DNA/RNA hybrids avoid channel gating that leads to the continued packaging of numerous hybrids into the phi29 protein shell.

Packaging of double-stranded DNA (dsDNA) into viral capsids is crucial in dsDNA viruses, including herpesviruses, adenoviruses, poxviruses, and bacteriophages. An ATPase motor compacts genomes. The phi29 DNA packaging motor, a model system, employs a hexameric pRNA (packaging RNA) ring and ATPase, sharing a revolving mechanism observed in herpesvirus genome packaging, bacterial DNA transport, Holliday junction resolution, and plasmid conjugation. Channel gating terminates translocation and readies a reversed pore for dsDNA exit; its mechanism is unclear. We report a packaging efficiency difference between dsDNA and RNA/DNA hybrids. Single-channel electrophysiology and sucrose gradient ultracentrifugation reveal that packaging fails if both ends are dsRNA, but succeeds if either 5' or 3' end is DNA. As long as one strand is DNA, RNA/DNA hybrids are packaged, with a higher copy number than dsDNA. Single-pore conductance assays show that this efficiency results from the absence of channel gating. The channel remains open during RNA/DNA translocation and does not close after hybrid packaging, implying dsDNA's role in gating and conformational changes. This gating arises from dsDNA's interaction with three flexible loops of the motor channel. These findings offer a structural and chemical foundation for designing containers to package RNA/DNA hybrids for gene/RNAi delivery, therapy, synthetic biology, nanotechnology, and single-particle sensing.