Molecular Biology

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.

RNA molecules adopt defined structural conformations that are essential to exert their function. During the course of evolution, the structure of a given RNA can be maintained via compensatory base-pair changes that occur among covarying nucleotides in paired regions. Therefore, for comparative, structural, and evolutionary studies of RNA molecules, numerous computational tools have been developed to incorporate structural information into sequence alignments and a number of tools have been developed to study covariation. The bioinformatic protocol presented here explains how to use some of these tools to generate a secondary-structure-aware multiple alignment of RNA sequences and to annotate the alignment to examine the conservation and covariation of structural elements among the sequences.

Compared to the recent dramatic growth in the numbers of genome-wide and functional studies of complex non-coding RNAs, mechanistic and structural analyses have lagged behind. A major technical bottleneck in the structural determination of large RNAs and their complexes is preparation of diffracting crystals. Empirically, a vast majority of such RNA crystals fail to diffract X-rays to usable resolution (~4 Å) due to their inherent disorder and non-specific packing within the crystals. Here, we present a protocol that combines post-crystallization cation replacement and dehydration that dramatically improved the diffraction quality of crystals of a large gene-regulatory mRNA-tRNA complex. This procedure not only extended the resolution limit of X-ray data from 8.5 to 3.2 Å, but also significantly improved the quality of the data, enabling de novo phasing and structure determination. Because it exploits the general importance of counterions and solvation in RNA structure, this procedure may prove broadly useful in the crystallographic analyses of other large non-coding RNAs.

This protocol describes the methodology for the determination of the secondary structure of an RNA fragment in solution using Selective 2´-Hydroxyl Acylation analyzed by Primer Extension, abbreviation SHAPE. It consists in the very fast chemical modification of flexible and therefore possibly single-stranded nucleotides in a sequence-independent manner using benzoyl cyanide (BzCN), forming 2´-O-adducts. The modifications in the RNA are then analyzed by primer extension. Reverse transcriptase is blocked by the 2´-O-adducts formed. The advantage of the method is, first, that not each RNA molecule studied but the primer used in the extension reaction is labelled and, second, that the resulting cDNA analyzed in sequencing gels is much more stable than the modified RNA.