The present invention is based, in part, upon the insight that compound DsiRNA agents can be generated using site-specific RNase H-cleavable double-stranded nucleic acid regions to attach, e.g., one DsiRNA moiety to another DsiRNA moiety and/or one DsiRNA moiety to a functional group and/or payload. Because such double-stranded nucleic acid joining sequences are site-specifically RNase H-cleavable, the bifunctional molecule is cleaved into DsiRNAs bearing terminal ends that orient dicer cleavage. Detrimental impacts of administering a single double-stranded nucleic acid RNAi agent of longer than 30-35 nucleotides (e.g., provocation of interferon response) is minimized, as once administered to a subject or RNase H-containing cell, RNase H cleavage produces a shortened, active DsiRNA agent(s). The invention provides bifunctional DsiRNA agents that are joined by double-stranded DNA extension joining sequences, which do not provoke RNase H cleavage.
