Endcaps for stabilizing short DNA duplexes

Article abstract of DOI:10.1081/NCN-120023088

The syntheses of endcaps for covalently linking the 3′ and 5′ hydroxyl groups of blunt end double-stranded DNA are described. Endcap diols were converted into DMTr protected phosphoramidites and incorporated between nucleotides 4 and 5 of a self-complementary octamer. The stabilizing effect of the endcaps on duplex DNA was determined by T_m experiments on the self-complementary octamer.

Full text of DOI:10.1081/NCN-120023088

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Endcaps for Stabilizing Short DNA Duplexes
Pei-Sze Ng a , Maneesh R. Pingle a , Ganesan Balasundarum a b , Alan Friedman b , Xiaolin Zu
a & Donald E. Bergstrom a c d
a Department of Medicinal Chemistry and Molecular Pharmacology , Purdue University , West
Lafayette, Indiana, USA
b Department of Biological Sciences , Purdue University , West Lafayette, Indiana, USA
c Walther Cancer Institute , Indianapolis, Indiana, USA
d Department of Medicinal Chemistry and Molecular Pharmacology , Purdue University , 401
Hansen Building, West Lafayette, IN, 47907-1333, USA
Published online: 31 Aug 2006.
To cite this article: Pei-Sze Ng , Maneesh R. Pingle , Ganesan Balasundarum , Alan Friedman , Xiaolin Zu & Donald E.
Bergstrom (2003) Endcaps for Stabilizing Short DNA Duplexes, Nucleosides, Nucleotides and Nucleic Acids, 22:5-8, 1635-1637,
DOI: 10.1081/NCN-120023088
To link to this article: http://dx.doi.org/10.1081/NCN-120023088
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, Nos. 5–8, pp. 1635–1637, 2003
Endcaps for Stabilizing Short DNA Duplexes
Pei-Sze Ng,1 Maneesh R. Pingle,1 Ganesan Balasundarum,1,2
Alan Friedman,2 Xiaolin Zu,1
and Donald E. Bergstrom1,3,
*
1Department of Medicinal Chemistry and Molecular Pharmacology and
2Department of Biological Sciences, Purdue University,
West Lafayette, Indiana, USA
3Walther Cancer Institute, Indianapolis, Indiana, USA
ABSTRACT
The syntheses of endcaps for covalently linking the 30 and 50 hydroxyl groups of
blunt end double-stranded DNA are described. Endcap diols were converted into
DMTr protected phosphoramidites and incorporated between nucleotides 4 and 5
of a self-complementary octamer. The stabilizing effect of the endcaps on duplex
DNA was determined by Tm experiments on the self-complementary octamer.
Key Words: Endcap; Amide; Terthiophene; Stabilization; DNA duplexes.
We have developed a series of endcaps, both aromatic hydrophobic and alipha-
tic hydrophilic, for stabilizing very short DNA duplexes. The aromatic hydrophobic
endcaps are based on either a naphthalene diimide core (1)[1] which provides the
greatest stability when incorporated adjacent to a CG base pair; or a terthiophene
core (2) that results in the highest stability when incorporated adjacent to an AT base
*Correspondence: Donald E. Bergstrom, Department of Medicinal Chemistry and Molecular
Pharmacology, Purdue University, 401 Hansen Building, West Lafayette, IN 47907-1333,
USA; Fax: þ1 765 494 9193; E-mail: bergstrom@purdue.com.
1635
DOI: 10.1081/NCN-120023088
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

1636
Ng et al.
Figure 1. Synthesis of endcap spacers. 1 N,N0-bis(3-hydroxypropyl)naphthalene-1,4,5,8-
tetracarboxylic diimide 2 5, 500-bis(3-hydroxypropyl)-2,20 : 50,200-terthiophene: i) nBuLi, ii)
BF3 ꢀ Et2O,oxetane 3 N,N0-bis(3-hydroxypropyl)-2, 20-oxyacetamide: iii) CH2Cl2, Et3N, t-
butyldimethylsilyl chloride, iv) CH2Cl2, Et3N, diglycolyl chloride, v) 2.9% HCl in EtOH;
4 4-Hydroxy-N-[3-(4-hydroxybutyrylamino)propyl]butyramide: vi) dimethylaminopyridine
(DMAP), g-butyrolactone, methanol DMTr-phosphoramidite synthesis: vii) Dimethoxytrityl
chloride, DMAP, pyridine, viii) 2-cyanoethyl N,N0-diisopropylchlorophosphoramidite or
2-cyanoethyl bis(N,N0 diisopropyl)phosphoramidite, tetrazole.
pair. The hydrophilic endcaps are based on aliphatic diamides (3, 4) that provide
a more rigid structure than the commonly used poly(ethylene glycol) linkers.[2]
Endcaps can be visualized as replacements for the loop region of stem-loop hairpin
oligonucleotides. Endcaps are synthesized as dimethoxytrityl protected phosphora-
midite derivatives that can be incorporated into the oligonucleotide during auto-
mated synthesis (Fig. 1). The distance spanned by the endcaps can be readily
tuned by using an appropriate amino alcohol or carboxylic acid during synthesis
of the endcap. We have investigated the thermal melting characteristics of endcapped
four base-pair duplexes comprising one of each type of base-pair (AT, TA, GC and
Table 1. Melting temperatures (Tm) of complementary sequences linked by spacers 1–4 (P).
Spacer TmꢁC
Sequence
1, n ¼ 3
1, n ¼ 4
2
3
4
GCTA-P-TAGC
GCAT-P-ATGC
TACG-P-CGTA
CGTA-P-TACG
ATGC-P-GCAT
62
53
66
41
48
45
44
51
44
53
50
48
56
75
66
62

Stabilizing Short DNA Duplexes
1637
Figure 2. Models of complementary tetramers linked by endcaps 2 and 3.
CG). The aromatic hydrophobic endcaps result in duplexes that have Tm values in
the range of 53 to 75ꢁC (Table 1). The aliphatic hydrophilic endcaps provide
duplexes that have Tm values in the range of 41 to 56ꢁC (Table 1). Molecular model-
ing indicates that the endcaps do not result in significant distortion of the helical
duplex structure (Fig. 2). Endcapped duplexes are being studied as components of
protein-DNA complexes for structural and biological studies.
ACKNOWLEDGMENTS
Grant support from the Showalter Trust Fund and assistance from the National
Cancer Institute Grant (P30 CA23168) awarded to Purdue University is gratefully
acknowledged.
REFERENCES
1. Bevers, S.; Schuttle, S.; McLaughlin, L.W. Naphthalene- and Perylene-based
linkers for the stabilization of hairpin triplexes. J. Am. Chem Soc. 2000, 122,
5905–5915.
2. O’Dea, T.; McLaughlin, L.W. Engineering specific cross-links in Nucleic acids
using glycol linkers. In Current Protocols in Nucleic Acid Chemistry; Beaucage,
S., Bergstrom, D.E., Glick, G., Jones, R., Eds.; John Wiley and Sons, 2000;
5.3.1–5.38.