Detection of nucleic acids is the cornerstone of biotechnology and molecular biology. Single-base pair mutations in a gene can be detected with great specificity and accuracy. The level of expression of a particular gene can be measured by detecting mRNA expression levels for the gene in a cell. Knowledge about the location, sequence, regulation, and expression of a gene yields information critical to the diagnosis and treatment of a variety of diseases.
Nucleic acid probes are typically short nucleic acid sequences used to detect, amplify, and quantify DNA and RNA for diagnostic and therapeutic applications. They are designed to specifically hybridize with particular complementary target nucleic acid sequences. Nucleic acid probes are labeled with radioactive or fluorescent tags in order to detect the presence or absence of the target nucleic acid sequence using a variety of techniques (e.g., fluorescent in-situ hybridization, Southern blot, Northern blot, and chromatography). While these techniques are useful for detecting the presence or absence of particular target sequences, their sensitivity depends on the amount of target nucleic acid present in the sample. Furthermore, living cells cannot be analyzed using these techniques since samples must be extracted, and fixed or frozen prior to analysis.
In order to increase the sensitivity of nucleic acid detection techniques, labeled probes are combined with amplification processes, such as PCR, to amplify and detect nucleic acids. The combination of probes and amplification processes has been especially useful in forensic applications where the nucleic acid of interest may only be present in extremely small quantities. Many disease conditions are diagnosed by comparing the amount of mRNA produced in a normal cell to a diseased cell. For example, increased expression of an oncogene may indicate the presence of a tumor cell. Amplification techniques, such as PCR, are non-linear and exponentially increase the amount of nucleic acid present in a sample. Thus, the amount of nucleic acid present in a sample subject to PCR is not representative of the amount of nucleic originally present in the sample. Nucleic acid amplification techniques are of limited use for quantifying the amount of nucleic acid present in a sample.
Previously, nucleic acids were thought to be a medium solely for storing, transporting, processing, and expressing genetic information. Recently, however, nucleic acids capable of additional functions have been identified. These “functional nucleic acids” can, for example, catalyze reactions or bind specifically to particular targets. The three-dimensional structure of functional nucleic acids provides the specificity necessary to bind other compounds much like the three-dimensional structure of an enzyme determines its specificity for a substrate. The small size, specificity, and ease of manipulation of nucleic acids can now be applied to functions traditionally associated with proteins (e.g., catalysis, receptors, and antibodies).
Molecular beacons are functional single-stranded DNA probes that can report the presence of specific nucleic acids. Molecular beacons are stem-loop shaped molecules containing a nucleotide sequence in the loop portion of the molecule complementary to a target DNA or RNA. Molecular beacons are labeled on one end with a fluorescent molecule and on the other with a quenching molecule. In its native hairpin structure, the quenching molecule is in close proximity with the fluorescent molecule and absorbs the light emission of the fluorescent molecule. When the complementary nucleotide sequence on the molecular beacon loop binds its target molecule, the molecular beacon undergoes a conformational change that opens up the stem-loop structure and causes the fluorescent molecule and the quenching molecule to move away from each other. The light emission from the fluorescent molecule is no longer quenched and the signal can be detected.
Molecular beacons are of limited use in generating amplified signals. The fluorescent signal of a molecular beacon is an integral part of the molecular beacon. Thus, the fluorescent molecule of the molecular beacon can generate only one signal in the presence of its target. The signal cannot be further amplified or altered by use of, for example, secondary labeled antibodies. The weak, unamplified signal generated by the molecular beacon is not able to penetrate living tissue sufficiently for use in non-invasive imaging.
Aptamers are functional synthetic nucleic acids optimized for high-affinity binding to targets (e.g., nucleic acids, proteins, and chemical compounds). Unlike naturally occurring nucleic acids, which are optimized with respect to transfer of genetic information, aptamers are selected on the basis of their ability to specifically bind their ligand. Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is a process of selecting aptamers directed to a chosen ligand. See Hermann and Patel, Science 287, pp 820–825 (2000); U.S. Pat. Nos. 5,475,096, 5,595,877, 5,660,985, and 6,180,348. The SELEX process selects aptamers by screening random sequence libraries, retaining sequences that bind the chosen target molecule, and repeating the cycle with increasing levels of binding stringency. The selected aptamer is capable of binding a chosen target but not other molecules.
Ribozymes are functional nucleic acid molecules with enzymatic capabilities, including the ability to cleave nucleic acid molecules in a sequence-specific manner. The three-dimensional structure of a ribozyme, like a protein enzyme, determines the specificity of its interaction with a particular target. Allosteric ribozymes are ribozyme nucleic acid constructs having a ribozyme portion and an antisense nucleic acid portion. The activation of the ribozyme is regulated by the binding of the antisense nucleic acid to a complementary nucleic acid target. In the absence of the antisense target, the ribozyme is inactive. In the presence of the antisense target, the ribozyme is activated and capable of catalyzing a reaction.
Allosteric ribozymes may also include an attenuator sequence that binds completely or partially to the antisense nucleic acid portion as well as to the ribozyme portion. The “overlapping” attenuator sequence and the antisense target sequence compete for the binding site on the antisense sequence. In the presence of a sufficient amount of the antisense target sequence, the attenuator nucleic acid strand is displaced from the antisense sequence and the ribozyme is able to fold into its active conformation. However, strand displacement of the attenuator by the antisense target sequence has significant disadvantages. For example, the attenuator sequence must be designed to bind both the ribozyme and the antisense target sequence. This requirement limits the availability of compatible ribozyme-antisense sequence combinations. In addition, the antisense target sequence must compete for an overlapping binding site on the antisense sequence. Therefore, a relatively large quantity of the antisense target sequence is required in order to successfully displace the attenuator from the antisense sequence, thus resulting in reduced sensitivity of the probe.
It should be appreciated that there is a need for highly sensitive, functional nucleic acid probes capable of being activated to bind a target only in the presence of a particular ligand without being subject to foregoing limitations.