Bioreactive allosteric polynucleotides

Abstract

Polynucleotides having allosteric properties that modify a function or configuration of the polynucleotide with a chemical effector and/or physical signal are employed primarily as biosensors and/or enzymes for diagnostic and catalytic purposes. In some preferred embodiments, the polynucleotides are DNA enzymes that are used in solution/suspension or attached to a solid support as biosensors to detect the presence or absence of a compound, its concentration, or physical change in a sample by observation of self-catalysis. Chemical effectors include organic compounds such as amino acids, amino acid derivatives, peptides, nucleosides, nucleotides, steroids, and mixtures of these with each other and with metal ions, cellular metabolites or blood components obtained from biological samples, steroids, pharmaceuticals, pesticides, herbicides, food toxins, and the like. Physical signals include radiation, temperature changes, and combinations thereof.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS [0001] This application is a continuation-in-part of co-pending U.S. application Ser. No. 09 / 331,809, filed Jun. 18, 1999 as a national phase entry of PCT / US97 / 24158, filed internationally Dec. 18, 1997 and claiming priority benefit of U.S. Provisional application Ser. No. 60 / 033,684, filed Dec. 19, 1996 and Ser. No. 60 / 055,039, filed Aug. 8, 1997.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH [0002] The invention was made with partial government support under NIH grant GM59343. The government has certain rights in the invention.BACKGROUND OF THE INVENTION [0003] 1. Field of the Invention [0004] This invention relates primarily to functional DNA polynucleotides that exhibit allosteric properties, and to catalytic RNA and DNA polynucleotides that have catalytic properties with rates that can be controlled by a chemical effector, a physical signal, or combinations thereof. Bioreactive allosteric polynucleotides of the invention are useful in ...

AI Technical Summary

Benefits of technology

[0043] Bioreactive polynucleotides of the invention exhibit allosteric properties that modify polymer function or configuration with a physical signal or a combination of a physical signal and a chemical effector in alternate embodiments. Physical signals include, but are not limited to, radiation (23a), temperature changes, movement, physical conformational changes in samples, and combinations thereof. Physical signals include, but are not limited to, tags, beacons, and the like allosteric reporters that respond to UV, IR, and/or visible light (23b, 44b). The effects are reversible. Chemical effectors binding to allosteric ribozymes and/or deoxyribozymes of the invention, for example, can enhance or inhibit the catalytic rate, or do both. It is an advantage of the invention that, because the molecules are truely allosteric, any type of allosteric interconversion is possible. Hence, a sample of allosteric polynucleotide enzymes can be fully active, partially active, or fully inactive. In other words, acting as a switch, they can be all “on” or all “off”, or exhibit any level of activity between “on” or “off”. (For a further discussion of switches, see Soukup and Breaker, 39c). Morever, because they are truly allosteric, the observable response time to an effector molecule or effect is immediate. The kinetics of allosteric polynucleotides are similar to what is observed with allosteric polypeptides. Illustrated hereafter are polynucleotides that react in less than 60 minutes, preferably inless than 6 minutes, and most preferably, in less than about a minute. (See, for example, FIGS. 7 and 8.) Most preferred allosteric polynucleotides respond to effectors within seconds.

[0044] Many embodiments employ bioreactive allosteric polynucleotides of the invention as biosensors in solution or suspension or attached to a solid support such as that illustrated in FIG. 1. Alone or as a component of a biosensor, the polynucleotides are used to detect the presence or absence of a compound or its concentration and/or a physical signal by contact with the polynucleotide. In a typical practice of these methods, a sample is incubated with the polynucleotide or biosensor comprising the polynucleotide as a sensing element for a time under conditions sufficient to observe a modification or configuration of the polynucleotide caused by the allosteric interaction. These are monitored using any method known to those skilled in the art, such as measurement and/or observation of polynucleotide self-cleavage; binding of a radioactive, fluorescent, or chromophoric tag; binding of a monoclonal or fusion phage antibody; or change in component concentration, spectrophotometric, or electrical properties. It is an advantage of the invention that current biosensor technology employing potentiometric electrodes, FETs, various probes, redox mediators, and the like can be adapted for use in conjunction with the new polynucleotide biosensors of the invention for measurement of changes in polynucleotide function or configuration.

[0045] The initial studies described in the Examples that follow have involved the creation and characterization of novel RNA- and DNA-cleaving enzymes that function with specific cofactors, or that can be regulated by specific small-molecule chemical effectors, physical signals, or combinations thereof. It is clear that additional molecules with similar sensor and biocatalytic properties can be created by similar means, thereby expanding the applications of such molecules. The creation and characterization of a prototype biosensor for ATP is given herein. One construct (H3) in particular shows ATP concentration-dependent catalytic activity, indicating that this ribozyme could be adapted for use in reporting the concentration

Problems solved by technology

One drawback to the use of existing enzymes as biosensors is that one is limited

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