The ability to modify but, more specifically, to enhance binding
between nucleotide bases has always been of critical importance in many
avenues of DNA and RNA research. The cytosine – guanine (C-G) base
pair with 3 hydrogen bonds is already much stronger than the adenine –
thymine (A-T) base pair, which has 2 hydrogen bonds. In this article,
we focus on the C-G base pair and a remarkable cytosine analogue, which
has been popularly named “G-clamp”.
G-clamp1 is a tricyclic Aminoethyl-Phenoxazine 2’- deoxyCytidine
analogue (AP-dC). The nucleoside structure and its relationship to dC
are shown in Figure 1, Page 2. The heterocyclic phenoxazine structure
provides a stable basis for the protonated amine of an aminoethyl chain
to interact with the O6 position of a complementary dG molecule, as shown
in Figure 2, Page 2. In principle, therefore, G-clamp should stabilize
a duplex due to its ability to interact with both the Watson-Crick and
Hoogsteen faces of the target G. In practice, G-clamp did indeed have
a very dramatic effect on duplex stabilization. A single G-clamp incorporation
in a poly-pyrimidine decamer resulted in a spectacular 18 °C enhancement
of the melting temperature of the duplex relative to a control containing
5-Me-dC at the same point.1
Since the G-clamp relies on the hydrogen bonding of its protonated amine
to the O6 position of the G target, specificity should, in theory, be
enhanced, and indeed it is. The sequence containing a single G-clamp residue
exhibited better discrimination between the perfect match with G in comparison
to the mismatches with A, C and T.1 In further experiments, it was shown1
that the enhanced binding of G-clamp was not affected by the nature of
the bases flanking the target G. It was also shown that the interaction
was with G rather than the phosphate backbone when it was determined that
the ionic strength of the buffer had little effect on the binding and
when uncharged linkages flanked the enhanced base pair.
The specific interaction between G-clamp and the guanine residue should
also be unaffected by the nature of the duplex. The melting temperature
of a B-form DNA-DNA duplex containing G-clamp was enhanced to about the
same extent as an A-form DNA-RNA hybrid.1
Oligonucleotides containing G-clamp have been evaluated2 for sequence-context
dependence, activity mismatch, sensitivity, RNAse-H cleavage, and hybridization
kinetics in antisense experiments. In previously optimized systems, oligophosphorothioates
(S-ON) containing a single G-clamp residue increased the potency of the
antisense oligonucleotide even in comparison to the most potent C5-propynyl-modified
S-ON previously tested by the researchers.2 Indeed, they conclude
that the G-clamp modification is a highly potent, mismatch-sensitive cytosine
analog that will find applications in elucidating gene function, in validating
gene targets, and in developing more potent antisense oligonucleotides.
Furthermore, a single incorporation of AP-dC at the 3' terminus
was shown3 to protect phosphodiester oligonucleotides from
Our own experiments confirm that AP-dC not only increases hybridization
efficiency but, at the same time, the increase is additive over more than
one addition of AP-dC. We have also confirmed that AP-dC increases mismatch
descrimination. These results are listed in Table 1 and Table 2.
These findings clearly support the conclusion that AP-dC (G-clamp) is
a very important addition to the modified nucleosides that enhance hybridization.
The product is clearly remarkable for being so stable to conditions of
oligonucleotide synthesis and deprotection. We especially envisage its
use in the development of short, easily prepared oligonucleotides for
use in highly specific single nucleotide polymorphism (SNP) and other
in vitro diagnostic assays.
We are indebted to Isis Pharmaceuticals for including AP-dC-CE
Phosphoramidite, (1) in Figure 1, to our License Agreement.
1. K.Y. Lin and M.D. Matteucci, J Am Chem Soc, 1998, 120, 8531-8532.
2. W.M. Flanagan, et al., Proc Nat Acad Sci USA, 1999, 96, 3513-3518.
3. M.A. Maier, et al., Biochemistry, 2002, 41, 1323-1327.
This product is covered by patents or patents pending owned by Isis
Pharmaceuticals, Inc. (“Isis”). Purchase of this product
includes a limited license to use this product solely for internal research.
This license specifically excludes (and you have no right to use this
product for): (a) therapeutic or diagnostic applications (including
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Neither this product nor any product created through its use may be
used in human clinical trials.
FIGURE 1: dC, AP-dC AND AP-dC-CE PHOSPHORAMIDITE
| dC AP-dC
|FIGURE 2: G - C AND G - AP-C BASE PAIRING
|G -------------- C
||G ------------- AP-C
TABLE 1: MELTING EXPERIMENTS WITH SHORT POLY-PYRIMIDINE OLIGOS
Tm measurements were carried out as described in Reference 1.
|TABLE 2: MELTING EXPERIMENTS WITH 19-MER
Tm measurements determined by thermal denaturation
in a Jasco V530 spectrophotometer. Each oligo was at a concentration
of 1 µM in 5mM Tris, 0.1 M NaCl pH 7.4