Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-10-26
2004-03-16
Siew, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S091510, C435S287100, C435S287200, C536S023100, C536S025320
Reexamination Certificate
active
06706481
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of biochemistry and nucleic acid chemistry. More specifically, the present invention relates to in vitro selection of RNA nucleic acid binding species containing from one to three identical fluorescently labeled nucleotides.
2. Description of the Related Art
Nucleic acid binding species (aptamers) that bind a wide range of targets are readily selected from random sequence populations.
3-6
Selected aptamers can recognize molecules as simple as amino acids
7
or as complex as red blood cell membranes
8
. The incorporation of chemically modified bases into aptamers drastically improves their stabilities and potentially renders them suitable for use in homogenous assays with sera or urine samples.
Reagentless biosensors that can directly transduce molecular recognition to optical signals can potentiate the development of sensor arrays for a wide variety of analytes. Pluripotent nucleic acid binding species, aptamers, are readily selected, but can be difficult to adapt to biosensor applications. The adaptation of selected nucleic acid binding species (aptamers) to function as biosensors can further potentiate numerous diagnostic applications.
1.2
Some nascent examples of aptamer biosensors have already been developed. Fluorescently labeled aptamers and capillary electrophoresis coupled to laser induced fluorescence (CE-LIF) have been used to sensitively detect IgE and thrombin in solution. A labeled anti-thrombin aptamer immobilized on a glass support can detect thrombin in solution by following changes in evanescent-wave-induced fluorescence anisotropy
10
. Labeled anti-CD4 aptamers are used to stain mouse T cells that express human CD4
11
. A labeled anti-human neutrophil elastase (HNE) aptamer is as effective as an anti-anti-human neutrophil elastase antibody for detecting human neutrophil elastase on beads
12
.
However, these analytical methods are essentially mimics of methods already developed with antibodies, and generally rely upon an indirect readout of binding following washing or other separation techniques. In contrast, molecules that can directly signal the presence of analytes are proving increasingly useful as biosensors
13
. For example, a mutant of the
E. coli
phosphate binding protein labeled with a fluorescent dye at the edge of its binding site
14
exhibited a large increase in fluorescence upon inorganic phosphate-binding.
15
A similarly labeled maltose binding protein quantitatively detects maltose in solution
16
, while a labeled glucose binding protein detects glucose
17
. The conjugation of both acceptor and donor fluorophores to cAMP-dependent protein kinase yielded a sensor in which fluorescence resonance energy transfer (FRET) is modulated by cAMP
18
.
Aptamers that bind small molecules have been shown to undergo conformational changes upon interactions with their cognate ligands
19,20
. A reporter fluorophore introduced into an aptamer in a region known to undergo conformational change can lead to a change in fluorescence intensity after the binding event. However, such an introduction of a fluorophore may result in a significant loss of binding energy, possibly due to steric hindrance or the perturbation of the conformational equilibrium.
Given that functional nucleic acids contain only four monomers with limited chemistries, and that nucleic acid structure is largely predicated on Watson-Crick pairs in which nucleotides are co-dependent, it is surprising that binding species and catalysts can b e selected from random sequence pools that are substantially depleted in a given nucleotide. However, Rogers and Joyce
22
have shown that ribozymes lacking cytidine can be selected following continuous evolution of the Bartel Class I ligase. While aptamers and ribozymes can be selected from pools depleted in one of the nucleotides, a functional price is apparently paid for the lost chemistry and structure. The C-less ribozyme is roughly 100 to 10,000-fold slower than comparable ribozymes that contain cytidine.
It is advantageous, therefore, to reduce the apparent dissonance between ligand-binding and fluorescent signaling. A b initio selection methods that yield signaling aptamers containing only a few identical residues of a fluorescent nucleotide provide such a means. These selected signaling aptamers couple the broad molecular recognition properties of their aptamers with signal transduction.
The prior art is deficient in the lack of in vitro selection methods for signaling aptamers. The present invention fulfills this long-standing need and desire in the art.
SUMMARY OF THE INVENTION
The present invention provides a method of selecting signaling aptamers in vitro comprising the steps: synthesizing a DNA pool so that the DNA has a random insert of nucleotides in a specific skewed mole ratio; amplifying the DNA pool; transcribing an RNA pool from the amplified DNA wherein a nucleotide used in the RNA transcription is fluorescently labeled; applying the fluorescently labeled RNA pool to an affinity column wherein high-affinity fluorescent RNA molecules are removed from the fluorescently labeled RNA pool; obtaining a cDNA pool from the high-affinity fluorescent RNA molecules; repeating the amplification and selection steps on the fluorescent RNA molecules and cloning the fluorescent RNA molecules where the clones comprise signaling aptamers.
The present invention also provides a method of selecting signaling aptamers in vitro comprising the steps of synthesizing a DNA pool, where said DNA has a random insert of nucleotides and said nucleotides comprise a skewed mole ratio; amplifying the DNA pool wherein a nucleotide used in the DNA amplification is labeled with one or more reporter molecules; isolating the labeled single-stranded DNA from the amplified DNA; applying the labeled single-stranded DNA pool to an affinity column wherein high-affinity labeled DNA molecules are removed from the labeled DNA pool; repeating steps (a) through (d) so as to retain the high-affinity labeled DNA pool on the affinity column; and cloning the retained labeled DNA molecules where the clones comprise signaling aptamers.
In another embodiment of the present invention, there is provided a signaling aptamer that transduces the conformational change upon binding a ligand to a change in fluorescence intensity of a fluorescently labeled nucleotide incorporated into the RNA sequence of the signaling aptamer. Additionally, the fluorescently labeled nucleotide may be incorporated into a DNA signaling aptamer.
In a preferred embodiment of the present invention there is provided a fluoresceinated RNA anti-adenosine signaling aptamer.
REFERENCES:
patent: 5688670 (1997-11-01), Szostak et al.
patent: 6399302 (2002-06-01), Lannigan et al.
patent: 6426409 (2002-07-01), Winnacker et al.
Huizenga, et al.A DNA Aptamer That Binds Adenosine and ATP. Biochemistry,vol. 34, 1995, pp. 656-665.
Burke, et al.RNA Aptamers to the Adenosine Moiety of S-Adenosyl Methionine: Structural Inferences from Variations on a Theme and the Reproducibility of Selex. Nucleic Acids Research,vol. 25, 1997, pp. 2020-2024.
Famulok, et al.Aptamers as Tools in Molecular Biology and Immunology. Curr. Top Microbiol. Immunol,vol. 243, 1999, pp. 123-136.
Jhaveri, et al.Designed Signaling Aptamers that Transduce Molecular Recognition to Changes in Fluorescence Intensity. American Chemical Society,vol. 122, 2000, pp. 2469-2473.
Potyrailo, et al.Adapting Selected Nucleic Acid Ligands(Aptamers_to Biosensors. Anal. Chem.,vol. 70, 1998, pp. 3419-3425.
Patel, et al.Structure, Recognition and Adaptive Binding in RNA Aptamer Complexes. J. Mol. Biol.,vol. 272, 1997, pp. 645-664.
Ellington Andrew D.
Jhaveri Sulay D.
Rajendran Manjula
Adler Benjamin Aaron
Research Development Foundation
Siew Jeffrey
Tung Joyce
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