i Motif Structures in the Human Genome: Formation and Function

Repetitive DNA sequences play a crucial role in genome function, and i-motif structures have gained attention for their regulatory significance. These four-stranded DNA secondary structures form under specific conditions, such as acidic pH, and are composed of cytosine-rich sequences. Unlike the widely studied G-quadruplexes, i-motifs adopt a distinct folding pattern that may influence various biological processes.

Understanding their role in genomic regulation is essential, as research suggests they contribute to gene expression control and cellular function. Recent advancements have provided insights into their stability and potential links to disease.

Structural Formation And Stability

The formation of i-motif structures is driven by the base-pairing properties of cytosine-rich sequences, which fold into an intercalated, four-stranded conformation stabilized by hemiprotonated C•C⁺ base pairs. This structure emerges under mildly acidic conditions, where protonation of cytosine at the N3 position facilitates stable hydrogen bonds. Unlike rigid G-quadruplexes, i-motifs are dynamic, transitioning between folded and unfolded states based on environmental factors such as pH, temperature, and molecular crowding. This suggests they may function as molecular switches, responding to cellular conditions.

Beyond pH sensitivity, stability is influenced by sequence composition and loop length. Shorter loops between cytosine tracts enhance stability, while longer loops introduce flexibility that can destabilize the fold. Adjacent guanine-rich sequences can also modulate i-motif formation, as G-quadruplexes and i-motifs often exist in equilibrium, potentially regulating genomic processes.

Cellular conditions further impact i-motif stability. Molecular crowding agents such as polyethylene glycol (PEG) and proteins enhance their persistence under near-neutral pH. Fluorescence-based probes have detected their presence in human cell nuclei, challenging earlier assumptions that i-motifs were only an in vitro phenomenon and reinforcing their biological relevance.

Detection Techniques

Identifying i-motif structures requires methodologies that capture their transient nature. Traditional biochemical techniques, such as gel electrophoresis and circular dichroism (CD) spectroscopy, have been instrumental in characterizing their formation in vitro. CD spectroscopy provides a distinctive signature, with a positive peak at approximately 285 nm and a negative peak near 260 nm, distinguishing i-motifs from other DNA structures. However, these approaches cannot directly visualize i-motifs in living cells.

To address this, researchers have developed fluorescence-based probes for real-time detection. One significant advancement is the single-chain fragment variable (scFv) antibody iMab, which selectively binds i-motif structures without disrupting their native conformation. Immunofluorescence microscopy studies using iMab have confirmed their presence in human nuclei, particularly in gene promoters.

Small-molecule probes also aid in detection. Thioflavin T (ThT), a dye traditionally used for amyloid detection, selectively binds i-motifs and fluoresces upon interaction, offering a non-invasive way to track i-motif dynamics in live cells. Förster resonance energy transfer (FRET)-based probes have been engineered to monitor i-motif folding and unfolding in response to environmental changes, providing insights into their role in genomic regulation.

Distribution In The Genome

i-Motif structures are not randomly distributed in the human genome but are enriched in regulatory regions, particularly gene promoters involved in cell cycle control, oncogenesis, and epigenetic regulation. High-throughput sequencing analyses have revealed their prevalence upstream of transcription start sites, suggesting they influence gene expression through DNA conformational changes.

They are also found in telomeric regions, where they coexist with G-quadruplex structures. Telomeres, composed of repetitive cytosine- and guanine-rich sequences, provide a favorable environment for these non-canonical DNA structures. The equilibrium between i-motifs and G-quadruplexes may contribute to telomere maintenance and genome stability. Given telomere dysfunction’s links to aging and cancer, i-motifs in these regions may play a role in cellular senescence and proliferative control.

Beyond promoters and telomeres, i-motif sequences appear in intronic and untranslated regions, suggesting their influence extends beyond direct transcriptional regulation. Their transient formation in response to environmental cues raises the possibility that they affect alternative splicing or RNA processing by modulating DNA topology. Their presence in enhancer elements implies a broader regulatory network where secondary DNA structures contribute to chromatin interactions.

Regulatory Interplay With Transcription

i-Motif structures in gene promoters position them as potential transcription regulators. Their folding and unfolding dynamics, influenced by cellular conditions such as pH and redox state, suggest they act as molecular switches modulating gene activity. When stabilized, i-motifs can alter DNA topology, creating structural barriers that affect transcriptional machinery recruitment. Conversely, their unfolding may facilitate access to transcription factors and RNA polymerase, promoting gene expression.

Studies have identified i-motif structures in promoters of genes regulating the cell cycle and oncogenesis, including c-MYC, BCL2, and KRAS. In c-MYC, a gene implicated in many cancers, an i-motif within its promoter influences transcription. Small molecules that stabilize this structure can suppress c-MYC expression, highlighting a potential therapeutic strategy. i-Motifs may also function in epigenetic regulation, interacting with histone modifications and chromatin remodeling factors to fine-tune transcriptional responses.

Protein Binding And Recognition

i-Motif structures interact with cellular proteins that modulate their stability and function. Certain DNA-binding proteins recognize i-motifs with high specificity, either stabilizing or destabilizing them depending on the cellular context. These proteins often belong to helicase families, transcription factors, or chromatin remodeling complexes, shaping local DNA architecture.

The heterogeneous nuclear ribonucleoprotein (hnRNP) family, including hnRNP K, preferentially binds i-motifs in gene promoters, potentially influencing transcription by stabilizing or resolving these structures. Nucleolin, a protein involved in ribosomal biogenesis and chromatin organization, also binds i-motif sequences, enhancing their stability in oncogene promoters.

Helicases such as DHX36 (RHAU) further underscore their regulatory potential. DHX36 unwinds non-canonical DNA structures, including G-quadruplexes and i-motifs, facilitating transitions between folded and unfolded states. This enzymatic activity suggests a mechanism for resolving i-motifs during replication, transcription, and chromatin remodeling. The interplay between i-motifs and their protein partners reinforces their role as active participants in genome regulation.

Potential Contributions To Disease Mechanisms

i-Motif structures in key regulatory regions have been implicated in disease mechanisms, particularly cancer and neurodegenerative disorders. Their role in gene expression modulation suggests a potential influence on tumorigenesis, where dysregulated transcription drives uncontrolled cell proliferation. Their enrichment in oncogene promoters, including c-MYC and BCL2, highlights their potential impact on cancer progression. Stabilizing i-motifs with specific proteins or small molecules can suppress oncogene expression, presenting a possible therapeutic strategy.

Beyond cancer, i-motifs may contribute to neurodegenerative diseases through their involvement in genome stability and transcriptional control. DNA secondary structures, including i-motifs, can influence the expression of genes associated with neurological function. Disruptions in i-motif dynamics, possibly due to oxidative stress or aging-related epigenetic changes, could alter gene expression linked to conditions such as Alzheimer’s and Parkinson’s disease. i-Motifs may serve as biomarkers for disease progression, as their formation could indicate broader genomic instability associated with pathological states.