Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-08-07
2001-01-09
Geist, Gary (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S024300, C536S024500, C536S026700, C536S026800, C536S027600, C536S027800, C536S027810, C536S028500, C536S028530, C536S025340, C558S070000, C435S088000, C435S091100
Reexamination Certificate
active
06172209
ABSTRACT:
FIELD OF THE INVENTION
This invention is directed to aminooxy-modified oligonucleotides, to oligonucleotides that elicit RNase H for cleavage in a complementary nucleic acid strand, and to oligonucleotides wherein at least some of the nucleotides are functionalized to be nuclease resistant, at least some of the nucleotides of the oligonucleotide include a substituent that potentiates hybridization of the oligonucleotide to a complementary strand of nucleic acid, and at least some of the nucleotides of the oligonucleotide include 2′-deoxy-erythro-pentofuranosyl sugar moieties. The inclusion of one or more aminooxy moieties in such oligonucleotide provides, inter alia, for improved binding of the oligonucleotides to a complementary strand. The oligonucleotides and macromolecules are useful for therapeutics, diagnostics and as research reagents.
BACKGROUND OF THE INVENTION
Oligonucleotides are known to hybridize to single-stranded RNA or single-stranded DNA. Hybridization is the sequence specific base pair hydrogen bonding of bases of the oligonucleotides to bases of target RNA or DNA. Such base pairs are said to be complementary to one another.
In determining the extent of hybridization of an oligonucleotide to a complementary nucleic acid, the relative ability of an oligonucleotide to bind to the complementary nucleic acid may be compared by determining the melting temperature of a particular hybridization complex. The melting temperature (T
m
) a characteristic physical property of double helices, denotes the temperature in degrees centigrade, at which 50% helical (hybridized) versus coil (unhybridized) forms are present. T
m
is measured by using the UV spectrum to determine the formation and breakdown (melting) of the hybridization complex. Base stacking which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity). Consequently, a reduction in UV absorption indicates a higher T
m
. The higher the T
m
, the greater the strength of the bonds between the strands.
Oligonucleotides can be used to effect enzymatic cleavage of a target RNA by using the intracellular enzyme, RNase H. The mechanism of such RNase H cleavage requires that a 2′-deoxyribofuranosyl oligonucleotide hybridize to a target RNA. The resulting DNA-RNA duplex activates the RNase H enzyme and the activated enzyme cleaves the RNA strand. Cleavage of the RNA strand destroys the normal function of the RNA. Phosphorothioate oligonucleotides operate via this type of mechanism. However, for a DNA oligonucleotide to be useful for cellular activation of RNase H, the oligonucleotide must be reasonably stable to nucleases in order to survive in a cell for a time period sufficient for RNase H activation. For non-cellular uses, such as use of oligonucleotides as research reagents, such nuclease stability may not be necessary.
Several publications of Walder et al. describe the interaction of RNase H and oligonucleotides. Of particular interest are: (1) Dagle et al.,
Nucleic Acids Research
1990, 18, 4751; (2) Dagle et al.,
Antisense Research And Development
1991, 1, 11; (3) Eder et al.,
J. Biol. Chem.
1991, 266, 6472; and (4) Dagle et al.,
Nucleic Acids Research
1991, 19, 1805. According to these publications, DNA oligonucleotides having both unmodified phosphodiester internucleoside linkages and modified phosphorothioate internucleoside linkages are substrates for cellular RNase H. Since they are substrates, they activate the cleavage of target RNA by RNase H. However, the authors further note that in Xenopus embryos, both phosphodiester linkages and phosphorothioate linkages are also subject to exonuclease degradation. Such nuclease degradation is detrimental since it rapidly depletes the oligonucleotide available for RNase H activation.
As described in references (1), (2) and (4), to stabilize oligonucleotides against nuclease degradation while still providing for RNase H activation, 2′-deoxy oligonucleotides having a short section of phosphodiester linked nucleotides positioned between sections of phosphoramidate, alkyl phosphonate or phosphotriester linkages were constructed. Although the phosphoramidate-containing oligonucleotides were stabilized against exonucleases, in reference (4) the authors noted that each phosphoramidate linkage resulted in a loss of 1.6° C. in the measured T
m
value of the phosphoramidate containing oligonucleotides. Such a decrease in the T
m
value is indicative of an decrease in hybridization between the oligonucleotide and its target strand.
Other authors have commented on the effect such a loss of hybridization between an oligonucleotide and its target strand can have. Saison-Behmoaras et al.,
EMBO Journal
1991, 10, 1111, observed that even though an oligonucleotide could be a substrate for RNase H, cleavage efficiency by RNase H was low because of weak hybridization to the mRNA. The authors also noted that the inclusion of an acridine substitution at the 3′ end of the oligonucleotide protected the oligonucleotide from exonucleases.
U.S. Pat. No. 5,013,830, issued May 7, 1991, discloses mixed oligomers comprising an RNA oligomer, or a derivative thereof, conjugated to a DNA oligomer via a phosphodiester linkage. The RNA oligomers also bear 2′-O-alkyl substituents. However, being phosphodiesters, the oligomers are susceptible to nuclease cleavage.
European Patent application 339,842, filed Apr. 13, 1989, discloses 2′-O-substituted phosphorothioate oligonucleotides, including 2′-O-methylribooligonucleotide phosphorothioate derivatives. The above-mentioned application also discloses 2′-O-methyl phosphodiester oligonucleotides which lack nuclease resistance.
U.S. Pat. No. 5,149,797, issued Sep. 22, 1992, discloses mixed phosphate backbone oligonucleotides which include an internal portion of deoxynucleotides linked by phosphodiester linkages, and flanked on each side by a portion of modified DNA or RNA sequences. The flanking sequences include methyl phosphonate, phosphoromorpholidate, phosphoropiperazidate or phosphoramidate linkages.
U.S. Pat. No. 5,256,775, issued Oct. 26, 1993, describe mixed oligonucleotides that incorporate phosphoramidate linkages and phosphorothioate or phosphorodithioate linkages.
Although it has been recognized that cleavage of a target RNA strand using an oligonucleotide and RNase H would be useful, nuclease resistance of the oligonucleotide and fidelity of hybridization are of great importance in the development of oligonucleotide therapeutics. Accordingly, there remains a long-felt need for methods and materials that could activate RNase H while concurrently maintaining or improving hybridization properties and providing nuclease resistance. Such oligonucleotides are also desired as research reagents and diagnostic agents.
BRIEF DESCRIPTION OF THE INVENTION
In accordance with one embodiment of this invention there are provided oligonucleotides formed from a sequence of nucleotide units. The oligonucleotides incorporate a least one nucleotide unit that is functionalized to increase nuclease resistance of the oligonucleotides. Further, at least some of the nucleotide units of the oligonucleotides are functionalized with a substituent group to increase binding affinity of the oligonucleotides to target RNAs, and at least some of the nucleotide units have 2′-deoxy-erythro-pentofuranosyl sugar moieties.
In one embodiment the first and third regions have the substituent group of the shown formula. In a further embodiment each of the substituent groups has the formula O—(CH
2
)
2
—O—NH
2
. In an even further embodiment each of the substituent groups has the formula O—(CH
2
)
2
—O—N(CH
3
)
2
. In another embodiment each of the substituent groups has the formula O—(CH
2
)
2
—O—N═CH
2
.
In preferred oligonucleotides of the present invention, nucleotide units which are functionalized for increased binding affinity are functionalized to include a 2′-substituent group. In preferred embodiments, the 2′-substituent group is a 2′-aminooxy group having one o
Cook Phillip Dan
Kawasaki Andrew M.
Manoharan Muthiah
Prakash Thazha P.
Crane Larson
Geist Gary
Isis Pharmaceuticals , Inc.
Woodcock Washburn Kurtz Mackiewicz & Norris LLP
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