Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-12-04
2003-07-01
Wang, Andrew (Department: 1635)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S006120, C435S091310, C435S320100, C435S375000, C536S023100, C536S023200, C536S024300, C536S024330, C536S024500
Reexamination Certificate
active
06586180
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to antisense agents. More particularly, the invention relates to compositions and methods for generation of directed antisense libraries and methods of use thereof wherein the antisense agents in the libraries can potentially bind to every binding site on a selected RNA transcript.
Antisense RNA, DNA, and ribozymes have been widely studied as research tools and potential therapeutic agents for inhibiting the expression of specific genes. These agents operate by binding to a complementary region on an RNA transcript produced from the gene of interest. On binding, the antisense agent can prevent expression of the RNA, and this can occur through a variety of different mechanisms. There are many sites on any given RNA for targeted inhibition by an antisense molecule. For a typical RNA transcript of 2000 nucleotides, just under 2000 target sites are available. Examination of a few to tens of randomly chosen target sites reveals a great variability in activity. Clearly, not all target sites are equivalent in their ability to permit antisense mediated inhibition. Consequently, identification of effective target sites on the RNA transcript for interaction with the antisense molecule is imperative for successful application of antisense technology. Methods currently available for this purpose include the use of computer algorithms to predict target accessibility based on the predicted secondary structure of the mRNA, the use of randomized oligonucleotide and ribozyme libraries in cell free systems, and the examination of a few to tens of antisense oligonucleotides, targeted to arbitrarily chosen sites, in cell culture assays. These approaches have met with limited success.
To identify the most effective target site(s), the following conditions should be met. First, all possible sites on the target RNA should be evaluated Second, evaluation should be carried out in the normal cellular milieu. This insures that the target is in its natural structure, associated with its normal complement of cellular factors. Additionally, the antisense agent has the opportunity to act on alternate structures that may arise as a result of the many RNA processing reactions.
To evaluate all target sites, antisense libraries must be used. These libraries should contain antisense molecules targeted to every site. One approach is the use of completely randomized DNA, RNA, or ribozyme libraries. The use of completely randomized libraries suffers from two major disadvantages. First, while such libraries may contain antisense molecules directed at all sites on the target RNA, they also contain antisense molecules directed at all sites of all potential RNA transcripts produced by the cell. Therefore, these random libraries potentially have the capability to inhibit expression of every gene in the cell. Because of this, random libraries are limited to in vitro use in cell free assays. Second, the complexity of these libraries is enormous. For example, a random library that uses 14 nucleotides to recognize its target must contain at least 2.6×10
8
(i.e., 4
14
) different members. Realistically, the size of the library must be at least 10- to 100-fold greater in size to insure representation of all sequences. The production and screening of such large libraries is likely beyond current capabilities.
Herein there is described a new method for identifying optimal antisense target sites against any desired RNA transcript This is a directed library approach. In other words, this approach uses an antisense library that targets every site on any selected RNA and only sites present on the selected RNA. This library, therefore, does not inhibit other non-target RNA transcripts. This approach is also an improvement over known methods because it uses relatively small libraries. For example, a library targeting an RNA transcript of 2000 nucleotides, and using 14 nucleotides to recognize its target, theoretically needs 1986 members. In practice, the library would need to be 10- to 50-times this size. At 50 times, or 99,300 members, this is still a relatively small library. These directed libraries can be used in both in vitro and in vivo assays for the detection of effective target sites for antisense mediated gene inhibition.
In view of the foregoing, it will be appreciated that a method for generating directed antisense libraries would be a significant advancement in the art. Herein is described a method for examining the entire length of any RNA transcript for sites that are accessible to antisense agents. This approach allows for the localization of the most effective sites for targeting with antisense agents.
BRIEF SUMMARY OF THE INVENTION
It is an object of the present invention to provide a simple and inexpensive method for producing directed antisense libraries against any selected RNA transcript.
It is also an object of the invention to provide a method of producing directed antisense libraries wherein such libraries contain antisense agents directed against all targets spanning the entire selected RNA transcript.
It is another object of the invention to provide a method of using directed antisense libraries for locating efficient target sites on the selected RNA transcript.
It is still another object of the invention to provide compositions for use in constructing directed antisense libraries.
It is yet another object to provide a method for making fragment libraries of a selected size of DNA fragment inserted in a cloning vector.
These and other objects can be addressed by providing a method for generating an antisense library targeted to a selected RNA transcript comprising:
(a) preparing a double-stranded cDNA, comprising a first end, a second end, and a central site thereof, from the selected RNA transcript and cloning the cDNA in a cloning vector comprising a promoter configured such that an antisense transcript of the cDNA is synthesized upon transcription mediated by the promoter, resulting in a cloned cDNA;
(b) creating a plurality of deletion derivatives of the cloned cDNA wherein each of the plurality of deletion derivatives has a deletion extending from the first end into the cloned cDNA such that the plurality of deletion derivatives comprises a deletion library comprising deletions extend serially into the cDNA;
(c) reducing the size of the cDNA contained in the deletion library to a preselected size by removing a portion of the cDNA from the second end thereof to result in a fragment library;
(d) inserting an antisense gene DNA into the central site of the cDNA in the fragment library, thereby obtaining the antisense library.
Preferred cloning vectors comprise multi-cloning sequences comprising SEQ ID NO:1 and a combination of SEQ ID NO:2 and SEQ ID NO:3. In a preferred embodiment of the invention, the deletion derivatives are created with exonuclease III resection of the cloned cDNA. The size of the cDNA contained in the deletion library is preferably reduced to a preselected size by digesting the deletion library with a type IIS restriction endonuclease. Further, inserting the antisense gene DNA into the central site of the cDNA in the fragment library preferably comprises digesting the fragment library with a type IIS restriction endonuclease, thereby creating the central site, and ligating the antisense gene DNA at the central site. A preferred antisense gene comprises a ribozyme catalytic core, more preferably, a hammerhead ribozyme catalytic core.
Another aspect of the invention relates to a method for generating a library of DNA fragments of a selected size wherein the fragments collectively span all possible sites of the selected size in a source DNA comprising a first end, a second end, and a central site thereof, comprising:
(a) cloning the source DNA in a cloning vector,
(b) creating a plurality of deletion derivatives of the cloned source DNA wherein each of the plurality of deletion derivatives has a deletion extending from the first end into the cloned DNA such that the plurality of deletion derivatives comprises a deletion library c
Chen Zhidong
Pierce Michael L.
Ruffner Duane E.
Clayton Howarth & Cannon, P.C.
Schultz James Douglas
University of Utah
Wang Andrew
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