Compositions and methods for the identification of inhibitors of protein synthesis

Inventors

GUENTHER, Richard H. • Newman, Winnell H. • Yenne, Samuel P. • Mitchell, Dan • Malkiewicz, Andrzej

Assignees

Trana Discovery Inc

Abstract

Compositions and methods for identifying inhibitors of RNA-target molecule interactions are provided as well as identifying inhibitors that block the role of tRNA in protein synthesis. The methods involve forming a mixture comprising a tRNA fragment molecule containing a modified nucleotide, a target molecule capable of binding to the tRNA fragment, and a test compound. The mixture is incubated under conditions that allow binding of the tRNA and the target molecule in the absence of the test compound. Assays can then be performed that detect whether or not the test compound inhibits the binding of the tRNA molecule and the target molecule. High throughput assays are also provided.

Core Innovation

The disclosed invention identifies inhibitors of tRNA-target molecule interaction by forming a mixture comprising a tRNA D-loop fragment with at least one modified nucleotide, a target molecule capable of binding to the tRNA D-loop fragment, and a test compound. The mixture is incubated under conditions that allow binding of the tRNA D-loop fragment and the target molecule in the absence of the test compound, and the method detects whether the test compound inhibits binding. The absence of binding of the tRNA fragment and the target molecule is indicative of the test compound being an inhibitor of tRNA-target molecule interaction.

The method is further implemented with an anticodon stem-loop fragment containing at least one modified nucleotide, where the target molecule is capable of binding to both the tRNA D-loop fragment and the ASL fragment. In other implementations, an inhibitor-identifying method forms a mixture comprising a biological sample containing a target molecule and at least one nucleic acid molecule derived from or corresponding to a tRNA loop having at least one modified nucleotide, where the tRNA loop is a D-loop or an anticodon loop. The nucleic acid molecule is a tRNA fragment having 7 to 20 continuous nucleotides, and binding is assessed by detecting whether the target molecule binds to the nucleic-acid fragment.

Another aspect identifies a target molecule that binds to an RNA molecule by forming a mixture comprising a biological sample and at least one nucleic acid molecule derived from or corresponding to a tRNA loop having at least one modified nucleotide, where the tRNA loop is a D-loop or an anticodon loop. After incubating to allow binding, binding is detected, with the at least one nucleic acid molecule attached to a microarray. The described nucleic-acid constructs include tRNA loop fragments and modified nucleotide-containing sequences, and the target interaction is assessed via a binding-detection readout.

Claims Coverage

The provided independent claims cover three related identification methods. The core coverage includes forming an incubation mixture, binding-detection, and using tRNA-loop nucleic acids with at least one modified nucleotide, with microarray attachment in the target-binding method.

Overall, the claim set focuses on binding-based identification of inhibitors and binding partners using tRNA-loop nucleic acids that contain modified nucleotides, specifically employing D-loop fragments with modified nucleotides, optionally combined with an anticodon stem-loop fragment also containing modified nucleotides, and using a microarray-attached format for target-molecule binding identification.

Stated Advantages

Documented Applications