Joining of long double-stranded RNA molecules through controlled overhangs

We describe two methods for creating long (>1 kb) dsRNA molecules with specific, user-controlled overhangs for efficient hybridization and ligation. The two methods create double-stranded RNA (dsRNA) molecules with 5′ overhangs or with 3′ overhangs using T7 RNA polymerase (T7 RNAP) in transcription reactions of carefully designed PCR products. Primers utilized in the PCR reactions provide the template for the desired dsRNA overhangs. These methods provide complete control of the length and the sequence of the overhangs. This supplies a tool which is particularly lacking in dsRNA biochemistry given the absence of restriction endonucleases active on these substrates