Glen Report 23.15: Product Update - Unnatural Base Pairs

For the last several years, Glen Research has collaborated with TagCyx Biotechnologies on the commercial development of an unnatural base pair that relies on hydrophobic interaction rather than the normal hydrogen bonding of the standard A – T and C – G base pairs. In this article, we will briefly outline the strategies for using the existing product line. We will also introduce a new highly fluorescent nucleoside analogue and suggest some applications for its use in this unnatural base pair environment.

Figure 1 shows all of the products that are currently commercially available for this technology, including dDss, the new fluorescent analogue. Although the array of products may be complicated at first glance, the set breaks down simply as they appear in the TagCyx name as y and x:

yx
DNA
dDs dPa
dDss dPa
ds dPa
RNA
sTP BiotinPaTP
10-1003
dDs
10-1003
ds
10-1003
dPa
Figure 1: Unnatural base pair phosphoramidites and triphosphates
10-1003
dDss
10-1003
sTP
10-1003
Biotin PaTP

Potential applications make use of the varying physical properties of these nucleoside analogues.

DNA Research

dDs is a weakly fluorescent and stable nucleoside analogue that can be readily incorporated into oligonucleotides for studies where dPa is the complementary “base”, as shown in Figure 2.1

Table 1
Figure 2: Unnatural base pairing due to hydrophobic interaction

ds is a fluorescent analogue which also pairs with dPa. Pac-ds CE Phosphoramidite has a phenoxyacetyl (Pac) protecting group so is best used with Pac anhydride as the capping reagent. The fluorescence of ds is very sensitive to its environment and this attribute makes ds very useful in structural analysis.2

As shown in Figure 3, dDss is strongly fluorescent and is useful as a fluorescent tag for DNA detection. dDss also forms a base pair with dPa.3

Figure 3
Figure 3: Comparison of fluorescence properties of Ds, s and Dss

RNA Research

Using template DNA containing dDs, RNA transcription using BiotinPaTP leads to RNA biotinylated at sites complementary to dDs. The presence of biotin can be used to capture and immobilize the transcribed RNA.4

Similarly, a DNA template containing dPa can be used along with sTP to generate transcribed RNA containing s at defined locations. Since the fluorescence of s is sensitive to its environment, as illustrated in Figure 4, partial structural analysis of the transcribed RNA can again be developed.5

Figure 4
Figure 4: Fluorescence of s in RNA transcripts varies with environment

Properties of dDss

dDss-CE Phosphoramidite is incorporated into oligonucleotides by standard procedures. The modified oligonucleotides can be deprotected by any of the popular procedures.3 dDss has an absorption maximum at 380nm with an extinction coefficient of 31,000 L/mole. With an emission maximum for dDss at 470nm, dabcyl would be a good quencher.

The future for this unnatural base pair family is promising and we expect to see further refinements in the base pairs for specific applications in the future.6

References

  1. I. Hirao, et al., Nucleic Acids Symp Ser (Oxf), 2006, 33-4.
  2. T. Mitsui, M.Kimoto, R.Kawai, S.Yokoyama, and I. Hirao, Tetrahedron, 2007, 35, 3528-3537.
  3. M. Kimoto, T. Mitsui, S. Yokoyama, and I. Hirao, J Am Chem Soc, 2010, 132, 4988-4988.
  4. K. Moriyama, M. Kimoto, T. Mitsui, S. Yokoyama, and I. Hirao, Nucl. Acids Res., 2005, 33, e129.
  5. Y. Hikida, M. Kimoto, S. Yokoyama, and I. Hirao, Nat Protoc, 2010, 5, 1312-23.
  6. M. Kimoto, R.S. Cox, 3rd, and I. Hirao, Expert Rev Mol Diagn, 2011, 11, 321-31.

Intellectual Property

This product is covered by patents or patents pending owned by TagCyx Biotechnologies. Purchase of this product includes a limited license to use this product solely for research. This license specifically excludes: (a) therapeutic or diagnostic applications (including products or services that incorporate this product), (b) any in vivo toxicity/safety study in support of an investigational new drug application (or foreign counterpart), (c) resale, or (d) gene functionalization activities (including products or services that incorporate data derived from gene functionalization activities) if such activities have commercial application. All of the above require a separate license from TagCyx Biotechnologies. Neither this product nor any product created through its use may be used in human clinical trials.

Ordering Information

The items in this article have been discontinued.