Control of Gene Expression by Triple Helix-Forming Oligonucleotides. The Antigene Strategy

Control of Gene Expression by Triple Helix-Forming Oligonucleotides

The Antigene Strategy"

CLAUDE HELENE Laboratoire de Biophysique

Muskum National d 'Histoire Naturelle 43, me Cuvier

75231 Paris Cedex 05, France

INSERM U. 201 - UA. 481

NGUYEN T. THUONG Centre de Biophysique Molkculaire

CNRS lA, avenue de la Recherche Scientifique

45071 Orlkans Cedex 02, France

ANNICK HAREL-BELLAN Laboratoire d'lmmunologie

Institut Gustave Roussy 94805 VillejuiJ France

LIRA - CNRS 1156

Oligonucleotides can be used to control gene expression according to several strategies. In the antisense approach the oligonucleotide is targeted to a messenger RNA, thereby inhibiting translation, or to a viral RNA (or single-stranded DNA) where it can control RNA or DNA replication or reverse transcription. An oligode- oxynucleotide is able to induce cleavage of the target RNA via activation of an endogenous RNase H. An oligoribonucleotide may also induce catalytic cleavage of RNA in the absence of any protein (ribozyme). Antisense RNAs and ribozymes can be synthesized within cells via genetic constructs. In the sense approach the oligonucleotide is used to bind a protein: a single-stranded oligoribonucleotide can be used to trap viral proteins, such as tat and rev from HIV; a double-stranded oligonucleotide can act as a trap for a transactivating factor involved in the control of transcription. In the antigene strategy the oligonucleotide binds to a specific sequence of DNA, thereby forming a triple helix that may inhibit transcription of the target gene. This review presents the latest developments of this new strategy to control gene expression. Reviews of the other strategies are presented elsewhere.'.2

'This work was supported in part by Rhhe-Poulenc-Rorer and by the Agence Nationale de Recherche contre le Sida (ANRS).

21

28 ANNALS NEW YORK ACADEMY OF SCIENCES

TRIPLE HELIX FORMATION BY OLIGONUCLEOTIDES BOUND TO THE MAJOR GROOVE OF DNA

Base Triplets

A homopurine-homopyrimidine sequence of duplex DNA can be recognized by an oligonucleotide containing the usual bases (FIG. 1). Recognition involves the formation of two hydrogen bonds with the purines of Watson-Crick base pairs within the major groove of the double helix. Thymine, cytosine, and guanine can adopt two different orientations; they will be called “Hoogsteen” and “reverse Hoogsteen” by analogy with the hydrogen-bonding scheme discovered by Hoogsteen in co-crystals of A and T derivatives (FIG. 1). In contrast, adenine and inosine can form two hydrogen bonds with an A.T base pair in a single orientation. It should be noted that in order to form two hydrogen bonds with G, cytosine must be protonated. Therefore, triplets involving C+ x G.C are more stable at acidic pH. Methylation at C-5 of cytosine shifts the pH range where triplexes can form to higher values, probably as a result of

!&

FIGURE 1. Base triplets formed by Watson-Crick A.T and G.C. base pairs with T, protonated C (C+), G, A, and I. If bases in the third strand are in the anti conformation with respect to the sugar ring, the triplets in the left column correspond to a parallel orientation of the third strand with respect to the homopurine sequence of DNA target; those in the righr column correspond to an antiparallel orientation. If bases in the third strand were in the syn conformation, the orientation would be reversed. For the T x A.T triplets the left- hand one corresponds to the so- called Hoogsteen hydrogen-bonding pattern; the right-hand one to reverse Hoogsteen.

HELENE et al.: ANTIGENE STRATEGY 29

0

CI' 1' Y

CI ' THIRD STRAND IiOOGSTEEN

G . 0 1

0

CI' PY

CI' THIRD STRAND REVERSE ffOO(;STEEN

@

c1 I'u

DOUBLE HELIX DOUBLE HELIX

FIGURE 2. Position of C 1' atoms in the plane of base triplets. The C 1 ' atoms of the Watson-Crick strands are lixed in order to show the variations in position of the C 1' atom of the third strand of the triple helix. Hoogsteen and reverse Hoogsteen refer to the type of hydrogen bonds formed by the third base with the Watson-Crick base pair. They correspond to the left and right columns of FIGURE 1 , respectively.

hydrophobic interactions between the methyl substituents of 5-methylcytosine and thymine which form a helical spine of hydrophobic group^.^.^

An important parameter governing the stability of triple helices (besides the intrinsic energy of triplet formation) is the energy penalty associated with the distortion of the phosphodiester backbone when one moves from one triplet to the next one. This is illustrated in FIGURE 2 where the position of the C1' atom of the nucleotide in the third strand is given with respect to the C1' atoms of the two nucleotides involved in Watson-Crick hydrogen bonding. Only two triplets are isomorphous: T x A.T and C+ x G.C in the Hoogsteen configuration. Therefore, only homopyrimidine oligonucleotides can form a third strand without backbone distortion. All other triplets (including T x A.T and C+ x G.C in the reverse Hoogsteen configuration) are not isomorphous. Any combination of these triplets will be associated with an energy penalty due to backbone distortion.

Third-Strand Orientation

The orientation of the third strand will be parallel to the homopurine target strand if the base triplets are in the Hoogsteen configuration and if nucleotides in the third strand have the anti conformation. Third-strand orientation is antiparallel to the homopurine strand when reverse Hoogsteen hydrogen bonding is involved with anti conformation. Changing the conformation of the nucleosides from anti to syn will change the orientation of the third strand.

Energy calculations suggest that Hoogsteen triplets T x A.T, C+ x G.C, and G x G.C are more stable than are those involving reverse Hoogsteen hydrogen b ~ n d i n g . ~ For oligonucleotides containing only T and C, the parallel orientation (with respect to the homopurine sequence) will always be favored. However, the distortion of third strand backbone is less in the reverse Hoogsteen configuration for T X A.T and G X G.C triplets (FIG. 2). Consequently the orientation of oligonucleotides containing G


and T is expected to depend on the sequence? a high number of GpT or TpG steps will be detrimental to parallel binding (with respect to the homopurine sequence) in the Hoogsteen configuration as a result of the higher energy penalty associated with backbone distortion. Therefore, oligo (G,T) with a large number of GpT and TpG steps will prefer an antiparallel orientation even though reverse Hoogsteen hydrogen bonding is not the lowest energy triplet configuration for both T x A.T and G x G.C triplets.

In contrast, an oligo (G,T) with few GpT and TpG steps will bind more strongly in a parallel orientation. We have exploited this observation to design oligonucleotides that will bind to two consecutive homopurine.homopyrimidine sequences where the two homopurine sequences belong to different strands of the double helix.5 The oligo (G,T) switches from one strand to the other without any interrup- tion of base triplet formation. This allows extension of the number of DNA sequences that can be recognized by triple helix-forming oligonucleotides. Other possibilities for switching from one strand of the double helix to the other exist, but they involve a linker between two oligonucleotide sequences and leave unrecognized one or several base pairs between the two target

SpeciJiCity of Triple Helix Formation

Mismatches in short oligonucleotide duplexes destabilize the hybrids. This destabilization has been exploited in the case of antisense oligonucleotides to discriminate between two messenger RNAs that differ by a single point mutation, as in ras oncogene activation.s

When a homopurine sequence in DNA is interrupted by a pyrimidine, destabilization of triplex structures is observed. The stability of triple helices has been estimated from the extent of cleavage induced by oligonucleotide-EDTA-Fe conju- gatesg and determined from Tm measurements.I0 Measurements of the superhelical twist required to induce intramolecular triplex formation (H-DNA) have also been used to determine the energy penalty associated with mismatches in triplexes.I1 The change in temperature of half dissociation (Tm) of the third strand from its duplex target depends on the nature and location of the mismatch.I0 The most destabilizing triplet in our experiments was C x T.A. A T.A base pair in the center of Pu.Py base pairs can be recognized by G in the third strand if both neighbors of T are adenines on each side,!' but a guanine on either (or both) side(s) of T does not allow recognition of T.A by G.I0 As expected, a terminal mismatch is less detrimental to triplex stability than is a central mismatch.

Kinetics and Thermodynamics of Triplex Formation

The association of an oligonucleotide with duplex DNA to form a triplex is much slower than is duplex f ~ r m a t i o n . ~ ~ J ~ Measurements of activation energies have led to the conclusion that the rate-limiting step (nucleation) involves the formation of 3 to 5 base triplets before zippering occurs. A high negative activation enthalpy is associated with the nucleation process.I3 Dissociation of a third strand from a duplex is also a slow process as compared to duplex dissociation. When formed, a triplex can be rather long-lived.

These kinetic phenomena have to be taken into account when competition experiments are carried out between a triple helix-forming oligonucleotide and another DNA ligand, such as a protein.


The stability of triplexes depends on base sequence and on p H if cytosines are involved in the third strand (as a result of the requirement for cytosine protonation). Thermodynamic studies are presently underway in several laboratories to determine thermodynamic parameters associated with each base triplet in its appropriate environment.

Cations stabilize triple helices as expected because three negatively charged backbones are involved in triplex formation. Polycations such as Mg2+ or Spermine4+ are more efficient than are monocations such as Na+. In the presence of Mg2+ or Spermine4+, Na+ ions destabilize triple helical structures. It should be noted that formation of G x G.C reverse Hoogsteen base triplets requires the presence of Mg2+; that of A x A.T base triplets appears to be favored by Zn2+.

a- Oligonucleotides

Oligonucleotides synthesized with the a-anomers of nucleotide units instead of natural P-anomers are assumed to adopt the opposite orientation. (In the a-ano- meric configuration the base is on the same side as the 3’-OH with respect to the deoxyribose, whereas in the natural P-anomers the base is on the same side as the 5’-OH). An oligo-a-T, was shown to bind in a parallel orientation with respect to the homopurine sequence, that is, the same orientation as the corresponding oligo-p- T,.I4 If in both cases thymidines have an anti conformation, this result implies that the T x A.T triplets adopt the reverse Hoogsteen configuration for the a-anomers and the Hoogsteen configuration for the P-anomers. Oligo-a-deoxynucleotides containing both C and T bind in an antiparallel orientation, while the corresponding oligo-p-deoxynucleotides bind in a parallel orientation with respect to the homopurine sequence.Is Therefore, in both cases the Hoogsteen configuration is preferred for both triplets T x A.T and C+ X G.C, as expected because these Hoogsteen triplets are isomorphous. Combination of bases other than C and T in the third strand have not yet been investigated for a-oligonucleotides.

Stabilization of Triple Helical Structures

As mentioned, substitution of 5-methylcytosine to cytosine increases the stability of triplex structures obtained with oligonucleotides containing C and T. This holds true for a-oligonucleotides as well as natural P-oligomers. The pK of cytosine is only slightly shifted (0.2 p H units) by methylation at the C-5 position. Therefore, it appears more likely that additional stabilization is provided by hydrophobic interactions between the methyl groups of 5-methylcytosine and thymine. When the third strand wraps around the double helix within the major groove, a helical “spine” of methyl groups is formed.

Strong stabilization of triplex structures was obtained when the third-strand oligonucleotide was substituted by an intercalating agent.4 The effect was greater when substitution occurred at the 5’ end. The substituent (an acridine derivative) was shown to be intercalated at the triplex-duplex junction. Intercalation provides additional binding energy that stabilizes the triple helical complex. Similar stabilization was observed when a homopyrimidine oligo-a-deoxynucleotide was substituted at its 3‘ end.Is As the a-oligomer was bound in a reverse orientation as compared to the corresponding p-oligomer, intercalation took place at the same triplex-duplex junction as with the 5’ substituted P-oligomer. A 16-bp homopurine.homopyrimidine


sequence repeated twice in the HIV proviral genome was chosen as a target for these oligonucleotides. l5

When a psoralen derivative was attached to the 5' end of an oligopyrimidine, cross-linking of the two strands of the duplex was observed at the triplex-duplex junction when the complex was UV irradiated.16 This cross-linking is much more efficient when a 5'TpA3' step is present on the homopurine-containing strand.

CONTROL OF TRANSCRIPTION BY TRIPLEX-FORMING OLIGONUCLEOTIDES

Published experiments aimed at inhibiting gene transcription by triplex-forming oligonucleotides are still rather limited (in contrast to the numerous examples of antisense inhibition of mRNA translation).

Several mechanisms exist by which triple helix formation can alter gene transcription:

1. Triple helix formation within the promoter region can change DNA conformation and therefore alter the rate and efficiency of RNA polymerase initiation. This can lead to either activation or inhibition of transcription.

2. Oligonucleotide binding to a DNA sequence overlapping a transcription factor binding site may inhibit its transactivating capacity.

3. Triplex formation within or adjacent to the region where RNA polymerase binds may inhibit transcription initiation even if RNA polymerase and transcription factors are still bound to the promoter.

4. Oligonucleotide binding downstream of the RNA polymerase recognition site might inhibit progression of the transcription machinery along the DNA and therefore block RNA elongation.

Inhibition of Transcription by Inhibition of Transcription Factor Binding

Cooney et al. l7 first described that a 28nt-long oligonucleotide bound to a purine-rich sequence (but not homopurine) in the c-myc gene promoter inhibited transcription in vitro. The same group later reported that the same oligonucleotide could inhibit c-myc gene transcription in cells in culture. (As a matter of fact, the oligonucleotide was bound to an overlapping sequence in the reverse orientation as compared to the original proposal).18 They also provided evidence for triple helix formation within cells by demonstrating that a DNase hypersensitive site located within the target sequence was protected in cells incubated with the oligonucleotide. It was proposed that the 28nt-long oligonucleotide inhibited c-myc mRNA synthesis via competition with a transcription factor recognizing part or all of the triplex target. l8

Subunit a of the interleukin-2 receptor (IL2-Ra) was also chosen as a target for triplex-forming oligonucleotides. A 30% inhibition of IL2-Ra expression was observed when cells were incubated in the presence of a 34nt-long purine-rich oligonucleotide expected to bind in an antiparallel orientation with respect to a purine-rich sequence within the promoter region.19 Grigoriev et aL20 used a 15-mer homopurine oligonucleotide that binds to a 15-bp homopurine.homopyrimidine sequence in the promoter region of the IL2-Ra gene (FIG. 3). This target sequence overlaps by 3 bp the recognition sequence of the NF-KB transcription factor. Triplex formation was shown to inhibit transcription factor binding in vitro. The stability of


the triplex was strongly increased by substituting 5-methylcytosine t o cytosine and by covalent attachment of an intercalating agent at the 5' end of the oligonucleotide. Oligonucleotide-intercalator conjugates exhibited much higher affinity due to additional binding energy provided by intercalation at the duplex-triplex j ~ n c t i o n . ~

The IL2-Ra gene promoter was fused to the bacterial reporter chloramphenicol acetyltransferase (CAT) gene. To demonstrate that the effects of triplex-forming oligonucleotides on CAT gene expression were due to triple helix formation, a mutant of the target sequence was constructed by inverting three base pairs within the homopurine . homopyrimidine sequence (FIG. 3). Footprinting studies showed

WILD TYPE

\ Triplex target

s i t e

\ NF KB

J , IAACGGC+GGGdTCTfjCCTCTCCTTTdAT '

Tr ip lex ____-_____-__-_____-__________________.

MUTATED

Mutat ion

FIGURE 3. The promoter region of the gene coding for subunit aof the interleukin-2 receptor (IL2-Ra) has been cloned upstream of the chloramphenicol acetyltransferase (CAT) gene. The binding site for the transcription factor NF-KJ3 and the site for triple helix formation are boxed. The mutant promoter used as a control does not allow for triple helix formation. The triplex-forming 15-mer oligonucleotide containing thymine and 5-methylcytosine (shown below the boxed sequence of the wild-type promoter) was substituted at its 5'-end by either an acridine derivative to stabilize the triple helix (refs. 4 and 15) or a psoralen derivative to photo-induce a cross-link between the two strands of the duplex at the 5'TpA3' step on the 3'-side of the homopyrimidine target sequence (ref. 16). See references 20 and 21 for experimental results.

that the triplex-forming 15 mer did not bind to the mutant sequence. This mutation, however, did not affect NF-KB-dependent transcription of the IL2-Ra-CAT construct.

When the IL2-Ra-CAT construct was transfected into human lymphocytes and exposed to the 15 mer containing T and "C, no inhibition was observed on either the wild-type (WT) or the mutated promoter. However, when the 15 mer was 5'- substituted by acridine, a strong reduction in CAT gene expression was seen with the WT sequence but not with the mutant. An acridine-substituted 11 mer had n o effect


on either construct, demonstrating that the effect of Acr-15-mer was not due to degradation products such as acridine itself.

When the 15 mer was S’-substituted with psoralen, we could easily show in vitro that the two strands of the plasmid carrying the WT promoter were cross-linked under UV irradiation, whereas no cross-linking was detected with the mutant. We first irradiated the plasmids in vitro and then transfected the cross-linked plasmids into recipient cells; CAT gene expression was strongly reduced with the WT construct, whereas no inhibition was seen with the mutant. In another set of experiments the plasmids were first transfected into cells in the presence of pso-15- mer, and then the transfected cells were irradiated. The plasmids were extracted and cut with restriction enzymes that generate a 2,400-bp fragment expected to contain the cross-link. After denaturation of the fragments those that are cross-linked migrate as dimers, because the two strands remain covalently attached to each other. The results clearly showed that the WT construct was cross-linked at the expected position, whereas the mutant was not. When CAT expression was measured in irradiated cells, the correlation between cross-linking and inhibition of CAT gene expression was very good. The experiments with the mutant clearly showed that the observed effects were due to triple helix formation at the expected target site.

Control of Transcription by Repressor Oligonucleotides

A 13-mer oligonucleotide was bound to a homopurine 1 homopyrine sequence adjacent to or slightly overlapping the RNA polymerase binding site on the Escheri- chiu coli p-lactamase gene promoter.22 Both RNA polymerase and the oligonucleotide could bind to the same DNA fragment, as shown by footprinting experiments. Triplex formation inhibited transcription initiation in a concentration- and temperature-dependent manner. However, this inhibition was only transiently observed. Dissociation of the oligonucleotide from its target sequence allowed fast initiation of transcription in the presence of the four NTPs. As reported above, dissociation and association reactions are slow processes for intermolecular triple helix formation from a duplex and a single-stranded oligomer. Transcription from the p-lactamase promoter is faster (when RNA polymerase is bound to its promoter before adding the four NTPs) than association of the oligonucleotide at the concentrations used in these studies. Therefore, only a temperature-dependent delay was seen in the synthesis of the full transcripts.

On the contrary, a time-independent inhibition was obtained when the oligonucleotide was covalently linked to a psoralen derivative and the complex irradiated before the start of transcription. Covalent linkage of the oligonucleotide-psoralen conjugate occurred on the nontranscribed strand. (No cross-link between the two DNA strands was made due to the absence of a 5‘TpM’ sequence at the triplex- duplex junction.) Nevertheless, transcription initiation was irreversibly arrested.

The 13-mer triple-helix-forming oligonucleotide targeted to the p-lactamase gene behaves as a repressor of transcription. An analogy can be drawn with the lac repressor which binds to a region overlapping the RNA polymerase binding site but does not prevent polymerase binding to the promoter sequence.23 When the repressor is tightly bound, transcription initiation does not occur. In the presence of an inducer the dissociation rate of the repressor is increased and transcription begins.


CONCLUSION

Formation of a triple helix resulting from oligonucleotide binding to the DNA double helix offers new possibilities to control gene expression at the transcriptional level. Two modes of action have been documented until now: ( 1 ) the oligonucleotide can behave as a negative regulatory factor by inhibiting the binding of a transcription- activating protein to its recognition sequence; ( 2 ) the oligonucleotide can act as a repressor when it binds to a site adjacent to or overlapping the binding site of RNA polymerase in the downstream region of the polymerase binding site. Additional modes of action can be expected, such as (1) alteration of DNA conformation with subsequent effects on transcription kinetics and efficiency even when the oligonucleotide binding site does not overlap any binding site of a component of the transcription machinery; and (2) transcription arrest when the oligonucleotide is bound within the transcribed region but not adjacent to the polymerase bound to its promoter. Oligonucleotide-intercalator conjugates might prove to be strong stoppers of RNA chain elongation, especially when the site of intercalation is located upstream of the triple helix and encountered first by the RNA polymerase moving along its template.

It should be kept in mind that triplex-forming oligonucleotides might also interfere with DNA r e p l i c a t i ~ n . ~ ~ When the oligonucleotide carries a reactive group, DNA alterations that are induced site-specifically are expected to be recognized by repair enzymes. Experiments are presently underway to analyze the mechanisms and biological consequences of DNA modifications induced on DNA within living cells by triple-helix-forming oligonucleotides.

Triple helices can be formed on single-stranded nucleic acids. A homopurine sequence can be recognized by two oligonucleotides, one forming Watson-Crick base pairs, the other one forming Hoogsteen hydrogen bonds with the Watson-Crick double helix. These two oligonucleotides can be linked together at either or both ends.26 The stability of the triple helix formed by such molecules is strongly increased as compared to the oligonucleotide forming only Watson-Crick base pairs. Nearly all hydrogen-bonding possibilities of the purines in the target sequences are used for r e ~ o g n i t i o n . ~ ~ Therefore, triple helices might also find applications in the antisense field by allowing strong complexes to be formed on selected homopurine sequences of a messenger RNA.

ACKNOWLEDGMENTS

We wish to thank all colleagues who have contributed to the work described in this review. Their names and contributions can be found in the reference list.

REFERENCES

1. HELENE, C. & J. J. TOULME 1990. Biochim. Biophys. Acta 1049: 99-125. 2. HELENE, C. 1991. Eur. J. Cancer 27: 1466-1471. 3. THOMAS, J. P. & P. DERVAN. 1989. J. Am. Chem. SOC. 111: 3059-3061. 4. SUN, J. S., J. C. FRAN~OIS , T. MONTENAY-GARESTIER, T. SAISON-BEHMOARAS, V. ROIG,

5. SUN, J. S., T. DE BIZEMONT, G. DUVAL-VALENTIN, T. MONTENAY-GARESTIER & C. N. T. THUONG & C. HELENE. 1989. Proc. Natl. Acad. Sci. USA 8 6 9198-9202.

HELENE. 1991. C. R Acad. Sci. Paris, t. 313, Serie 111: 585-590.


6. 7. 8.

9. 10.

11.

12. 13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23. 24.

25.

26.

HORNE, D. A. & P. DERVAN. 1990. J. Am. Chem. SOC. 112 2435-2437. ONO, A., C. N. CHEN & L. S. KAN. 1991. Biochemistry 30: 9914-9921. SAISON-BEHMOARAS, T., B. TOCQUE, 1. REY, M. CHASSIGNOL, N. T. THUONG & C. HLLENE.

GRIFFIN, L. C. & P. DERVAN. 1989. Science 254 967-971. MERGNY, J. L., J. S. SUN, M. ROUGEE, T. MONTENAY-CARESTIER, F. BARCELO, J.

CHOMILIER & C. HELENE. 1991. Biochemistry 3 0 9791-9798. BELOTSERKOWSKII, B. P., A. G . VESELKOV, S. A. FILIPPOV, V. N. DOBRYNIN, S. M. MIRKIN

& M. D. FRANK-KAMENETSKII. 1990. Nucleic Acids Res. 18: 6621-6624. MAHER, L. J., P. B. DERVAN & B. J. WOLD. 1990. Biochemistry 2 9 8820-8826. ROUGEE, M., B. FAUCON, J. L. MERGNY, F. BARCELO, C. GIOVANNANGELI, T. MONTENAY-

GARESTIER & C. HELENE. 1992. Biochemistry. Submitted. LE DOAN, T., L. PERROUAULT, D. PRASEUTH, N. HABHOUB, J. L. DECOUT, N. T. THUONG,

J. LHOMME & C. H~LENE. 1987. Nucleic Acids Res. 15: 7749-7760. SUN, J. S., C. GIOVANNANGELI, J. C. FRANCOIS, R. KURFURST, T. MONTENAY-GARESTIER,

T. SAISON-BEHMOARAS, N. T. THUONG & C. HELENE. 1991. Proc. Natl. Acad. Sci. USA 8 8 6023-6027.

TAKASUGI, M., A. GUENDOUZ, M. CHASSIGNOL, J. L. DECOUT, J. LHOMME, N. T. THUONG & C. HELBNE. 1991. Proc. Natl. Acad. Sci. USA 88: 5602-5606.

COONEY, M., G. CZERNUSZEWICZ, E. H. POSTEL, S. J. FLINT& M. E. HOGAN. 1988. Science 241: 456-459.

POSTEL, E. H., S. J. FLINT, D. J. KESSLER & M. E. HOGAN. 1991. Proc. Natl. Acad. Sci.

ORSON, F. M., D. W. THOMAS, W. M. MCSHAN, D. J. KESSLER & M. E. HOGAN. 1991. Nucleic Acids Res. 19 3435-3441.

GRIGORIEV, M., D. PRASEUTH, P. ROBIN, A. HEMAR, T. SAISON-BEHMOARAS, A. DAUTRY- VARSAT, N. T. THUONG, C. HELENE & A. HAREL-BELLAN. 1992. J. Biol. Chem.

GRIGORIEV, M., D. PRASEUTH, P. ROBIN, A. L. GUYESSE, N. T. THUONG, C. HELENE & A.

DUVAL-VALENTIN, G., N. T. THUONG & C. HELENE. 1992. Proc. Natl. Acad. Sci. USA

HELENE, C. & G . LANCELOT. 1982. Prog. Biophys. Molec. Biol. 39 1-68. BIRG, F., D. PRASEUTH, D. ZERIAL, N. T. THUONG, U. ASSELINE, T. LE DOAN & C.

GIOVANNANGELI, C., T. MONTENAY-GARESTIER, M. ROUG~E, M. CHASSIGNOL, N. T.

KOOL, E. T. 1991. J. Am. Chem. SOC. 113: 6265-6266.

1991. EMBO J. 1 0 1111-1118.

USA 1991.88: 8227-8231.

267: 3389-3395.

HAREL-BELLAN. 1992. Submitted.

89 504-508.

HELENE. 1990. Nucleic Acids Res. 1 8 2901-2908.

THUONG & C. HELENE. 1991. J. Am. Chem. SOC. 113: 7775-7777,

Control of Gene Expression by Triple Helix-Forming Oligonucleotides. The Antigene Strategy

Documents

Transcript of Control of Gene Expression by Triple Helix-Forming Oligonucleotides. The Antigene Strategy