Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-10-25
2002-07-23
Ponnaluri, Padmashri (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S007100, C435S091500
Reexamination Certificate
active
06423493
ABSTRACT:
TECHNICAL FIELD OF INVENTION
This invention relates generally to the generation of aptamers and to the use of aptamers as diagnostic and therapeutic agents. More particularly, the present invention relates to a method of using combinatorial chemistry to prepare novel oligonucleotide sequences having at least one thiophosphate replacement in the phosphate backbone and that have enhanced target binding properties.
BACKGROUND OF THE INVENTION
Without limiting the scope of the invention, its background is described in connection with oligonucleotide agents and with methods for the isolation and generation thereof.
Oligonucleotide agents have been shown to have functional activity in vitro and thus the promise of therapeutic potential. Some of these agents are believed to operate via mechanisms such as the sequence-specific antisense translation arrest of mRNA expression or through direct binding to protein targets where they function as “decoys”. While oligonucleotide agents show therapeutic promise, various pharmacological problems must first be overcome.
Oligonucleotide agents have been used as high specificity therapeutic agents in vitro. High sensitivity to nuclease digestion, however, makes oligonucleotide agents unstable and thus impracticable for in vivo administration by either intravenous or oral routes.
From the foregoing it is apparent the there is a need in the art for methods for generating high binding, nuclease resistant oligonucleotide that retain their specificity. Also needed are compounds and methods that permit the generation of high binding, high specificity, nuclease resistant oligonucleotide agents that have an improved half-life and are target specific.
SUMMARY OF THE INVENTION
Aptamers may be defined as nucleic acid molecules that are selected from random or high-sequence diversity libraries due to their ability to bind with a target. An iterative process known as in vitro selection is used to enrich the library for species with high affinity to the target. The iterative process involves incubation of the library with the target, separation of target bound oligonucleotide (“TBO”) sequences from free TBO and amplification of the bound and thus selected TBO population to enrich the library. Amplification may be effected enzymatically, for example, using a thermostable DNA polymerase in a polymerase chain reaction (“PCR”). The result is a sub-library populated with a small subset of sequences that have a high affinity for the target. The library is then subcloned to sample and preserve the target specific DNA, RNA or mixed sequences selected. These “lead compounds” may then be studied in further detail to elucidate the mechanisms by which they interact with the target.
The present invention is directed to the generation of aptamers that are nuclease resistant and therapeutically effective. In one embodiment of the present invention, a method is provided of selection of modified oligonucleotide aptamers wherein the modification of constituent nucleotides confers nuclease resistance to the oligomer. In a first step, a random single-stranded combinatorial library is chemically synthesized wherein the synthetic library includes at least one set of 5′ and 3′ primer segments flanking a randomized region. Next, the random combinatorial library is amplified and modified using enzymatic synthesis, for example, by PCR. The synthetic reaction is provided with a pool of four nucleotides including dA, dT, dG and dC, in which at least one, for example dA is used. Not all four nucleotides, however, are chemically modified prior to use in the reaction. The modified oligonucleotide library is then placed into contact with the target molecule. Nucleotides that bind the specific target are isolated from those which do not. Next, oligonucleotides binding to the target molecule are again amplified enzymatically in the presence of the desired modified nucleotide substrates. The isolation and amplification steps are repeated iteratively until at least one oligonucleotide population of defined sequence is obtained. Sequencing and cloning of target-selected aptamers is used to isolate, preserve and enhance the conformation of the aptamer.
In one embodiment of the present invention the nucleotide modification conferring nuclease resistance is thiolation of one or both of the non-bridging oxygens around the phosphorus. The present inventors have shown that various backbone modifications, such as the phosphorothioates and phosphorodithioates, may render aptamers more nuclease resistant while still permitting efficient uptake by cells (Wang, S., Lee, R. J., Cauchon, G. G., Gorenstein, D. G. & Low, P. S.,
Proc. Natl. Acad. Sci., U.S.A
. (1995) 92: 3318).
Unfortunately, it has become apparent that oligonucleotides possessing high thiophosphate backbone substitutions appear to be “stickier” towards proteins than normal phosphate esters, attributable to non-specific interactions. (Cho, Y. S., Zhu, F. C., Luxon, B. A. & Gorenstein, D. G., J. Biomol. Struct. Dyn.(1993) 11: 685). Similarity, thiosubstitution may lead to structural perturbations in the structure of the duplex. Id. Even in specific protein-nucleic acid contacts, sulfurization of the internucleotide linkages leads to their enhanced binding (Milligan & Uhlenbeck,
Biochemistry
(1989) 28: 2849; Marshall & Caruthers, Science (1993) 259: 1564). As most of the direct contacts between DNA binding proteins and their binding sites are to the phosphate groups (Otwinowski et al., Nature (1988) 335: 321) the extent of incorporation of modified nucleotides must be controlled for thiosubstituted aptamers to retain target specificity. To this end, the present inventors have developed combinatorial library techniques where the phosphorothioate groups are controllably incorporated during library amplification rather than synthesized into “post library” selection oligonucleotide sequences. The method of the present invention provides optimization of the total number of thiophosphates incorporated into the aptamer. Thus, forming novel phosphorothioate oligonucleotides that are nuclease resistant yet have high specificity and high binding is achieved.
In another embodiment of the present invention, a method is provided for thiophosphate selection of nuclease resistant aptamers. In the first step, a random single-stranded combinatorial library is chemically synthesized where at least one set of 5′ and 3′ primer segments flanking a randomized region is included in the sequence. Next, the random combinatorial library is concurrently amplified and modified using enzymatic synthesis, e.g. PCR. The synthetic reaction is provided with a pool of four nucleotides including dA, dT, dG and dC, in which at least one is a thiophosphate.
Although a variety of methods may be employed for isolating aptamers, in one embodiment, target-oligonucleotide complexes are separated from non-binding oligonucleotides by filtration. Oligonucleotides binding to the target molecule are again amplified enzymatically in the presence of the desired thiophosphate nucleotide. The isolation and amplification may be repeated iteratively until at least one oligonucleotide population of defined sequences is obtained.
The method of the present invention was applied to NF-IL6 as a target. A specific binding sequence was obtained using the present invention to obtain a sequence that differs from the known consensus NF-IL6 binding domain. Thus, the present method provides for the generation of novel aptamers that are specific yet nuclease resistant. Although the present NF-IL6 aptamers were generated using monothiophosphate nucleotides, either mono or di-thiophosphate monomers may be used in a PCR reaction or by split synthesis (“mix and separate”), to incorporate these backbone modifications into the aptamer. Depending on whether monothiophosphate or dithiophosphate nucleotides are provided, similar or different sequences may be obtained thus expanding the repertoire of target specific aptamers to any given target.
In other aspects, the present invention is direct
Brasier Allan R.
Gorenstein David G.
King David J.
Ventura Daniel A.
Board of Regents the University of Texas System
Flores Edwin S.
Friend Thomas
Gardere Wynne & Sewell LLP
Ponnaluri Padmashri
LandOfFree
Combinatorial selection of oligonucleotide aptamers does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Combinatorial selection of oligonucleotide aptamers, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Combinatorial selection of oligonucleotide aptamers will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2912697