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Duplex Stability Design and Protocols

Introduction to Duplex Stability Duplex Stability Applications Duplex Stability Design/Protocol Duplex Stability Literature Order Online

Duplex Stability Design / Protocol
Modulating Oligonucleotide Duplex Stability--Design Considerations.

Specific and stable hybridization of an oligo to its complementary sequence is the desired outcome of a successful hybridization protocol. The melting temperature (Tm) of an oligo indicates the strength of the affinity, and thus the stability, of the hybridization. Manipulation of the oligo sequence to increase its duplex stability or, in some cases, to decrease the duplex stability in certain loop structures will lead to oligos with increased affinity for the target molecule.

Duplex stability can be modulation by incorporation of modifications that are altered on the nucleotide base (e.g., 5-methyl-dC) or sugar (e.g., 2-F or 2-O-Methyl RNA bases). In addition, the phosphodiester backbone also can be altered (e.g., by phosphorthiolation).

For oligonucleotides having the same base sequences, duplex stability (and Tm) proceeds in the following order:


For a DNA:DNA duplex, the sugars are 2-endo, resulting in a B-form duplex. By contrast, in RNA:RNA duplexes, the sugars are 3-endo, and the resulting duplex is A-form. The latter is more stable than the former. DNA:RNA duplexes have an intermediate sugar conformation, and thus a stability between those of DNA:DNA and RNA:RNA, with the specifics dependent on the composition and ratio of deoxyribo- to ribonucleotides (5).

2-O-methyl and 2-fluoro RNA bases are common duplex-stabilizing modifications that serve to modify the sugars of an oligo. 2-O-methyl RNA bases are particularly useful in anti-sense applications, where their stabilizing effect can counteract the de-stabilizing effect of phosphorothiolation, which is used to confer nuclease resistance onto an oligo. Incorporating combinations of 2-O-methyl and 2-fluoro RNA bases is often a cost-effective way to significantly increase duplex stability in lieu of using highly expensive modifications.

5-methyl-dC, 2-amino-dA, and C5-pyrimidines are commonly used duplex stabilizing moieties that are modified on the nucleotide base.

Phosphorothiolation and methylphosphonate linkages are two ways of modifying the backbone of an oligonucleotide that affect the stability of a duplex. Unlike the other modifications however, these two, when incorporated into an oligo, actually lower the Tm of the corresponding duplex, and thus are de-stabilizing, with the methylphosphonate being the more de-stabilizing of the two. These two modifications can potentially be used in conjunction with other modifications to fine-tune the Tm of a duplex formed by a probe and its target (6,7). More commonly, phosphorothiolate and methylphosphonate linkages are used to confer nuclease resistance to an oligo slated for in vivo studies (e.g., anti-sense work). The duplex de-stabilizing effect of these modifications must be taken into account when designing such oligos.


(1) Swayze, E.E., Balkrishen, B. The Medical Chemistry of Oligonucleotides. In: Antisense drug technology: principles, strategies, and applications, 2nd Ed., Crooke, S.T. (Ed), CRC Press, Boca Raton (FL), 2008, 143-182.
(2) Kurreck, J. Antisense technologies. Improvement through novel chemical modifications. Eur. J. Biochem. (2003), 270: 1628-1644.
(3) Schulz, R.G., Gryaznov, S.M. Oligo-2-fluoro-2-deoxynucleotide N3P5phosphoramidites: synthesis and properties. Nucleic Acids Res. (1996), 24: 2966-2973.
(4) Lebedev, Y.; Akopyants, N.; Azhikina, T.; Shevchenko, Y.; Potapov, V.; Stecenko, D.; Berg, D.; Sverdlov, E.. Oligonucleotides containing 2-aminoadenine and 5-methylcytosine are more effective as primers for PCR amplification than their nonmodified counterparts. Genet Anal. (1996), 13: 15-21.
(5) Saenger, W. in Principles of Nucleic Acid Structure, C.R. Cantor, Ed., Springer Advanced Texts in Chemistry, Springer-Verlag, New York, 1984.
(6) Tidd, David M. Specificity of antisense oligonucleotides. in Perspectives in Drug Discovery and Design, Vol. 4, ESCOM Science Publishers, B.V., 1996, pp. 51-60.
(7) Takagi-Sato, M., Tokuhiro, S., Kawaida, R., Koizumi, M. Fine-Tuning of ENA Gapmers as Antisense Oligonucleotides for Sequence-Specific Inhibition. Oligonucleotides (2007), 17: 291-301.

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