Noncovalent probes for the investigation of structure and dynamics of protein‐nucleic acid assemblies: The case of NC‐mediated dimerization of genomic RNA in HIV‐1

Abstract

The nature of specific RNA-RNA and protein-RNA interactions involved in the process of genome dimerization and isomerization in HIV-1, which is mediated in vitro by the stemloop 1 (SL1) of the packaging signal and by the nucleocapsid (NC) domain of the viral Gag polyprotein, was investigated by using archetypical nucleic acid ligands as non-covalent probes. Smallmolecule ligands make contact with their target substrates through complex combinations of Hbonds, salt bridges, and hydrophobic interactions. Therefore, their binding patterns assessed by electrospray ionization (ESI) mass spectrometry can provide valuable insights into the factors determining specific recognition between species involved in biopolymer assemblies. In the case of SL1, dimerization and isomerization create unique structural features capable of sustaining stable interactions with classic nucleic acid ligands. The binding modes exhibited by intercalators and minor groove binders were adversely affected by the significant distortion of the duplex formed by palindrome annealing in the kissing-loop (KL) dimer, whereas the modes observed for the corresponding extended duplex (ED) confirmed a more regular helical structure. Consistent with the ability to establish electrostatic interactions with highly negative pockets typical of helix anomalies, polycationic aminoglycosides bound to the stem-bulge motif conserved in all SL1 conformers, to the unpaired nucleotides located at the hinge between kissing hairpins in KL, and to the exposed bases flanking the palindrome duplex in ED. The patterns afforded by intercalators and minor groove binders did not display detectable variations when the corresponding NC-SL1 complexes were submitted to probing. In contrast, aminoglycosides displayed the ability to compete with the protein for overlapping sites, producing opposite effects on the isomerization process. Indeed, displacing NC from the stem-bulges of the KL dimer induced inhibition of stem melting and decreased the efficiency of isomerization. Competition for the hinge region, instead, eliminated the NC stabilization of a grip motif formed by nucleobases of opposite strands, thus facilitating the strand-exchange required for isomerization. These non-covalent probes provided further evidence that the structural context of the actual binding sites has significant influence on the chaperone activities of NC, which should be taken in account when developing potential drug candidates aimed at disrupting genome dimerization and isomerization in HIV-1.

Higher-order structure of nucleic acids in the gas phase: Top-down analysis of base-pairing interactions

“…In this direction, we have employed sustained off-resonance irradiation (SORI) 24 CID to characterize the unique structural features exhibited by DNA-RNA hybrid duplexes 25, 26 and RNA stemloop structures 27, 28 involved in viral replication. The observation that backbone fragmentation could be inhibited by the contact of non-covalent ligands 27 prompted us to propose the utilization of classic nucleic acid ligands as non-covalent structural probes.…”

“…The observation that backbone fragmentation could be inhibited by the contact of non-covalent ligands 27 prompted us to propose the utilization of classic nucleic acid ligands as non-covalent structural probes. 28 This general strategy relies on firm knowledge of the binding profiles of such ligands and counts on the ability to locate their specific sites by top-down MS. We have also shown that SORI-CID can be effectively employed to interrogate the conformational state of RNA complexes, such as those involved in the dimerization and packaging of the HIV-1 genome. 29, 30 In this case, isomeric dimers that could not be recognized from their identical molecular mass were readily distinguished on the basis of the different gas-phase stabilities afforded by their interstrand pairing.…”

The Design of RNA Binders: Targeting the HIV Replication Cycle as a Case Study

“…Indeed, aminoglycosides are positively charged compounds that will readily interact with any RNA by electrostatic interactions, especially in low salt conditions. Several RNA-aminoglycoside interactions reported in the literature, including HIV-1 subtype B DIS, [123][124][125][126] are very likely relevant such nonspecific interactions.…”

MS analysis of nucleic acids in the post-genomic era

“…In this case, the KL- and ED-specific mutants exhibited very different binding patterns with both intercalators and minor groove binders, which are sensitive to the integrity of helical structures. 50 The observed differences were attributed to the significant distortion of the palindrome duplex in the KL conformer as compared to the ED form, in which it assumes a more regular helical structure. Neither class of ligand affected the ability of SL1 dimers to bind NC, thus suggesting that protein interactions did not significantly alter their distinctive conformations.…”