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As a non-canonical structure, iMs are indicated to play an important role in the genome.

Complementary G-rich and C-rich sequences can form G4s and iMs interdependently during distinct cellular processes ( 4). iMs are also four-stranded structures, but formed from cytosine C-rich regions that are stabilised by hemi-protonated C-C base pairs (C +:C) ( 2, 3). G4s are four-stranded structures formed from G-rich sequences and are stabilised by Hoogsteen hydrogen bonding between guanines ( 1). Besides the canonical Watson-Crick double-helical B-form structure, DNA can form non-canonical secondary structures such as G-quadruplexes (G4s) and i-Motifs (iMs). In addition to this transfer of information, DNA and RNA can form complex structures, which can play crucial functional roles in organisms. DNA encodes genetic information, which is transcribed to mRNA and then translated to protein. Nucleotides are the basic units that form DNA and RNA, two key molecules in the central dogma. Model interpretation confirms that the nucleotide composition of the C-rich sequence significantly affects iM stability, with a positive correlation with sequences containing cytosine and thymine and a negative correlation with guanine and adenine. iM-Seeker predicts DNA iM folding status with a classification accuracy of 81% and estimates the folding strength with coefficient of determination ( R 2) of 0.642 on the test set. The programme iM-Seeker incorporates a Balanced Random Forest classifier trained on genome-wide iMab antibody-based CUT&Tag sequencing data to predict the folding status and an Extreme Gradient Boosting regressor to estimate the folding strength according to both literature biophysical data and our in-house biophysical experiments. Here, we introduce a machine learning pipeline, iM-Seeker, to predict both folding status and structural stability of DNA iMs. However, there are no dedicated computational tools for predicting the folding status and strength of iM structures. Much previous research on iM has focused on assessing the iM folding properties using biophysical experiments. Although putative iM forming sequences are widely distributed in the human genome, the folding status and strength of putative iMs vary dramatically.

I-Motifs (iMs), are secondary structures formed in cytosine-rich DNA sequences and are involved in multiple functions in the genome.

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