Development of RNA G-quadruplex (rG4)-targeting L-RNA aptamers by rG4-SELEX

Abstract

RNA G-quadruplex (rG4)-SELEX is a method that generates L-RNA aptamers to target an rG4 structure of interest, which can be applied to inhibit G-quadruplex-mediated interactions that have important roles in gene regulation and function. Here we present a Protocol Extension substantially modifying an existing SELEX protocol to describe in detail the procedures involved in performing rG4-SELEX to identify rG4-specific binders that can effectively suppress rG4–peptide and rG4–protein associations. This Protocol Extension improves the speed of aptamer discovery and identification, offering a suite of techniques to characterize the aptamer secondary structure and monitor binding affinity and specificity, and demonstrating the utility of the L-RNA aptamer. The previous protocol mainly describes the identification of RNA aptamers against proteins of interest, whereas in this Protocol Extension we present the development of an unnatural RNA aptamer against an RNA structure of interest, with the potential to be applicable to other nucleic acid motifs or biomolecules. rG4-SELEX starts with a random D-RNA library incubated with the L-rG4 target of interest, followed by binding, washing and elution of the library. Enriched D-aptamer candidates are sequenced and structurally characterized. Then, the L-aptamer is synthesized and used for different applications. rG4-SELEX can be carried out by an experienced molecular biologist with a basic understanding of nucleic acids. The development of rG4-targeting L-RNA aptamers expands the current rG4 toolkit to explore innovative rG4-related applications, and opens new doors to discovering novel rG4 biology in the near future. The duration of each selection cycle as outlined in the protocol is ~2 d.

Introduction

Guanine (G)-rich sequences of single-stranded DNA and RNA can fold into stable, intra- or intermolecular secondary structures called G-quadruplexes (dG4s and rG4s). These nucleic acid structure scaffolds are composed of stacks of G-quartets and can be further stabilized in the presence of monovalent ions, preferentially K⁺ or Na⁺ but not Li^+1,2,3. Earlier findings have shown that G4s play important roles in various cellular events, including but not limited to DNA replication, DNA damage repair, transcription, translation, RNA metabolism and epigenetic remodeling^{3,4,5,6,7,8,9}. The ability to regulate fundamental biological processes, as well as their chemically interesting structures, makes G4s promising targets for potential cancer, antimicrobial and antiviral treatments^{10,11,12,13,14,15,16,17,18,19}. With the mounting interests in the biological role of G4s, more structure-specific, sensitive and low-cytotoxicity tools are needed to not only differentiate between G4s and duplexes, but also among different subtypes of G4s, to allow gene/transcript control and manipulation by selective targeting of specific G4 structure in any gene/transcript of interest.

Naturally occurring nucleic acids are homochiral and are built from the monomers of D-DNA and D-RNA nucleotides. Earlier studies have shown that D-DNA/RNA oligonucleotides are incapable of forming contiguous Watson–Crick base pairing with their enantiomeric counterparts, L-DNA/RNA oligonucleotides²⁰. As they are unnatural, L-nucleic acids are unrecognizable by natural nucleases, which enables them to have extended half-lives for cellular and in vivo studies²⁰. These special properties motivate researchers to develop various biological tools based on L-nucleic acids²⁰. One of the common examples is an L-RNA aptamer, also known as a spiegelmer. Speigelmers, first reported by Furste and colleagues^21,22, evolved from a modified version of systematic evolution of ligands through exponential enrichment (SELEX)^23,24,25. To date, spiegelmers have been selected to recognize a range of targets, including small molecules, peptides and proteins²⁰. In addition, a few spiegelmers are currently under clinical trial phases I or II²⁶. Inspired by the earlier works of Sczepanski and Joyce to use spiegelmers to target canonical RNA structure motifs such as hairpin and stem-loop RNAs^27,28, and also by the protocol of Lorenz et al. to use natural RNAs to target proteins of interest²⁹, we were motivated to investigate whether such a strategy can be adopted, refined and generally applicable in developing a new class of targeting tool for noncanonical RNA structures such as rG4 motifs. The rationale behind structured RNA targeting spiegelmers is based on the non-Watson–Crick base-pairing principle of nucleic acids with opposite chirality. The experimental details shown below have been demonstrated and reported in our recent publications that employed (UUAGGG)₄, which is part of the telomeric repeat-containing RNA (TERRA) sequence³⁰, and the human telomerase RNA (hTERC rG4)³¹ as our targets, and in this Protocol Extension we have summarized this information as a protocol and resource for the scientific community.

Applications of the method

As we have successfully showcased the validity of rG4-SELEX using different rG4 targets in our proof-of-concept studies^30,31, we believe this method can be a novel strategy to create highly specific, noncytotoxic and nuclease-resistant rG4-targeting probes. The unique tertiary interaction between each L-aptamer (L-Apt.) and D-RNA G4 can potentially achieve an unprecedented specificity in G4 targeting, i.e., distinguishing an individual rG4 from other RNA structure motifs such as hairpins and stem loops, or even between dG4s and rG4s³⁰. In terms of potential biological applications, we have demonstrated that these spiegelmers can interfere with the binding of the target rG4s with biologically relevant peptides or proteins^30,31, with a half-maximal inhibitory concentration (IC₅₀) comparable to state-of-the-art small-molecule G4 ligands³¹, which may be used as a strategy for G4 targeting therapeutics. The nuclease-resistant nature of these rG4-targeting L-RNA aptamers also allows them to be promising probes for developing trackers of G4 folding and unfolding dynamics, or high-specificity vehicles for delivery in cells by coupling them with fluorophores or other moieties of interest, respectively. Besides that, these rG4-targeting L-RNA aptamers can also be employed as rG4 ligands to regulate rG4-mediated gene expression and RNA metabolism, as well as control of gene activity for diverse applications³². Recently, Tolnai et al. developed a sandwich detection assay based on two different speigelmers that bind to the C- and N- termini of cardiac troponin I, respectively, which suggests spiegelmers can be developed into highly sensitive and specific sensors for biopolymers³³. A similar strategy may be designed for biosensing of rG4 using rG4-targeting spiegelmers.

Even though our method was developed and optimized for rG4s, it is likely that our strategy can be applied to other noncanonical nucleic acids structures such as pseudoknots³⁴ and i-motifs³⁵, potentially by refining the selection conditions to meet the folding of these specific types of structure motifs. In particular, pseudoknot RNAs were reported to be a highly conservative motif in the 3′ untranslated region (3′UTR) of coronaviruses³⁶ and other regions that regulate gene expressions in viruses through mechanisms like frameshifting³⁷. They were also found to be an essential element for standby-mediated translation in bacteria³⁸ and regulation of human telomerase RNA activity^39,40,41,42. DNA i-motifs, whose in vivo existence has long been controversial due to their structural instability at physiologically relevant pH range³⁵, were recently shown to be detectable in the nuclei of living cells^43,44. This noncanonical structure generally appears in the complementary strand of dG4s with high proximity, although its biological role is still elusive and requires further investigation⁴⁵. While discovery of small-molecule ligands for these noncanonical nucleic acid structural motifs is feasible and ongoing^46,47, it may be a challenging process, and often requires laborious chemical synthesis, purification and characterization; thus, we think that our spiegelmer approach may serve as an alternative and practical option, especially for biochemists and molecular biologists.

Comparison with other methods

A large collection of G4 detection methods is available, with many that show specificity toward G4 over non-G4 structural motifs such as duplexes and hairpins^3,48,49,50. One of the most commonly employed G4 detection tools is to use small-molecule ligands—low molecular weight (LMW) organic or inorganic compounds that typically show notable fluorescence enhancement or increase in thermostability in the form of melting temperature (T_m), or stronger binding affinity (lower dissociation constant K_d), while interacting with G4s^49,51. The use of G4 ligands has been widely adopted both in vitro and in cells for diverse chemical and biological applications^52,53,54,55, and some of these G4 ligands can stabilize the formation of G4s and dissociate G4–G4 binding protein complexes, which may be further developed as therapeutics^13,49,56,57. For instance, commercially available ligands like N-methyl mesoporphyrin IX (NMM)^58,59,60 and Thioflavin T (ThT)^61,62 have shown promising G4 sensitivity in vitro; thus, their fluorescence signal has widely been applied as one of the G4 detection methods³. Yet, unlike the L-RNA aptamers that we are presenting here, most ligands lack the ability to achieve selective G4 structure binding, i.e., selectively bind to an individual G4 target over other closely related G4s; recently, however, a few small-molecule ligands were reported to have a higher specificity toward an individual G4 target⁴⁹, such as the telomeric multimeric dG4⁶³ and the c-MYC dG4^64,65,66. These ligands showed preferred binding preference toward one specific G4 over other G4s, highlighting that individual G4 targeting should be feasible with G4 ligands, potentially by rationally designing ligand sidechains to recognize the groove and loops of G4 to provide additional specificity. However, the rationale behind such a property is not fully explained and whether such a strategy can be applied easily to any other G4 targets is unclear given the limited data. Apart from ligands, other G4 detection approaches using G4-specific antibodies and G4-specific peptides are also available^{67,68,69,70,71,72,73,74,75,76}. Similar to G4 ligands, they showed excellent binding toward G4s over non-G4s^{67,68,69,70,71,72,73,74,75,76}; however, so far there are only limited data on their ability to distinguish individual G4s over other G4s⁷⁶. Notably, Chen et al. utilized a supramolecular host–guest sensing array that allows efficient distinction of G4s of different topologies⁷⁷, while a few recent studies have reported guanine- or guanine analog–conjugated antisense oligonucleotides or peptides as new strategies to achieve greater G4 target specificity^78,79,80,81. Interestingly, some innovative methods like targeting G4 with small-molecule ligand-labeled oligonucleotides or G-rich displacing oligonucleotides with either modified nucleic acids or its analogs have shown some success in distinguishing G4 targets with high sequence specificity⁸¹. However, these conjugate probes require additional recognition sites with the G4 targeting module to achieve such specificity. Compared with these above-mentioned approaches, the unique tertiary interaction between an L-RNA aptamer and D-RNA G4 provides uncharted territory to explore the possibility of achieving selective rG4 targeting through specific interaction between D- and L-nucleic acids, potentially with higher G4 specificity than other currently available approaches without the additional recognition sites.

Limitations

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