Likelihood of Nonspecific Activity of Gapmer Antisense Oligonucleotides Is Associated with Relative Hybridization Free Energy

Watt, Andrew T.; Swayze, Grant; Swayze, Eric E.; Freier, Susan M.

doi:10.1089/nat.2020.0847

Cited by 25 publications

(19 citation statements)

References 63 publications

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“…However, modified DNA is generally used as ASO drugs to improve bio-stability and cellular uptake of the oligonucleotides. The stability of hybridization with ASO is reported higher than that with unmodified DNA ( 68 , 69 ). Therefore, truncated ASO with a much shorter length than the sgRNA can be used to obtain optimal binding affinity.…”

Section: Resultsmentioning

confidence: 92%

“…Therefore, truncated ASO with a much shorter length than the sgRNA can be used to obtain optimal binding affinity. A recent report clearly suggested that duplex stability of modified ASO can be correlated with the stability of unmodified duplex in 100 mM NaCl solution ( 68 ). The stability changes of hybrid duplexes in physiological salt concentration resulting from replacing DNA with different kinds of chemically-modified ASO were published earlier ( 68 , 69 ).…”

Section: Resultsmentioning

confidence: 99%

“…A recent report clearly suggested that duplex stability of modified ASO can be correlated with the stability of unmodified duplex in 100 mM NaCl solution ( 68 ). The stability changes of hybrid duplexes in physiological salt concentration resulting from replacing DNA with different kinds of chemically-modified ASO were published earlier ( 68 , 69 ). Increases in melting temperature (Δ T m ) per modification were reported for several modified ASO in comparison to unmodified DNA.…”

Section: Resultsmentioning

confidence: 99%

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Improved nearest-neighbor parameters for the stability of RNA/DNA hybrids under a physiological condition

Banerjee

Tateishi‐Karimata

Ohyama

et al. 2020

Nucleic Acids Research

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The stability of Watson–Crick paired RNA/DNA hybrids is important for designing optimal oligonucleotides for ASO (Antisense Oligonucleotide) and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)–Cas9 techniques. Previous nearest-neighbour (NN) parameters for predicting hybrid stability in a 1 M NaCl solution, however, may not be applicable for predicting stability at salt concentrations closer to physiological condition (e.g. ∼100 mM Na+ or K+ in the presence or absence of Mg2+). Herein, we report measured thermodynamic parameters of 38 RNA/DNA hybrids at 100 mM NaCl and derive new NN parameters to predict duplex stability. Predicted ΔG°37 and Tm values based on the established NN parameters agreed well with the measured values with 2.9% and 1.1°C deviations, respectively. The new results can also be used to make precise predictions for duplexes formed in 100 mM KCl or 100 mM NaCl in the presence of 1 mM Mg2+, which can mimic an intracellular and extracellular salt condition, respectively. Comparisons of the predicted thermodynamic parameters with published data using ASO and CRISPR–Cas9 may allow designing shorter oligonucleotides for these techniques that will diminish the probability of non-specific binding and also improve the efficiency of target gene regulation.

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Section: Resultsmentioning

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Improved nearest-neighbor parameters for the stability of RNA/DNA hybrids under a physiological condition

Banerjee

Tateishi‐Karimata

Ohyama

et al. 2020

Nucleic Acids Research

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show abstract

“…Notably, the interaction between the nucleic acids and proteins, including polymerase, must be considered to more precisely understand the reaction. The parameters can also be beneficial in antisense oligonucleotide (ASO) technology for selecting effective DNA oligonucleotides in crowded environments because a recent report showed that NN parameters are also suitable predictor for chemically modified ASO/RNA duplexes (46).…”

Section: Resultsmentioning

confidence: 99%

Nearest-neighbor parameters for predicting DNA duplex stability in diverse molecular crowding conditions

Ghosh

Takahashi

Ohyama

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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The intracellular environment is crowded and heterogeneous. Although the thermodynamic stability of nucleic acid duplexes is predictable in dilute solutions, methods of predicting such stability under specific intracellular conditions are not yet available. We recently showed that the nearest-neighbor model for self-complementary DNA is valid under molecular crowding condition of 40% polyethylene glycol with an average molecular weight of 200 (PEG 200) in 100 mM NaCl. Here, we determined nearest-neighbor parameters for DNA duplex formation under the same crowding condition to predict the thermodynamics of DNA duplexes in the intracellular environment. Preferential hydration of the nucleotides was found to be the key factor for nearest-neighbor parameters in the crowding condition. The determined parameters were shown to predict the thermodynamic parameters (∆H°, ∆S°, and ∆G°37) and melting temperatures (Tm) of the DNA duplexes in the crowding condition with significant accuracy. Moreover, we proposed a general method for predicting the stability of short DNA duplexes in different cosolutes based on the relationship between duplex stability and the water activity of the cosolute solution. The method described herein would be valuable for investigating biological processes that occur under specific intracellular crowded conditions and for the application of DNA-based biotechnologies in crowded environments.

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“…They calculated the edit distance, a count of the number of mismatches or gaps between the ASO and RNA sequence, to all genes, and found that 139 / 256 (54%) of all downregulated genes were at an edit distance of zero or one. Others have used qPCR dose-response experiments to investigate off-targets nominated from in silico screens of 96 high viability gapmers (Watt et al, 2020). In this study 97/ 832 predicted off-target sites had a reduction in gene expression with potency within 10-fold of the intended target transcript.…”

Section: Introductionmentioning

confidence: 99%

Transcriptome-Wide Off-Target Effects of Steric-Blocking Oligonucleotides

Holgersen

Gandhi

Zhou

et al. 2020

Preprint

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Steric-blocking oligonucleotides (SBOs) are short, single-stranded nucleic acids designed to modulate gene expression by binding to mRNA and blocking access from cellular machinery such as splicing factors. SBOs have the potential to bind to near-complementary sites in the transcriptome, causing off-target effects. In this study, we used RNA-seq to evaluate the off-target differential splicing events of 81 SBOs and differential expression events of 46 SBOs. Our results suggest that differential splicing events are predominantly hybridization-driven, while differential expression events are more common and driven by other mechanisms. We further evaluated the performance of in silico screens for off-target events, and found an edit distance cutoff of three to result in a sensitivity of 14% and false discovery rate of 99%. A machine learning model incorporating splicing predictions substantially improved the ability to prioritize low edit distance hits, increasing sensitivity from 4% to 26% at a fixed FDR. Despite these large improvements in performance, the approach does not detect the majority of events at a false discovery rate below 99%. Our results suggest that in silico methods are currently of limited use for predicting the off-target effects of SBOs.

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Likelihood of Nonspecific Activity of Gapmer Antisense Oligonucleotides Is Associated with Relative Hybridization Free Energy

Cited by 25 publications

References 63 publications

Improved nearest-neighbor parameters for the stability of RNA/DNA hybrids under a physiological condition

Improved nearest-neighbor parameters for the stability of RNA/DNA hybrids under a physiological condition

Nearest-neighbor parameters for predicting DNA duplex stability in diverse molecular crowding conditions

Transcriptome-Wide Off-Target Effects of Steric-Blocking Oligonucleotides

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