disease-causing mutations at the RNA level. Here we use structural information available for ADAR2-RNA complexes to guide the design of nucleoside analogs for the position in the guide strand that contacts a conserved glutamic acid residue in ADARs (E488 in human ADAR2), which flips the adenosine into the ADAR active site for deamination. Mutating this residue to glutamine (E488Q) results in higher activity because of the hydrogen bond donating ability of Q488 to N3 of the orphan cytidine on the guide strand. We describe the evaluation of cytidine analogs for this position that stabilize an activated conformation of the enzyme-RNA complex and increase catalytic rate for deamination by the wild-type enzyme. A new crystal structure of ADAR2 bound to duplex RNA bearing a cytidine analog revealed a close contact between E488, stabilized by an additional hydrogen bond and altered charge distribution when compared to cytidine. In human cells and mouse primary liver fibroblasts, this single nucleotide modification increased directed editing yields when compared to an otherwise identical guide oligonucleotide. Our results show that modification of the guide RNA can mimic the effect of hyperactive mutants and advance the approach of recruiting endogenous ADARs for site-directed RNA editing.

Site-directed RNA editing: recent advances and open challenges

“…Interestingly, the Beal lab could show that a combination of a guide RNA that is abasic opposite the target A and an engineered ADAR2 that puts a phenylalanine at position 488 the target specificity can be dramatically increased while decreasing off-target editing, as the phenylalanine prevents the base flipping required for editing [ 70 ]. Similarly, introduction of a deoxycytidine opposing the target A can boost editing with a hyperactive variant of ADAR2 [ 71 ]. Still, both approaches require the use of an engineered ADAR enzyme that needs to be introduced to cells.…”

“…Still, both approaches require the use of an engineered ADAR enzyme that needs to be introduced to cells. Most interestingly, introduction of a Benner’s base Z opposing the A also leads to a boost in editing in combination with endogenous ADAR, thus precluding the need for an engineered ADAR [ 71 ].…”

“…(Stability and durability) The low stability of RNA and transient nature of the treatment needs to be improved. Thanks to intensive research on the therapeutic use of small RNAs, numerous chemical modifications that improve stability are known today [ 14 , 40 , 82 , 83 ]. Most recently, embedding of guide RNAs in circular RNAs has been shown to increase their stability by weeks [ 84 ].…”

“…Although the transient nature of off-target effects will vanish with time it is still important to prevent undesired changes in the transcriptome [ 13 ]. However, as outlined above, altering guide RNA-sequences and their chemical modifications can help to decrease off-target editing [ 14 , 40 , 46 , 70 , 71 ].…”

Nucleoside analogs in ADAR guide strands targeting 5′-UA̲ sites

“…8,[10][11][12] Chemical modifications to the guide strand are used to facilitate uptake into target cells, to increase metabolic stability, and to increase efficiency and selectivity of the ADAR reaction. 8,10,[12][13][14] Our group has used structures of ADAR2 bound to RNA bearing an adenosine deamination transition state analog 15 to inform the design of modifications that increase the efficiency 11 and selectivity 16 of the ADAR reaction. Here we focus on a nucleotide position in the guide strand near the editing site that adopts a highly unusual conformation in ADAR2-RNA structures.…”