Role of Pseudoisocytidine Tautomerization in Triplex-Forming Oligonucleotides: In Silico and in Vitro Studies

Hartono, Yossa Dwi; Pabón-Martínez, Y. Vladimir; Uyar, Arzu; Wengel, Jesper; Lundin, Karin E.; Zain, Rula; Smith, C. I. Edvard; Nilsson, Lennart; Villa, Alessandra

doi:10.1021/acsomega.7b00347

Cited by 10 publications

(8 citation statements)

References 67 publications

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“…In addition, the contribution of tautomeric forms that are unproductive for binding in the ψ C base further diminish stability. [ 116 ] The native bases are unique in their relative chemical stability and tautomeric purity; unnatural WC‐shuffled bases like those examined by Benner and coworkers are limited to some degree by the contribution of nonproductive tautomers [ 59 ] and can be susceptible to epimerization, oxidation and hydrolysis. [ 66 ] Benner has reported that one such WC‐shuffled base, [ 117 ] a pyrazine derivative (pyDDA) mimicking C + , can target the GC base pair via the purine Hoogsteen face [ 118 ] at neutral pH (Figure 5).…”

Section: Unnatural Bases In Noncanonical Interactionsmentioning

confidence: 99%

Unnatural bases for recognition of noncoding nucleic acid interfaces

et al. 2020

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The notion of using synthetic heterocycles instead of the native bases to interface with DNA and RNA has been explored for nearly 60 years. Unnatural bases compatible with the DNA/RNA coding interface have the potential to expand the genetic code and co‐opt the machinery of biology to access new macromolecular function; accordingly, this body of research is core to synthetic biology. While much of the literature on artificial bases focuses on code expansion, there is a significant and growing effort on docking synthetic heterocycles to noncoding nucleic acid interfaces; this approach seeks to illuminate major processes of nucleic acids, including regulation of transcription, translation, transport, and transcript lifetimes. These major avenues of research at the coding and noncoding interfaces have in common fundamental principles in molecular recognition. Herein, we provide an overview of foundational literature in biophysics of base recognition and unnatural bases in coding to provide context for the developing area of targeting noncoding nucleic acid interfaces with synthetic bases, with a focus on systems developed through iterative design and biophysical study.

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Section: Unnatural Bases In Noncanonical Interactionsmentioning

confidence: 99%

Unnatural bases for recognition of noncoding nucleic acid interfaces

et al. 2020

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“…Previous studies have indicated that due to tautomeric forms, J may form stable J-G base pairs (Figure 4a). 36 We propose that M disfavors Watson−Crick pairing with G because it forms only two hydrogen bonds in the unprotonated state and has repulsive interactions when protonated (Figure 4a).…”

Section: ■ Resultsmentioning

confidence: 98%

The 2-Aminopyridine Nucleobase Improves Triple-Helical Recognition of RNA and DNA When Used Instead of Pseudoisocytosine in Peptide Nucleic Acids

et al. 2021

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Pseudoisocytosine (J), a neutral analogue of protonated cytosine, is currently the gold standard modified nucleobase in peptide nucleic acids (PNAs) for the formation of J•G-C triplets that are stable at physiological pH. This study shows that triple-helical recognition of RNA and DNA is significantly improved by using 2-aminopyridine (M) instead of J. The positively charged M forms 3-fold stronger M+•G-C triplets than J with uncompromised sequence selectivity. Replacement of six Js with Ms in a PNA 9-mer increased its binding affinity by ∼2 orders of magnitude. Mmodified PNAs prefer binding double-stranded RNA over DNA and disfavor off-target binding to single-stranded nucleic acids. Taken together, the results show that M is a promising modified nucleobase that significantly improves triplex-forming PNAs and may provide breakthrough developments for therapeutic and biotechnology applications.

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“…This could be due to the ability of the pseudoisocytidine base to tautomerize such that N3 would be either a hydrogen bond donor or acceptor (Figure S2c). These results suggest that base-flipping by ADAR2 is most efficient when a complementary hydrogen bond donor–acceptor pair exists between residue 488 and N3 of the orphan base.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2021

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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.

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Role of Pseudoisocytidine Tautomerization in Triplex-Forming Oligonucleotides: In Silico and in Vitro Studies

Cited by 10 publications

References 67 publications

Unnatural bases for recognition of noncoding nucleic acid interfaces

Unnatural bases for recognition of noncoding nucleic acid interfaces

The 2-Aminopyridine Nucleobase Improves Triple-Helical Recognition of RNA and DNA When Used Instead of Pseudoisocytosine in Peptide Nucleic Acids

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

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