Studying aminoglycoside antibiotic binding to HIV-1 TAR RNA by electrospray ionization mass spectrometry

The recognition of the aminoglycosides neomycin and streptomycin by HIV-I T AR RNA was studied by electrospray ionization mass spectrometry (ESI-MS). M embers of the aminoglycoside family of antibiotics are known to target a wi de variety of RNA molecules. Neomycin and streptomycin inhibit the formatio n of the Tat protein-TAR RNA complex, an assembly that is believed to be ne cessary for HIV replication. The noncovalent complexes formed by the bindin g of aminoglycosides to TAR RNA and the Tat-TAR complex were detected by ES I-MS. Neomycin has a maximum binding stoichiometry of three and two to TAR RNA and to the Tat-TAR complex, respectively. Data from the ESI-MS experime nts suggest that a high affinity binding site of neomycin is located near t he three-nucleotide bulge region of TAR RNA. This is consistent with previo us solution phase footprinting measurements [H.-Y. Mei et al., Biochemistry 37 (1998) 14204]. Neomycin has a higher affinity toward TAR RNA than strep tomycin, as measured by ESI-MS competition binding experiments. A noncovale nt complex formed between a small molecule inhibitor of TAR RNA, which has a similar solution binding affinity as the aminoglycosides, and TAR RNA is much less stable than the RNA-aminoglycoside complexes to collisional disso ciation in the gas phase. It is believed that the small molecule inhibitor interacts with TAR RNA via hydrophobic interactions, whereas the aminoglyco sides bind to RNAs through electrostatic forces. This difference in gas pha se stabilities may prove useful for discerning the types of noncovalent for ces holding complexes together. (C) 1999 Elsevier Science B.V.