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New parameters for accurate prediction of RNA/DNAhybrid duplex stability and their advantage in
CRISPR-Cas9 technique
Dipanwita Banerjee,1 Hisae Tateishi-Karimata,1 Tatsuya Ohyama,1 SaptarshiGhosh,1 Tamaki Endoh,1 Shuntaro Takahashi,1 Naoki Sugimoto1,2*
1 Frontier Institute for Biomolecular Engineering Research (FIBER), 2Graduate School ofFrontiers of Innovative Research in Science and Technology (FIRST), Konan University, Kobe,
650-0047, Japan
ABSTRACT
Prediction of thermodynamic stability for RNA/DNA hybrid duplex under a physiologicalcondition is essential to better insight biological reactions and improve gene modifying techniques
related to hybrid duplex formation. Previous parameters for predicting hybrid duplex stability
were derived in much higher cation concentration (10 times) compared to the physiologicalcation concentration. Therefore, new parameters were determined that predict the stability in aphysiological condition with significant accuracy. Moreover, the use of new parameters to design
efficient guide RNA for CRISPR (Clustered Regularly Interspaced Short PalindromicRepeats)-Cas9 technique was demonstrated.
INTRODUCTION
RNA/DNA hybrid duplex forms not only as the intermediate in cellular reactions like replication,transcription, reverse transcription, but also in therapeutic techniques, for example, gene editing
CRISPR-Cas9 technique.1 In vivo development of the technique requires precise prediction of hybrid
duplex stability in the physiological condition. Duplex stability can be predicted well using
nearest-neighbor (NN) model.2 According to the model, thermodynamic stability of duplex can be
determined by the summation of the free energy changes due to the formation of all the NN base pairs
and the helix initiation. Based on the model, NN parameters for hybrid duplex were developed that canpredict well the duplex stability in 1 M NaCl solution.3 The salt concentration, however, is almost 10times higher than the physiological salt concentration. Since cation concentrations affect duplex stability
largely, the previous parameters may not be useful under an intracellular salt condition. Here, weinvestigated the applicability of previous parameters, and therefore improved new NN parameters in thesolution closer to physiological environment for the development of the gene editing technique.
RESULTS AND DISCUSSION
Intracellular environment comprises monovalent (Na+, K+) and divalent cations (Mg2+, Ca2+).4 To
mimic the environment, we prepared a cellular cation condition containing 120 mM KCl, 10 mM NaCl,

1 mM MgCl2, 0.2 µM CaCl2, and 10 mM K2HPO4 (pH 7.0). The melting curves of hybrid duplex in thecellular cation condition was compared with the melting curves in the buffer solutions containing 1 M
NaCl and 100 mM NaCl. The comparison revealed that the free energy change for duplex formation in
the cellular condition at 37 °C (G37) shows
close proximity with the G37 in 100 mM
NaCl solution although G37 in 1 M NaClsolution was quite different from its value underphysiological conditions. Next, we verified thevalidation of the NN model for the hybrid
duplex in 100 mM NaCl solution. Then, based

Figure 1. Schematic presentation about the advantage of the precise
prediction for hybrid duplex stability in CRISPR-Cas9 technique.

on the thermodynamic parameters measured for
38 hybrid duplexes in the 100 mM NaClsolution, new NN parameters were derived. Theparameters predicted well the G37 of the 38hybrid duplexes with an average prediction error of only 2.9 % and also help to improve designing of
single guide RNA (sgRNA) in CRISPR-Cas9 technique. Length shortening of sgRNA helps to decrease
the off-target binding and increase the efficiency of the drug.5 The length of sgRNA should be optimal sothat it can form hybrid duplex with target site of optimal stability. Figure 1 showed the advantage of ourparameters over the previous parameters in the prediction of hybrid duplex stability in CRISPR
technique using a typical example.
CONCLUSION
The validity of NN model was verified and new NN parameters were determined for hybrid duplex in
100 mM NaCl solution. New parameters predicted well the stability of hybrid duplex in a physiologicalsalt condition. In the improvement of CRISPR technique, the application of our new parameters was also
demonstrated.
REFERENCES
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D. Banerjee, H. Tateishi-Karimata, T. Ohyama, S. Ghosh, T. Endoh, S. Takahashi, and N. Sugimoto, Nucleic
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I. Tinoco, O. C. Uhlenbeck, and M. D. Levine, Nature, 1971, 230, 362−367.

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N. Sugimoto, S. Nakano, M. Katoh, A. Matsumura, H. Nakamuta, T. Ohmichi, M. Yoneyama, and M. Sasaki,Biochemistry, 1995, 34, 11211−11216.

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H. Lodish, A. Berk, S. L. Zipursky et al. (2000) Molecular cell biology 4th ed. W. H. Freeman, (ed).

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Y. Fu, J. D. Sander, D. Reyon, V. M. Cascio, and J. K. Joung, Nat. Biotechnol. 2014, 32, 279–284.

*Corresponding author(s): sugimoto@konan-u.ac.jp