Guanine quadruplex structures in DNA and RNA affect normal cellular processes such as replication, recombination, and translation. Thus, controlling guanine quadruplex structures could make it possible to manipulate the biological function of nucleic acids. Here, we report a novel antisense strategy using guanine-tethered antisense oligonucleotides (g-ASs) that introduces an RNA-DNA heteroquadruplex structure on RNA templates in a predictable and sequence-specific manner, which in practice effectively inhibited reverse transcription on a variety of RNA sequences, including the HIV-1 RNA genome. Reverse transcriptase-mediated enzymatic analysis, together with other biophysical analyses, elucidated a cooperative binding of duplex and quadruplex in g-AS-RNA complexes. The remarkable ability of g-ASs to inhibit reverse transcription could make possible the development of novel anti-retroviral gene therapies based on blocking the replication of RNA genomes to complementary DNA, which is a critical step for integration into the host's genome.