The synthesis of 2'-O-methyl G-clamp containing oligonucleotides and their inhibition of the HIV-1 Tat-TAR interaction.

Article abstract of DOI:10.1081/NCN-120022850

We have synthesised a 2'-O-methyl riboside phosphoramidite derivative of the cytosine analogue 9-(2-aminoethoxy)-phenoxazine ('G-clamp') and successfully incorporated it into a series of small steric blocking 2'-O-methyl oligonucleotides targeting the stem-loop region of HIV-1 TAR RNA. The 'G-clamp' containing oligonucleotides show significant increases in binding to a model TAR RNA system when the 'G-clamp' is positioned opposite the loop region. The oligonucleotides also display dose-dependent inhibition of Tat-dependent transcription of an HIV DNA template in HeLa cell nuclear cell extract.

Full text of DOI:10.1081/NCN-120022850

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The Synthesis of 2′-O-Methyl G-Clamp Containing
Oligonucleotides and Their Inhibition of the HIV-1 Tat-
TAR Interaction
Stephen C. Holmes a & Michael J. Gait a b
a Medical Research Council Laboratory of Molecular Biology, Cambridge, UK
b Medical Research Council Laboratory of Molecular Biology, Hills Rd., Cambridge, CB2 2QH,
UK
Published online: 31 Aug 2006.
To cite this article: Stephen C. Holmes & Michael J. Gait (2003) The Synthesis of 2′-O-Methyl G-Clamp Containing
Oligonucleotides and Their Inhibition of the HIV-1 Tat-TAR Interaction, Nucleosides, Nucleotides and Nucleic Acids, 22:5-8,
1259-1262, DOI: 10.1081/NCN-120022850
To link to this article: http://dx.doi.org/10.1081/NCN-120022850
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, Nos. 5–8, pp. 1259–1262, 2003
The Synthesis of 20-O-Methyl G-Clamp Containing
Oligonucleotides and Their Inhibition of the
HIV-1 Tat-TAR Interaction
*
Stephen C. Holmes and Michael J. Gait
Medical Research Council Laboratory of Molecular Biology,
Cambridge, UK
ABSTRACT
We have synthesised a 20-O-methyl riboside phosphoramidite derivative of the
cytosine analogue 9-(2-aminoethoxy)-phenoxazine (‘G-clamp’) and successfully
incorporated it into a series of small steric blocking 20-O-methyl oligonucleotides
targeting the stem-loop region of HIV-1 TAR RNA. The ‘G-clamp’ containing
oligonucleotides show significant increases in binding to a model TAR RNA sys-
tem when the ‘G-clamp’ is positioned opposite the loop region. The oligonucleo-
tides also display dose-dependent inhibition of Tat-dependent transcription of an
HIV DNA template in HeLa cell nuclear cell extract.
Key Words: Steric block; Oligonucleotides; G-clamp; Nucleoside analogue.
HIV-1 transcription is regulated by the trans-activator protein, Tat, binding to
its RNA recognition sequence, TAR, a stem loop structure that occurs at the 50-end
of all HIV RNA transcripts.[1] As such, the Tat-TAR interaction has become an im-
portant drug target and model system for the development of RNA-protein inhibitors.
*Correspondence: M. Gait, Medical Research Council Laboratory of Molecular Biology,
Hills Rd., Cambridge CB2 2QH, UK.
1259
DOI: 10.1081/NCN-120022850
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

1260
Holmes and Gait
Scheme 1. Synthesis: A: Markiewicz Reagent, Pyridine (95% yield); B: i. TMSCl, Et3N,
DCM. ii. MesCl, Et3N, DMAP. iii. s-NO2-phenol, DBO. iv. 2% Tosic acid, DCM (80%)
C: MeI, Ag2O, Acetone (85%) D: 4-nitrobenzaldoxime, tetramethylguanidine, Dioxane=Water
(75%) E: i. CCl4, PPh3, DCM, ii. 2-aminoresorcinol (75%) F: Benzyl-N-(2-hydroxyethyl)car-
bamate, DEAD, PPh3 (75%) G: KF, EtOH (60%) H: i. H2, Pd=C, DMF ii. Ethyltrifluoro-
acetate, DMAP (65%) I. DmtCl, Pyr. (85%) J. Phosphitylating agent, DIPEA, DCM (85%).
Small molecules and peptidomimetics have been previously shown to inhibit the
Tat-TAR interaction by binding to TAR-RNA in the region of a U-rich bulge.[2]
Furthermore, the system has been used to investigate the ability of short oligonucleo-
tides (and analogues) to strand invade and sterically block TAR RNA function.[3–5]
A recent development within the field of steric blocking and antisense molecules
has been the synthesis of a cytosine analogue, 9-(2-aminoethoxy)-phenoxazine (‘G-
clamp’), capable of enhanced binding and specificity towards its complementary tar-
get.[6] A single analogue incorporated into a decamer DNA sequence was found to
increase the Tm of its duplex with complementary DNA by 18ꢀC.
We have chemically synthesised a novel 20-O-methyl riboside phosphoramidite
derivative of the ‘G-clamp’ (Sch. 1) and incorporated it into a series of small steric
blocking 20-O-methyl oligonucleotides targeting the stem-loop region of TAR
RNA by solid phase methods previously described.[4,5]
The results from a gel mobility shift assay indicated that the 20-O-methyl ‘G-
clamp’ containing oligonucleotides showed significant increases in binding to a
model TAR RNA system when the base analogue was positioned opposite the
TAR RNA loop region. However, positioning the ‘G-clamp’ analogue to target
guanosine residues in the duplex region of TAR RNA resulted in poorer binding
than that obtained using an unmodified 20-O-methyl oligoribonucleotide (Table 1).

20-O-Methyl G-Clamp Containing Oligonucleotides
1261
Table 1. 20-O-methyl oligonucleotide binding to TAR RNA (Kd) and to complementary
DNA and RNA (Tm).
Gel mobility shift
binding assay
Tmc
Sequence 50-30
‘G-clamp’ analogue ¼ C
DNA
(ꢀC 1)
RNA
(ꢀC 1)
Name
Kda
Kdb
12 TAR OMe
12 TAR P1
12 TAR P3
12 TAR P4
12 TAR P5
12 TAR P9
12 TAR P11
50- CUC CCA GGC UCA
50- CUC CCA GGC UCA
50- CUC CCA GGC UCA
50- CUC CCA GGC UCA
50- CUC CCA GGC UCA
50- CUC CCA GGC UCA
50- CUC CCA GGC UCA
12 1.5
20 0.8
6.5 0.6
1.0 0.1
0.9 0.4
15 2.0
27 1.1
3.2 0.7
3.3 1.6
1.5 0.4
0.8 0.2
0.2 0.1
2.4 1.0
3.1 0.7
58
64
71
75
73
70
67
79
85
90
94
94
87
88
Gel Mobility Shift Assay Buffer Conditions:
a(TK80 buffer) 50 mM Tris HCl pH 7.4, 80 mM KCl.
b(Transcription buffer) 20 mM HEPES, 2 mM DTT, 10 mM ZnSO4, 80 mM KCl, 3 mM MgCl2,
10 mM creatine phosphate.
Thermal Melting experiment conditions:
c20 mM KCl, 5 mM Na2HPO4 pH 7.2, 10 mM MgCl2.
The stabilities of duplexes formed by the modified oligonucleotides with their
complementary DNA or RNA targets were also investigated. All duplexes contain-
ing a 20-O-methyl ‘G-clamp’ analogue showed Tm increases ranging from 4–17
degrees. The magnitude of the increase is dependent on the position of the ‘G-clamp’
within the duplex and the most stable duplexes are those where the analogue is
placed in the interior of the oligonucleotide.
The series of 20-O-methyl ‘G-clamp’ containing oligonucleotides all displayed
dose-dependent inhibition of Tat promoted transcription of an HIV DNA template
in HeLa cell nuclear cell extract.[5] Fifty percent transcription inhibition occurred at
between 50-200 nM oligonucleotide concentration with the oligonucleotide 12BRU
P5 having the greatest inhibition of in vitro transcription. The investigation revealed
a complex relationship between the binding strength of an oligonucleotide and its
ability to inhibit in vitro transcription.
We are currently investigating the incorporation of multiple ‘G-clamp’ substitu-
tions into an oligonucleotide and its effect on binding strength and inhibition of in
vitro transcription. The ability of 20-O-methyl ‘G-clamp’ modified oligonucleotides
to inhibit Tat-dependent transcription in cells is also under investigation.
REFERENCES
1. Karn, J. Tackling Tat. J. Mol. Biol 1999, 293, 235–254.
2. Wilson, W.D.; Li, K. Targeting RNA with small molecules. Curr. Med. Chem.
2000, 7 (1), 73–98.

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Manoharan, M.; Zounes, M. Pseudo half-knot formation with RNA. Science
1992, 257 (5072), 958–961.
4. Mestre, B.; Arzumanov, A.; Singh, M.; Boulme, F.; Litvak, S.; Gait, M.J.
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to a guanine in helical nucleic acids. J. Am. Chem. Soc. 1998, 120 (33), 8351–
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14,645–14,654.