Selection and identification of an RNA aptamer that specifically binds the HIV-1 capsid lattice and inhibits viral replication

Abstract

The HIV-1 capsid core participates in several replication processes. The mature capsid core is a lattice composed of capsid (CA) monomers thought to assemble first into CA dimers, then into ∼250 CA hexamers and 12 CA pentamers. CA assembly requires conformational flexibility of each unit, resulting in the presence of unique, solvent-accessible surfaces. Significant advances have improved our understanding of the roles of the capsid core in replication; however, the contributions of individual CA assembly forms remain unclear and there are limited tools available to evaluate these forms in vivo. Here, we have selected aptamers that bind CA lattice tubes. We describe aptamer CA15-2, which selectively binds CA lattice, but not CA monomer or CA hexamer, suggesting that it targets an interface present and accessible only on CA lattice. CA15-2 does not compete with PF74 for binding, indicating that it likely binds a non-overlapping site. Furthermore, CA15-2 inhibits HIV-1 replication when expressed in virus producer cells, but not target cells, suggesting that it binds a biologically-relevant site during virus production that is either not accessible during post-entry replication steps or is accessible but unaltered by aptamer binding. Importantly, CA15-2 represents the first aptamer that specifically recognizes the HIV-1 CA lattice.

Differentiation SELEX approach identifies RNA aptamers with different specificities for HIV-1 capsid assembly forms

“…The starting library for this work was the HIV-1 assembled CA lattice round 15 library described previously ( 45 ). To initiate the differentiation selection, double-stranded DNAs from this library were first PCR-amplified using Taq DNA polymerase for 25 cycles and then transcribed using the Y639F mutant T7 RNA polymerase purified in house ( 76 ), in vitro transcription buffer (50 mM Tris-HCl pH 7.5, 15 mM MgCl 2 , 5 mM DTT, 2 mM spermidine), and 2 mM of each NTP (ATP, CTP, GTP, and UTP).…”

“…Thus, it is feasible that aptamers selected to differentiate among CA assembly forms could be developed as biochemical tools to improve our understanding of the roles and interactions of CA assembly forms during viral replication. We recently described an RNA aptamer selection against the assembled HIV-1 CA hexamer lattice ( 45 ). While the selected libraries demonstrated dose-dependent binding to the CA hexamer lattice but not the soluble CA hexamer or CA monomer, we reasoned that other low-abundant sequences capable of binding other CA assembly forms may be present within the libraries (Figure 1A).…”

RNA-Based Sensor Systems for Affordable Diagnostics in the Age of Pandemics

“…RNA structure is a key determinant of its diverse biological functions. − Therefore, creating RNA molecules with specific structures and functions is of great value to research, where computational methods for modeling RNA secondary structures come in handy. Thus far, this approach has been utilized for synthetic biology and biotechnology applications to design RNA-based devices, including riboregulators, ,,− aptamers, − and CRISPR guides. − Current advanced approaches for in-silico RNA design rely on free energy minimization algorithms, which estimate the thermodynamic stability of different structures. − To enhance the prediction accuracy, some methods also incorporate experimental mapping data or structural conservation information from a set of homologous sequences. − Several prominent software tools have been developed for in-silico RNA design based on these approaches, including but not limited to NUPACK, RNAstructure, ViennaRNA, UNAfold, and the deep-learning framework. These tools offer a variety of features, such as analysis, prediction, comparison, and synthesis of nucleic acid structures and interactions, each with its own advantages and drawbacks.…”

Smart Approach for the Design of Highly Selective Aptamer-Based Biosensors

“…They are chemically synthesized short single-stranded deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) that serve as selective biorecognition elements [ 17 ]. The specific sequence of the aptamers gives them a 3D folding shape that allows them to bind to their targets with high selectivity [ 18 , 19 , 20 , 21 , 22 , 23 ]. With respect to antibodies, aptamers are cost-effective, easier to synthesize, thermally stable, and simpler to use.…”