PNA-DNA-PNA chimeric macromolecules

Details PNA-DNA-PNA chimeric macromolecules PNA-DNA-PNA chimeric macromolecules

1998-07-01

2001-08-21

Yucel, Remy (Department: 1635)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S006120, C435S325000, C435S366000, C536S023100, C536S024500, C536S024300, C514S04400A

Reexamination Certificate

active

06277603

ABSTRACT:

FIELD OF THE INVENTION
This invention is directed to the synthesis and use of chimeric molecules having a PNA-DNA-PNA structure wherein each “PNA” is a peptide nucleic acid and the “DNA” is a phosphodiester, phosphorothioate or phosphorodithioate 2′-deoxyoligonucleotide. In a cell, a cellular extract or a RNase H containing diagnostic test system, a chimeric macromolecule of the invention having a base sequence that is hybridizable to a RNA target molecule can bind to that target RNA molecule and elicit a RNase H strand cleavage of that RNA target molecule.
BACKGROUND OF THE INVENTION
It is well known that most of the bodily states in mammals including most disease states, are effected by proteins. Such proteins, either acting directly or through their enzymatic functions, contribute in major proportion to many diseases in animals and man. Classical therapeutics has generally focused upon interactions with such proteins in an effort to moderate their disease causing or disease potentiating functions. Recently, however, attempts have been made to moderate the actual production of such proteins by interactions with messenger RNA (mRNA) or other intracellular RNA's that direct protein synthesis. It is generally the object of such therapeutic approaches to interfere with or otherwise modulate gene expression leading to undesired protein formation.
Antisense methodology is the complementary hybridization of relatively short oligonucleotides to single-stranded RNA or single-stranded DNA such that the normal, essential functions of these intracellular nucleic acids are disrupted. Hybridization is the sequence specific hydrogen bonding via Watson-Crick base pairs of the heterocyclic bases of oligonucleotides to RNA or DNA. Such base pairs are said to be complementary to one another.
Naturally occurring events that provide for the disruption of the nucleic acid function, as discussed by Cohen in
Oligonucleotides: Antisense Inhibitors of Gene Expression
, CRC Press, Inc., Boca Raton, Fla. (1989) are thought to be of at least two types. The first is hybridization arrest. This denotes the terminating event in which an oligonucleotide inhibitor binds to a target nucleic acid and thus prevents, by simple steric hindrance, the binding of essential proteins, most often ribosomes, to the nucleic acid. Methyl phosphonate oligonucleotides (see, e.g., Miller, et al.,
Anti
-
Cancer Drug Design
1987, 2, 117) and &agr;-anomer oligonucleotides are the two most extensively studied antisense agents that are thought to disrupt nucleic acid function by hybridization arrest.
In determining the extent of hybridization arrest of an oligonucleotide, the relative ability of an oligonucleotide to bind to complementary nucleic acids may be compared by determining the melting temperature of a particular hybridization complex. The melting temperature (T
m
), a characteristic physical property of double helixes, 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 hybridization. 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 binding of the strands. Non-Watson-Crick base pairing, i.e. base mismatch, has a strong destabilizing effect on the T
m
.
The second type of terminating event for antisense oligonucleotides involves the enzymatic cleavage of the targeted RNA by intracellular RNase H. The mechanism of such RNase H cleavages requires that a 2′-deoxyribofuranosyl oligonucleotide hybridize to a targeted RNA. The resulting DNA-RNA duplex activates the RNase H enzyme; the activated enzyme cleaves the RNA strand. Cleavage of the RNA strand destroys the normal function of the RNA. Phosphorothioate oligonucleotides are one prominent example of antisense agents that operate by this type of terminating event. For a DNA oligonucleotide to be useful for activation of RNase H, the oligonucleotide must be reasonably stable to nucleases in order to survive in a cell for a time sufficient for the RNase H activation.
Several recent publications of Walder, et al. further 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. In these papers, Walder, et al. note that 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 the 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 their oligonucleotides against nuclease degradation while still providing for RNase H activation, Walder, et al. constructed 2′-deoxy oligonucleotides having a short section of phosphodiester linked nucleotides positioned between sections of phosphoramidate, alkyl phosphonate or phosphotriester linkages. While 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 decrease in the T
m
value is indicative of an undesirable decrease in the hybridization between the oligonucleotide and its target strand.
Other authors have commented on the effect such a loss of hybridization between an antisense oligonucleotide and its targeted strand can have. Saison-Behmoaras, et al.,
EMBO Journal
1991, 10, 1111, observed that even through 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. 5,149,797 to Pederson et. al., that issued on Sep. 22, 1992 describes further oligonucleotides that operate by a RNase H mechanism. The oligonucleotides as claimed in this patent consist of an internal segment composed of phosphorothioate nucleotides flanked by methyl phosphonate, phosphoromorpholidates, phosphoropiperazidates or phosphoramidates. Since all of the components of these oligonucleotides, i.e. phosphorothioate, methyl phosphonates, phosphoromorpholidates, phosphoropiperazidates or phosphoramidates when used as oligonucleotide linkages individually decrease the hybridization between the oligonucleotide and its target strand, the comments of Saison-Behmoaras et al., could be equally applicable to the oligonucleotides described in this patent.
While it has been recognized that cleavage of a target RNA strand using an antisense oligonucleotide and RNase H would be useful, nuclease resistance of the oligonucleotide and fidelity of the hybridization are also of great importance. Heretofore, there have been no suggestion in the art of methods or materials that could both activate RNase H while concurrently maintaining or improving hybridization properties and providing nuclease resistance even though there has been a long felt need for such methods and materials. Accordingly, there remains a long-felt need for such methods and materials.
OBJECTS OF THE INVENTION
It is an object of this invention to provide chimeric macromolecules that hybridize with a target strand with improved binding a

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