Biology subjects

Guneri, D.

Publications and source records attributed to Guneri, D..

6 recordsLinked to original sources

bioRxiv2026

RNA i-Motif Formation at Neutral pH

i-Motifs are non-canonical nucleic acid secondary structures formed in sequences rich in cytosine. Previous work has shown that DNA i-motifs may form at neutral pH and provided evidence to suggest they influence biological functions. RNA i-motifs are less stable than DNA i-motifs, so more questions surround the biological relevance of these structures in RNA. Using biophysical methods, we found that increasing cytosine tract lengths resulted in increased thermal stability but not pH stability. In ensemble solution experiments RNA i-motifs appear globally unfolded at neutral pH. However, single molecule experiments revealed that 1% of RNA i-motifs remain folded in solution. This has implications for the potential of formation of RNA i-motifs in cells.

biophysics↗

bioRxiv2025

Affinity-selected peptide ligands specifically bind i-motif DNA and modulate c-Myc gene expression

c-Myc is an oncogene that is dysregulated in ~70% of cancers. Its multifaceted function complicates effective drug targeting of the protein. i-Motif DNA structures in gene promotor regions have gained attention for their potential role in modulation of gene expression. These include the i-motif formed by the cytosine-rich sequence that lies upstream of the key P1 promotor of the c-Myc gene. Currently, selective ligands interacting with i-motif structures are limited. Here, peptide ligands for the i-motif from the promoter of c-Myc were identified via phage display. Hit peptides were filtered for selective binding to i-motif structures over other DNA structures using displacement assays and DNA melting experiments. Two lead peptides were found to produce dose-dependent changes on c-Myc gene expression after delivery into HEK293 cells expressing a c-Myc luciferase reporter construct. These leads may be used as chemical tools for the manipulation of c-Myc i-motif in vitro and have potential to be developed into cell-permeable peptidomimetics for delivery in vivo. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/656635v2_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@18a4e9aorg.highwire.dtl.DTLVardef@12e72b0org.highwire.dtl.DTLVardef@6ba866org.highwire.dtl.DTLVardef@1fcd338_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

G-Quadruplexes Act as an On/Off Switch While i-Motifs Regulate Insulin Expression in Reporter Gene Assays

The insulin-linked polymorphic region (ILPR) is a variable number tandem repeat located in the promoter of the human insulin gene. This G-rich sequence can fold into four-stranded G-quadruplex DNA structures, while its complementary C-rich strand forms i-motifs. The ILPR varies in repeat number and sequence composition, but the relationship between sequence diversity, DNA structure, and insulin gene regulation remains poorly understood. Although both G-quadruplexes and i-motifs have been implicated in transcriptional control, their relative contributions, particularly when formed on complementary strands of the same locus, are unclear. Here, we characterised the structure and stability of nine ILPR-based sequences using biophysical techniques and luciferase reporter assays. We demonstrate that transcriptional activation in response to high glucose occurs only when both G-quadruplex and i-motif structures can form. Other combinations of structures do not induce transcription. Moreover, promoter activity correlated positively with i-motif stability, but not with G-quadruplex stability. These results suggest a model in which G-quadruplexes function as an on/off switch, while i-motifs act as modulators of gene expression. Our findings underscore the importance of treating G-quadruplexes and i-motifs as a dynamic, interdependent system in both the regulation of gene expression and also the potential of these structures as therapeutic targets. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC="FIGDIR/small/651924v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@16ae322org.highwire.dtl.DTLVardef@65e893org.highwire.dtl.DTLVardef@884ab3org.highwire.dtl.DTLVardef@1e0311e_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Prediction of DNA i-Motifs Via Machine Learning

i-Motifs (iMs), are secondary structures formed in cytosine-rich DNA sequences and are involved in multiple functions in the genome. Although putative iM forming sequences are widely distributed in the human genome, the folding status and strength of putative iMs vary dramatically. Much previous research on iM has focused on assessing the iM folding properties using biophysical experiments. However, there are no dedicated computational tools for predicting the folding status and strength of iM structures. Here, we introduce a machine learning pipeline, iM-Seeker, to predict both folding status and structural stability of DNA iMs. 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. iM-Seeker predicts DNA iM folding status with a classification accuracy of 81% and estimates the folding strength with coefficient of determination (R2) of 0.642 on the test set. 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. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/571121v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@11f4398org.highwire.dtl.DTLVardef@18eb7e9org.highwire.dtl.DTLVardef@cc49c6org.highwire.dtl.DTLVardef@1ff91a8_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2023

Structural Insights into Regulation of Insulin Expression Involving i-Motif DNA Structures in the Insulin-Linked Polymorphic Region.

The insulin linked polymorphic region (ILPR) is a variable number of tandem repeats (VNTR) region of DNA in the promoter of the insulin gene that regulates transcription of insulin. This region is known to form the alternative DNA structures, i-motifs and G-quadruplexes. Individuals have different sequence variants of VNTR repeats and although previous work investigated the effects of some variants on G-quadruplex formation, there is not a clear picture of the relationship between the sequence diversity, the DNA structures formed, and the functional effects on insulin gene expression. Here we show that different sequence variants of the ILPR form different DNA secondary structures and insulin expression is dependent on formation of i-motif and G-quadruplex structures. The first crystal structure and dynamics of an intramolecular i-motif also reveal sequences within the loop regions forming additional stabilising interactions, which are critical to formation of the stable i-motif structures that modulate insulin expression. The outcomes of this work reveal the detail in formation of stable i-motif DNA structures, with potential for rational based drug design for compounds to alter insulin gene expression.

biophysics↗

bioRxiv2022

The Response of the Eukaryotic Replisome to G-quadruplex- and i-motif-forming Sequences

Sequences that can form DNA secondary structures, such as G-quadruplexes (G4s) and intercalated-Motifs (iMs), are abundant in the human genome and play a range of physiological roles. However, they can also pose a challenge to the replication machinery and in turn threaten genome stability. Multiple lines of evidence suggest G4s interfere with replication, but the underlying mechanism remains unclear. Moreover, there is a lack of evidence of how iMs affect the replisome. Here, we reconstitute replication of physiologically derived structure-forming sequences to find that a single G4 or iM is sufficient to arrest DNA replication. Direct single molecule structure detection within solid-state nanopores reveals structures form as a consequence of replication. A combination of genetic and biophysical characterisation establishes that structure forming capacity is a key determinant of replisome arrest. Mechanistically, replication fork arrest is caused by impaired synthesis, resulting in helicase-polymerase uncoupling. Significantly, iMs also induce breakage of nascent DNA. Finally, stalled forks are only rescued by a specialised helicase, Pif1, but not Sgs1 or Chl1. Altogether, this study provides a potential mechanism for quadruplex structure formation and resolution during replication and highlights G4s and iMs as endogenous sources of replication stress, which may explain their genomic instability and mutation frequencies in cancer.

molecular biology↗