Molecular Biology

The genomes of RNA viruses can fold into dynamic structures that regulate their own infection and immune evasion processes. Proximity ligation methods (e.g., SPLASH) enable genome-wide interaction mapping but lack specificity when dealing with low-abundance targets in complex samples. Here, we describe HiCapR, a protocol integrating in vivo psoralen crosslinking, RNA fragmentation, proximity ligation, and hybridization capture to specifically enrich viral RNA–RNA interactions. Captured libraries are sequenced, and chimeric reads are analyzed via a customized computational pipeline to generate constrained secondary structures. HiCapR generates high-resolution RNA interaction maps for viral genomes. We applied it to resolve the in vivo structure of the complete HIV-1 RNA genome, identifying functional domains, homodimers, and long-range interactions. The protocol's robustness has been previously validated on the SARS-CoV-2 genome. HiCapR combines proximity ligation with targeted enrichment, providing an efficient and specific tool for studying RNA architecture in viruses, with broad applications in virology and antiviral development.

Engineered aptamers for new compounds are typically produced by using in vitro selection methods. However, aptamers that are developed in vitro might not function as expected when introduced into complex cellular environments. One approach that addresses this concern is the design of initial RNA pools for selection that contain structural scaffolds from naturally occurring riboswitch aptamers. Here, we provide guidance on design and experimental principles for developing riboswitch-inspired aptamers for new ligands. The in vitro selection protocol (based on Capture-SELEX) is generalizable to diverse RNA scaffold types and amenable to multiplexing of ligand candidates. We discuss strategies to avoid propagation of selfish sequences that can easily dominate the selection. We also detail the identification of aptamer candidates using next-generation sequencing and bioinformatics, and subsequent biochemical validation of aptamer candidates. Finally, we describe functional testing of aptamer candidates in bacterial cell culture.

Key features

• Develop riboswitch-inspired aptamers for new ligands using in vitro selection.

• Ligand candidates can be multiplexed to conserve time and resources.

• Test aptamer candidates in bacterial cells by grafting the aptamer back onto its expression platform.

Graphical overview

Atypical DNA and RNA secondary structures play a crucial role in simple sequence repeat (SSR) diseases, which are associated with a class of neurological and neuromuscular disorders known as “anticipation diseases,” where the age of disease onset decreases and the severity of the disease is increased as the intergenerational expansion of the SSR increases. While the mechanisms underlying these diseases are complex and remain elusive, there is a consensus that stable, non-B-DNA atypical secondary structures play an important – if not causative – role. These structures include single-stranded DNA loops and hairpins, G-quartets, Z-DNA, triplex nucleic acid structures, and others. While all of these structures are of interest, structures based on nucleic acid triplexes have recently garnered increased attention as they have been implicated in gene regulation, gene repair, and gene engineering. Our work here focuses on the construction of DNA triplexes and RNA/DNA hybrids formed from GAA/TTC trinucleotide repeats, which underlie Friedreich’s ataxia. While there is some software, such as the Discovery Studio Visualizer, that can aid in the initial construction of DNA triple helices, the only option for the triple helix is constrained to be that of an antiparallel pyrimidine for the third strand. In this protocol, we illustrate how to build up more generalized DNA triplexes and DNA/RNA mixed hybrids. We make use of both the Discovery Studio Visualizer and the AMBER simulation package to construct the initial triplexes. Using the steps outlined here, one can – in principle – build up any triple nucleic acid helix with a desired sequence for large-scale molecular dynamics simulation studies.