In HIV-1 reverse transcription, the nucleocapsid protein, NC, induces secondary structure fluctuations in specific DNA and RNA hairpins. Time-resolved single-molecule fluorescence resonance energy transfer was used to study NC chaperoned opening of DNA hairpins over a broader range of conditions and in more depth than in previous studies. The experiments reveal a complex mechanism for secondary structure fluctuations with dynamic processes occurring over a wide time range, i.e., similar to 5 to > 250 ms and with the involvement of long-lived intermediates. The dynamic role of DNA loop regions and NC binding/dissociation events are discussed.