10-23 Deoxyribozyme Design Optimization

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10-23 deoxyribozyme [1] is a widely used and powerful application of nanotechnology capable of site-specifically cleaving substrate ssRNA (or ssDNA with a single RNA linkage). 10-23 deoxyribozymes can be designed to target any given sequences as specified by Watson–Crick recognition of its 5' and 3' binding arms. However, proper design of the deoxyribozyme can be a tricky task. If the stability of the binding arms is too weak, catalysis will proceed very slowly due to rate-limiting association/dissociation kinetics. If the stability of the binding arms is too strong, dissociation of the cleaved product strands can become rate-limiting as the system becomes strongly product-inhibited. Furthermore, because DNA hybridization is a concentration-dependent process, the concentration at which the reaction is run will affect the range of optimal binding arm sequences. Thus binding sequences must be carefully optimized by analyzing the kinetic behavior of the overall system with different varying binding arm sequences.

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To obtain nearest-neighbor thermodynamic hybridization energies, either specific sequences can be entered or, in the absence of sequence information, lengths can be entered with average parameters by assigning a length for each arm.

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Contributed by: Brian M. Frezza (March 2011)
Open content licensed under CC BY-NC-SA


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Details

Kinetic parameters for 10-23 were taken from [2] and [3]. Nearest-neighbor thermodynamic parameters for DNA/DNA and RNA/DNA hybridization were taken from [4] and [5], respectively.

Buffer conditions for these parameters are 50mM , 150mM NaCl, 50mM EPPS. Catalytic rates can also vary in the presence of different divalent cations (such as or ) and concentrations of these salts (see [3] for more details).

The author of this Demonstration is a doctoral candidate at The Scripps Research Institute in La Jolla, CA, with thesis adviser M. Reza Ghadiri.

[1] S. W. Santoro and G. F. Joyce, "A General-Purpose RNA-Cleaving DNA Enzyme," Proc. Natl. Acad. Sci. U.S.A., 94(9), 1997 pp. 4262–4266.

[2] G. F. Joyce, "RNA Cleavage by the 10-23 DNA Enzyme," Methods Enzymol, 341, 2001 pp. 503–517.

[3] S. W. Santoro and G. F. Joyce, "Mechanism and Utility of an RNA-Cleaving DNA Enzyme," Biochemistry, 37(38), 1998 pp. 13330–13342.

[4] H. T. Allawi and J. SantaLucia, Jr., "Thermodynamics and NMR of Internal G-T Mismatches in DNA," Biochemistry, 36(34), 1997 pp. 10581–10594.

[5] N. Sugimoto, S. Nakano, M. Katoh, A. Matsumura, H. Nakamuta, and O. Ohmichi, "Thermodynamic Parameters to Predict Stability of RNA/DNA Hybrid Duplexes," Biochemistry, 34(35), pp. 11211–11216.



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