Methods and compositions for retroviral preintegration complex-based assays
OwnerFrederic Bushman, Julie Mullininventors
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Abstract
Disclosed are solution-based methods of screening for molecules that modulate the integration of retroviral cDNA into target DNA molecules. These methods can be used to identify molecules that modulate (e.g., inhibit) the integration of unintegrated retroviral cDNA. Such PIC activity modulators may have therapeutic application, for example, to treat or prevent diseases caused by or associated with retroviral infection.
Claims20
A method of detecting integration of an engineered viral cDNA into a free-floating target DNA, comprising:
- (i) in an aqueous reaction, contacting said free-floating target DNA with a retroviral preintegration complex comprising said engineered viral cDNA under conditions that allow integration of viral cDNA from an active retroviral preintegration complex into said target DNA;
- and
- (ii) detecting whether said engineered viral cDNA integrated into said target DNA.
A method of identifying a compound that modulates integration of a provirus, comprising:
- (i) in solution, contacting a test compound with an active retroviral preintegration complex that comprises an engineered viral cDNA;
- (ii) concurrent with or after contacting the test compound with the active retroviral preintegration complex, adding a free-floating target DNA to the solution of part (i) under conditions that allow integration of a viral cDNA from an active retroviral preintegration complex into the target DNA;
- and
- (iii) determining whether said viral cDNA integration into the target DNA is modulated by the test compound and, if so, identifying the test compound as a modulator of integration of a provirus.
A method of detecting integration of an engineered viral cDNA into a free-floating target DNA, comprising:
- (i) in an aqueous reaction, contacting said free-floating target DNA with a retroviral preintegration complex comprising said engineered viral cDNA under conditions that allow integration of viral cDNA from an active retroviral preintegration complex into said target DNA;
- and
- (ii) detecting whether said engineered viral cDNA integrated into said target DNA, wherein the detecting does not require pre-treatment with an exonuclease.
Dependencies are inferred from the claim text.