Triple - helix formation by a oligodeoxynucleotides and a oligodeoxynucleotide - intercalator conjugates ( human inmunodeficiency virus )

Abstract

Base-pair sequences in double-stranded DNA can be recognized by homopyrimidine oligonucleotides that bind to the major groove at homopurine-homopyrimidine sequences thereby forming a local triple helix. To make oligodeoxynucleotides resistant to nucleases, we replaced the natural (*8) anomers of the nucleotide units by the synthetic (a) anomers. The li-mer a oligodeoxynucleotide 5'-d(TCTCCTCCTTT)-3' binds to the major groove ofDNA in an antiparallel orientation with respect to the homopurine strand, whereas a 13 oligonucleotide adopts a parallel orientation. When an intercalating agent was attached to the 3' end of the a oligodeoxynucleotide, a strong stabilization of the triple helix was observed. A 16-base-pair homopurine-homopyrimidine sequence of human immunodeficiency virus proviral DNA was chosen as a target for a 16-mer homopyrimidine a oligodeoxynucleotide. A restriction enzyme that cleaves DNA at the junction of the homopurine'homopyrimidine sequence was inhibited by triple-helix formation. The 16-mer a oligodeoxynucleotide substituted by an intercalating agent was Z20 times more efficient than the unsubstituted oligomer. Nucleaseresistant a oligodeoxynucleotides offer additional possibilities to control gene expression at the DNA level. Sequence-specific recognition of the major groove ofDNA at homopurine-homopyrimidine sequences can be achieved by homopyrimidine oligonucleotides (1-7). Thymine and protonated cytosine form Hoogsteen-type hydrogen bonds with Watson-Crick ART and G-C base pairs, respectively. The homopyrimidine oligonucleotide is bound in a parallel orientation with respect to the homopurine-containing strand of duplex DNA. Oligonucleotide binding to the major groove of DNA is expected to interfere with biological processes-e.g., by preventing binding of regulatory proteins that are involved in controlling gene expression. It has been shown recently that triple-helix formation could inhibit restriction endonuclease cleavage or transcription factor binding at specific sequences (8-10). However, the potential use of oligonucleotides in vivo is limited by their sensitivity to nucleases that hydrolyze the phosphodiester backbone. There are several ways in which oligonucleotides can be made resistant to nucleases. The phosphodiester backbone can be modified: methylphosphonates (11), phosphotriesters (12), phosphorothioates (13), and phosphoramidates (14) have been described. Alternatively, the anomeric configuration of the nucleoside units can be changed. The 8 anomers (Fig. LA) are found in all natural nucleic acids. Oligonucleotides can also be synthesized with the unnatural (a) anomers (15-18) (Fig. lA). Such a oligodeoxynucleotides are resistant to nucleases both in vitro (18, 19) and in vivo (20). a oligodeoxynucleotides form right-handed double helices with complementary natural (*8) DNA sequences (21-23). As expected on the basis of model building (16), the two strands of the a-18 hybrid adopt a parallel orientation. An a octathymidylate was shown to bind to the major groove of duplex DNA at an (A)8-(T)8 sequence (1, 3). However, it was bound with the same orientation as its /8 analogue-i.e., parallel to the (A)8-containing strand. This orientation suggested that triple-helix formation by a T8 involved reverse Hoogsteen hydrogen bonding between thymines and ART base pairs in contrast to Hoogsteen base pairing when i thymidine was used (Fig. 1B). To determine whether this was a general property ofa oligonucleotides, we investigated the binding of an il-mer homopyrimidine a oligomer containing both cytosines and thymines. Protonated cytosine can form two hydrogen bonds with a G'C base pair. Two orientations ofcytosine are possible as shown in Fig. 1B. In one ofthem [Fig. 1B(c)], the position ofthe glycosidic bond is identical to that of thymine in the Hoogsteen mode [Fig. 1B(a)]. In the second [Fig. 1B(d)], the glycosidic bond does not occupy the same position as that ofthymine in the reverse Hoogsteen mode [Fig. 1B(b)]. This is due to the fact that thymine is changed from Hoogsteen to reverse Hoogsteen by a 1800 rotation around the N-3-C-6 axis, whereas changing cytosine from Hoogsteen to "pseudo" reverse Hoogsteen requires both a 180° rotation and a translation. Using absorption spectroscopy and footprinting assays, we show here that only the a il-mer synthesized in an antiparallel orientation with respect to the homopurine sequence binds to the major groove of duplex DNA containing a homopurine-homopyrimidine sequence. We then show that triplexes can be strongly stabilized by attaching an intercalating agent to the 3' end of the a oligomer. An a oligonucleotide binds to a 16-base-pair (bp) homopurine-homopyrimidine sequence ofproviral human immunodeficiency virus (HIV) DNA and inhibits cleavage by a restriction enzyme. The a oligonucleotide-intercalator conjugate is much more efficient than the unsubstituted one. MATERIALS AND METHODS The unmodified p oligonucleotides were obtained from the Pasteur Institute. They were purified by HPLC on reversephase columns. The a oligonucleotides were synthesized on a Pharmacia automatic synthesizer as described (24). The a anomer of 5-methylcytosine was obtained from a thymidine according to a procedure analogous to that described for the p anomer (24). The phosphoramidite derivative of 2-methoxy-6-chloro-9-(w-hydroxypentylamino)acridine was attached at the 5' end of the 8 oligonucleotides during solidphase synthesis (25). Attachment of the same acridine derivAbbreviation: HIV, human immunodeficiency virus. 6023 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 88 (1991)

6 Figures and Tables

Cite this paper

@inproceedings{SunTripleH, title={Triple - helix formation by a oligodeoxynucleotides and a oligodeoxynucleotide - intercalator conjugates ( human inmunodeficiency virus )}, author={Jian Sun and Christophe Giovannangeli and J . C . FRAN OIS and R . KURFURSTt and Th{\'e}r{\`e}se Garestier and U . ASSELINEt and T . SAISONBEHMOARAS and N . T . THUONGt and C . HfLtNE} }