RNA G-quadruplexes (rG4s) are four-stranded structures formed by the folding of G-rich RNA sequences. Four guanine bases associate via Hoogsteen hydrogen bonding to form a planar G-quartet, and the stacking of multiple G-quartets connected by loop nucleotides creates the G4 structure. This structure is stabilized in the presence of monovalent cations, with potassium being the most stable. DNA G-quadruplexes (dG4s) have been more extensively studied, while with the advances in technology and increased interest in RNA structure and biology have led to a growing understanding of the prevalence, formation, dynamics, and functions of rG4s. rG4s have been identified across various species and in different RNA classes, including mRNA, miRNA, and lncRNA. Emerging evidence suggests that rG4s play important regulatory roles, affecting processes such as translation, alternative polyadenylation, alternative splicing, transport, and stability. Additionally, rG4s have been associated with health conditions such as cancer and neurodegenerative diseases.

The primary tools employed for G4 targeting are G4-specific small molecule ligands, peptides, and antibodies, and so on. However, selectively targeting specific G4 structures by these tools has proven challenging. A new tool, the L-RNA aptamer (Spiegelmer) composing of unnatural L-nucleotides, has been demonstrated to specifically recognize RNA structures through tertiary interactions. Located in the 3′-untranslated region (3′UTR) of the amyloid precursor protein (APP), APP rG4 can form both in vitro and in cells, and it regulates the translation of APP. To target APP rG4, we developed an L-RNA aptamer, L-Apt.8f, by the rG4-systematic evolution of ligands by exponential enrichment (rG4-SELEX) protocol. We refined and analyzed the aptamer's structure and evaluated its binding strength and discernment for the APP rG4. Moreover, we showed that an L- Apt.8f aptamer can be delivered into cells and regulate gene expression by targeting the APP rG4 in both the luciferase reporter gene and native transcript.

However, two off-targets were observed among a panel of structures, showing cross-reactivity with Kras1 rG4 and Bcl2 rG4, albeit with slightly weaker binding. To obtain more specific tools that can recognize an individual rG4 structure, we designed an innovative approach to address this challenge by harnessing the power of multiple recognition modules. rG4s are structurally similar, but rG4 flanking sequences are divergent. Therefore, we rationally integrated an antisense oligonucleotide (ASO) complementary to the rG4 flanking sequence to enhance the specificity of the L-aptamer in recognizing rG4. Using APP rG4 and the general rG4 binder L-Apt.4-1c as an example, we ligate the ASO DNA and L-Apt.4-1c through click reaction to generate the first L-aptamer – ASO conjugate, L-Apt.4-1c-ASO15nt_(APP), to specifically recognize APP rG4 region in vitro and in cells over other rG4s. We also apply L-Apt.4-1c-ASO15nt_(APP) to cells and find that it can inhibit endogenous APP gene expression. We investigate the working mechanisms of L-Apt.4-1c-ASO15nt_(APP) in controlling gene activity, revealing that it controls gene expression by preventing DHX36 protein from unwinding the rG4 structure to enhance translational inhibition and recruiting RNase H to knockdown mRNA level.

Apart from L-aptamers, rG4 binding peptide is also a powerful tool to target rG4s. For example, a DHX36 protein-derived peptide, G4P, was reported to target and map genome dG4s. Increasing evidence has shown the presence of rG4s in cells. However, very limited studies showed the high-throughput, transcriptome-wide identification of rG4s in cells. In our study, we find that G4P can bind rG4s well. Therefore, we try to utilize G4P together with next generation sequencing (NGS) to map the rG4s landscape in cells. We develop the rG4PIP-seq (rG4 mapping by G4P-based immunoprecipitation and sequencing) method and identify about ~19,800 potential rG4 peaks in human cells. They are widely distributed in protein-coding RNAs (5'UTR, CDS and 3'UTR) and non-coding RNAs. We also selected some rG4s and found they can regulate gene expression validated by luciferase reporter assay.

In summary, we have developed a novel L-aptamer, L-Apt.8f, to target APP rG4 and control gene expression. To improve binding specificity, we designed an L-aptamer-ASO conjugate to recognize an individual rG4 and suppress gene expression by translation inhibition and RNase H-mediated mRNA knockdown. In addition, we developed a novel high-throughput rG4 mapping method, rG4PIP-seq, to profile transcriptome-wide rG4s in human cells.