Identification of Substrates for Adenosine Deaminases That Act on RNA

Part of the Methods in Molecular Biology book series (MIMB, volume 265)


Adenosine deaminases that acts on RNA (ADARs) are RNA-editing enzymes that convert adenosine to inosine in double-stranded RNA. This chapter provides a detailed protocol for identifying inosine-containing RNAs. Candidate ADAR substrates are identified by cleaving poly (A)+ RNA specifically after inosine and using differential display to detect cleaved molecules. To confirm the presence of inosine, each individual candidate substrate is amplified by reverse transcriptase polymerase chain reaction (RT-PCR) and the PCR product is directly sequenced. Sites that contain inosine at the RNA level appear as a mixture of adenosine and guanosine in the cDNA. The relative peak areas provide an estimate of the extent of editing at each site.

Key Words

Adenosine deaminases that act on RNA RNA editing inosine substrate differential display RNase T1 reverse transcriptase polymerase chain reaction 


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Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  1. 1.Chemistry DepartmentUnited States Naval AcademyAnnapolis

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