Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-01-16
2003-09-02
Campbell, Eggerton A. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100
Reexamination Certificate
active
06613517
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an assay and kit for screening a ligand for its ability to bind to one or more nucleotide sequences in a nucleic acid target. The invention also relates to a selection method employing this assay and a kit for performing the selection method.
BACKGROUND OF THE INVENTION
In order to discover new compounds capable of controlling the expression of levels of a selected protein, it is necessary to (i) identify one or more unique promoter-region sequences that can serve as a target for regulation of gene expression under the control of that promoter, and (ii) identify compounds capable of binding to such unique promoter sequences. It would be desirable, therefore, to provide a simple, reliable assay for screening compounds for their ability to bind to particular nucleic acid sequences, especially duplex base sequences (e.g., specific sequences or sequences rich in particular bases) in double-stranded DNA, and to provide kits including nucleic acid reagents for carrying out such assays.
SUMMARY OF THE INVENTION
The invention provides a nucleic acid binding assay for screening a ligand for its ability to bind to a nucleotide sequence in a nucleic acid molecule. The binding assay employs a nucleic acid molecule having a first region and second region, adjacent to the first region. The first region includes a first site that is susceptible to modification by a modifying agent, wherein the modification is effective to block strand-directed duplication by a polymerase enzyme at the first site. The binding assay entails contacting the nucleic acid molecule with a ligand, under conditions suitable for sequence-dependent binding of the ligand to the nucleic acid molecule. The resultant reaction mixture is treated with the modifying agent, under conditions such that nucleic acid molecules having a ligand bound to the second region are substantially protected from modification at the first site, and nucleic acid molecules that do not have a ligand bound to the second region are substantially modified at the first site. After treatment with the modifying agent, the nucleic acid molecule is contacted with the polymerase enzyme, under conditions wherein only unmodified nucleic acid strands are amplified, and this amplification therefore indicates that the ligand binds to a nucleotide sequence in the second region. If desired, the amplification product can be subjected to an additional round of ligand binding, modification, and amplification.
In preferred embodiments, the nucleic acid molecule is a double-stranded nucleic acid molecule, the first and second regions are first and second duplex regions, the first site is on a first strand of the nucleic acid molecule, and only the first strand is amplified.
In one embodiment, the first site includes a non-standard nucleotide that is modified by the modifying agent, and the modification is effective to block strand-directed duplication by the polymerase enzyme at the first site. In this embodiment, where the nucleic acid molecule is a DNA molecule, the modifying agent can be a prokaryotic or eukaryotic repair enzyme that recognizes and removes the non-standard nucleotide from duplex DNA or DNA/RNA duplexes.
In a variation of this embodiment, the nucleic acid molecule includes a second strand including one or more non-standard nucleotides that can be modified by a modifying agent. The modifying agent can be the same as or different from that used to modify the first site. Treatment with this modifying agent is effective to modify the non-standard nucleotide(s) in the second strand, which blocks strand-directed duplication of the second strand by the polymerase enzyme.
In an alternative embodiment, the modifying agent is a restriction enzyme and the first site includes a recognition site for the restriction enzyme.
The modifying agent(s) is/are preferably removed or inactivated prior to amplification of unmodified nucleic acid strands, which is preferably carried out by primer-directed PCR amplification.
In one embodiment, a set of different nucleic acid molecules that include different nucleotide sequences in the second region is assayed. In this embodiment, the method preferably includes determining the nucleotide sequence of the amplification product in the second region to determine whether the ligand shows binding specificity for one or more nucleotide sequences in the second region. This embodiment can be used for high-throughput screening of ligands and nucleic acid molecules.
The nucleic acid binding assay of the invention can be used to assess the binding affinity of the ligand-nucleic acid molecule interaction. In this case, the method additionally includes determining the concentration of the ligand that protects approximately 50 percent of the first sites from modification as an indication of the dissociation constant (K
d
) of the ligand. Where the amplification of unmodified nucleic acids is carried out by primer-directed PCR amplification, approximately 50 percent protection can determined by determining the concentration of ligand that gives a Ct value about 2 higher than the Ct value obtained when the nucleic acid molecule amplified without prior exposure to ligand or modifying enzyme.
The invention also provides a nucleic acid selection method that is based on the nucleic acid binding assay of the invention. The selection method can be used to screen a set of ligands for the ability to bind to nucleotide sequence(s) in a set of nucleic acid molecules, allowing, in effect, co-selection of ligands and their cognate binding sites. Each member of the set of nucleic acid molecules has a first region and a second region, adjacent to the first region. In the first region, the nucleic acid molecules include a first site that is susceptible to modification by a modifying agent, and such modification is effective to block strand-directed duplication by a polymerase enzyme at the first site. The nucleic acid molecules include different nucleotide sequences in the second region.
The selection method entails contacting the nucleic acid molecule with a ligand, under conditions suitable for sequence-dependent binding of one or more ligands to one or more nucleic acid molecules. The resultant reaction mixture is treated with the modifying agent, under conditions such that nucleic acid molecules having a ligand bound to the second region are substantially protected from modification at the first site, and nucleic acid molecules that do not have a ligand bound to the second region are substantially modified at the first site. After this treatment, the modifying agent is typically removed or inactivated before continuing on with the method.
Ligands that bind to the nucleic acid molecules are then separated from unbound ligands, and the ligands that bind to the nucleic acid molecules are recovered. In one embodiment, bound/unbound separation is facilitated by employing nucleic acid molecules linked to a substrate; unbound ligands are removed from the substrate by washing, and then bound ligands are eluted off of the substrate. Also, after treatment with the modifying agent, the set of nucleic acid molecules is contacted with the polymerase enzyme, under conditions wherein only unmodified nucleic acid strands are amplified. The selection method thus produces a pool of selected ligands that bind in a sequence-dependent manner to one or more nucleotide sequences present in a pool of selected nucleic acid molecules.
In preferred embodiments of the selection method, the ligands are heterologous peptides, expressed on the surface of phage particles, each phage particle includes a nucleic acid molecule encoding the heterologous peptide expressed on its surface, and thus the pool of selected ligands is a pool of selected phage particles. In one such embodiment, the method includes an additional round of selection that entails amplifying the pool of selected phage particles to produce amplified, selected phage particles, and contacting the pool of selected nucleic acid molecules with the amplified, selected
Campbell Eggerton A.
Genelabs Technologies, Inc.
LandOfFree
Nucleic acid binding assay and selection method does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Nucleic acid binding assay and selection method, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Nucleic acid binding assay and selection method will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3035350