Glen Research is pleased to offer two new products for
the synthesis of oligonucleotides. The new products allow for the preparation
of hybridization probes with increased affinity for complementary sequences.
Both are phosphoramidites that can be readily introduced via automated
DNA synthesis at the end of solid phase syntheses. The caps favor the
formation of stable Watson-Crick duplexes by stacking on the terminal
base pair (Figure 1). Melting point increases of over 10 °C per
modification can be realized for short duplexes.1,2
The caps fit canonical Watson-Crick base pairs and do not stack well
on mismatched base pairs. This leads to increased base pairing selectivity
at the terminal and the penultimate position of oligonucleotides featuring
the caps. Base pairing fidelity is usually low at the termini, where
fraying occurs frequently in the absence of caps. The beneficial effects
of the caps are also realized when longer target strands are bound,
so there is no need for blunt ends for the duplexes formed.1,2 The caps,
when attached to the terminus of an oligonucleotide, also facilitate
purification as their lipophilicity leads to prolonged retention on
reversed phase columns or cartridges. Finally, capping of termini may
discourage the degradation of oligonucleotides by exonucleases. Figure
2 shows the structures of the phosphoramidites producing the caps.
Trimethoxystilbene
Stilbenes have been successfully employed for covalently bridging the
termini of oligonucleotide hairpins.3 The trimethoxystilbene cap that
is now available is the result of a recent study that focused on stilbenes
that are covalently linked to only one of the two strands forming a
duplex.1 The three methoxy substituents interact with the 2’-methylene
group of the nucleoside in the target strand (Figure 3), as shown in
a recent high resolution structure.4 Together with the stacking on the
terminal base pair, this leads to much-improved mismatch discrimination.
The effect is observed for any of the four possible base pairs at the
terminus.1
When employed for hybridization probes immobilized on a glass surface
in the form of a DNA microarray, the trimethoxystilbene cap increases
the signal for the fully complementary target strand.1 This feature
is particularly important for A/T-rich sequences that often cause false
negatives. The selective stabilization of neighboring Watson-Crick base
pairs helps to suppress cross hybridization that would otherwise lead
to stronger false positive results.1
Figure 4 shows a recent example of a hybridization result involving
a DNA microarray, where enhanced target signal as well as improved mismatch
discrimination at the very terminus of the probe:target duplex are demonstrated.
Pyrenylmethylpyrrolindol
This phosphoramidite (2), when employed in the last step of an oligonucleotide
synthesis, will produce a cap that is more lipophilic than the trimethoxystilbene.
The aromatic stacking moiety is linked to the terminus of the DNA through
a more rigid, cyclic linker than in the case of (1). This feature may
prove advantageous for researchers interested in exploiting the special
photophysical properties of the pyrenyl substituent. The pyrrolindol
linker is stereoregular, leading to a single product that can be readily
purified by HPLC. The pyrenyl cap is the lead compound discovered in
a recent combinatorial study that evaluated over 40 different caps.2
The cap proved particularly successful for hybridization probes with
a 5’-terminal deoxyadenosine residue.2 Again, its duplex-stabilizing
effect does not require blunt ends. The tertiary amino group can be
expected to be protonated at physiological pH, producing a cationic
functionality that may help to attract target strands electrostatically.
The five membered ring presenting the pyrenyl stacking unit mimics the
deoxyribose of natural nucleosides, making duplexes terminating in this
cap more similar in shape to unmodified DNA than those capped with the
trimethoxystilbene.
We are indebted to Professor Clemens Richert, Institute of Organic Chemistry,
University of Karlsruhe, for sharing with us the information included
in this article.
References:
1. Dogan, Z.; Paulini, R.; Rojas Stütz, J. A.;
Narayanan, S.; Richert, C. 5’-Tethered stilbene derivatives as
fidelity- and affinity-enhancing modulators of DNA duplex stability.
J. Am. Chem. Soc. 2004, 126, 4762-4763.
2. Narayanan, S.; Gall, J.; Richert, C. Clamping down on weak terminal
base pairs: oligonucleotides with molecular caps as fidelity-enhancing
elements at the 5’- and 3’-terminal residues. Nucleic Acids
Res. 2004, 32, 2901-2911.
3. (a) Wu, T.; Burch, E. L.; Bassani, D. M.; Yang, J.-S.; Schneider,
S.; Jäger, W.; Letsinger, R. L. Hybrid oligonucleotides containing
sStilbene units. Excimer fluorescence and photodimerization J. Am. Chem.
Soc. 1995, 117, 8785-8792.
(b) Lewis, F.D.; Wu, T.; Zhang, Y.; Letsinger, R.L., Greenfield, S.R.;
Wasielewski, M.R. Distance-dependent electron transfer in DNA hairpins.
Science 1997, 277, 673-676. (c) Lewis, F. D.; Liu, X.; Wu, Y.; Miller,
S. E.; Wasielewski, M. R.; Letsinger, R. L.; Sanishivili, R.; Joachimiak,
A.; Tereshko, V.; Egli, M. Structure and photoinduced electron transfer
in exceptionally stable synthetic DNA hairpins with stilbenediether
linkers. J. Am. Chem. Soc. 1999, 121, 9905-9906. (d) Lewis, F. D.; Wu,
Y.; Liu, X. Synthesis, structure, and photochemistry of exceptonally
stable synthetic DNA hairpins with stilbene diether linkers. J. Am.
Chem. Soc. 2002, 124, 12165-12173.
4. Tuma, J.; Paulini, R.; Rojas Stütz, J. A.; Richert, C. How much
pi-stacking do DNA termini seek? Solution structure of a self-complementary
DNA hexamer with trimethoxystilbenes capping the terminal base pairs.
Biochemistry 2004, 43, 15680-15687
5. Dombi, K. L.; Griesang, N.; Richert, C. Oligonucleotide arrays from
aldehyde-bearing glass with coated background. Synthesis 2002, 816-824.
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