pharmaceuticals Review The i-Motif as a Molecular Target: More Than a Complementary DNA Secondary Structure Susie L. Brown and Samantha Kendrick * Biochemistry and Molecular Biology Department, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA; [email protected] * Correspondence: [email protected] Abstract: Stretches of cytosine-rich DNA are capable of adopting a dynamic secondary structure, the i-motif. When within promoter regions, the i-motif has the potential to act as a molecular switch for controlling gene expression. However, i-motif structures in genomic areas of repetitive nucleotide sequences may play a role in facilitating or hindering expansion of these DNA elements. Despite research on the i-motif trailing behind the complementary G-quadruplex structure, recent discoveries including the identification of a specific i-motif antibody are pushing this field forward. This perspective reviews initial and current work characterizing the i-motif and providing insight into the biological function of this DNA structure, with a focus on how the i-motif can serve as a molecular target for developing new therapeutic approaches to modulate gene expression and extension of repetitive DNA. Keywords: i-motif; DNA quadruplex structures; transcription regulation; cancer therapeutics 1. Introduction Citation: Brown, S.L; Kendrick, S. In 1953, Watson and Crick first published the structure of the DNA double helix [1]. The i-Motif as a Molecular Target: They described a DNA molecule as a right-handed, twisted coil composed of a purine and More Than a Complementary DNA pyrimidine inner core held together by hydrogen bonds, with a sugar–phosphate back- Secondary Structure. Pharmaceuticals bone that extended from these paired bases. The x-ray diffraction photograph produced 2021, 14, 96. https://doi.org/ by Rosalind Franklin is one of the most readily identifiable images from scientific litera- 10.3390/ph14020096 ture. Initially, two variations of the double helix were identified, the A- and B-forms [2]. These two conformations have distinct positions of the deoxyribose sugar that result in Academic Editor: Mary Meegan different spacing between nucleotide bases, with DNA most commonly found in the Received: 28 December 2020 B-form [3]. In the 1970s, Z-DNA, the left-handed duplex, was observed under certain Accepted: 22 January 2021 Published: 27 January 2021 laboratory conditions [4]. This discovery gradually led to the realization that DNA can adopt different configurations. Since this early work uncovering the varied helices of Publisher’s Note: MDPI stays neutral duplex DNA, continued investigations demonstrated that nucleic acids also form sequence- with regard to jurisdictional claims in dependent secondary structures, such as triplex DNA, hairpins, and cruciforms, as well published maps and institutional affil- as tetraplex structures: the guanine-quadruplex (G4) and the intercalated-motif (i-motif) iations. (reviewed in [5]). G4 and i-motif structures are formed from stacked tetrads of guanine or cytosine nucleotides, respectively, that can occur in both single strands of DNA and where two or four strands assemble together. G4s were first identified in 1962 and biologically relevant structures in human telomeres were first discovered in 1987 [6,7] to arise from Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. sequences of contiguous guanines stabilized by cations such as K+ [8]. Extensive research This article is an open access article has explored the formation of G4 structures, their role in transcriptional regulation and distributed under the terms and telomere maintenance, and therapeutic potential, particularly in the area of silencing conditions of the Creative Commons oncogene expression in the treatment of cancer [9–15]. Nearly 30 years later in 1993, another Attribution (CC BY) license (https:// quadruplex-based DNA structure, the i-motif, was shown in foundational studies using creativecommons.org/licenses/by/ nuclear magnetic resonance (NMR), but unlike the G4, this structure is built from adjacent 4.0/). runs of cytosines [16]. Specifically, the i-motif consists of intercalated hemi-protonated Pharmaceuticals 2021, 14, 96. https://doi.org/10.3390/ph14020096 https://www.mdpi.com/journal/pharmaceuticals Pharmaceuticals 2021, 14, x FOR PEER REVIEW 2 of 25 Pharmaceuticals 2021, 14, 96 2 of 24 of cytosines [16]. Specifically, the i-motif consists of intercalated hemi-protonated cytosine (C-C+)cytosine base pairs (C-C+) with loop base regions pairs with composed loop regions of various composed bases intervening of various between bases intervening the cytosinebetween runs (Figure the cytosine 1). Similar runs to (Figurethe guanines1). Similar in the toG4, the base guanines pairing of in cytosines the G4, base occurs pairing via Hoogsteenof cytosines hydrogen occurs bonds; via Hoogsteen however, hydrogen since the bonds;formation however, of i-mo sincetif structures the formation also of i- requiresmotif the structuresprotonation also of cytosines, requires the i-motifs protonation form more of cytosines, readily at i-motifs acidic pH. form This more pH- readily dependentat acidic formation pH. This of i-motifs pH-dependent and the formationinitial lack ofof i-motifsevidence and that the i-motifs initial could lack ofform evidence that i-motifs could form in solutions at physiological pH led to skepticism within the in solutions at physiological pH led to skepticism within the DNA scientific community DNA scientific community as to whether i-motifs existed in vivo. Contrary to the belief as to whether i-motifs existed in vivo. Contrary to the belief that these structures may not that these structures may not form in cellular nuclei, the prevalence of C-rich regions in form in cellular nuclei, the prevalence of C-rich regions in telomeres and promoter regions telomeres and promoter regions suggest that the i-motif serves a biological function [17]. suggest that the i-motif serves a biological function [17]. Shortly after the discovery of the Shortly after the discovery of the i-motif, structures are now recognized to form from i-motif, structures are now recognized to form from C-rich sequences within telomeres as C-rich sequences within telomeres as well as those found in the promoters of primarily well as those found in the promoters of primarily oncogenes, including c-MYC [17]. Be- oncogenes, including c-MYC [17]. Beginning in 1997 with i-motif structure formation in ginning in 1997 with i-motif structure formation in the insulin-linked polymorphic region, the insulin-linked polymorphic region, studies showing that i-motifs have the ability to studies showing that i-motifs have the ability to block DNA replication furthered the idea block DNA replication furthered the idea that i-motifs may regulate nuclear processes [18]. that i-motifs may regulate nuclear processes [18]. The continuing research pursuits to The continuing research pursuits to demonstrate their formation and role in physiological demonstraterelevant their contexts formation led toand several role in breakthroughs physiological in relevant the i-motif contexts field, led particularly to several in the breakthroughslast few years,in the i-motif and posit field, these particularly structures in asthe the last next few likelyyears, DNAand posit structure these tostruc- target for tures astherapeutic the next likely development. DNA structure to target for therapeutic development. This reviewThis reviewfocuses focuses on the proposed on the proposed models fo modelsr the biological for the biological role of i-motifs role of in i-motifs the in contextthe of contextselect diseases. of select We diseases. discuss We how discuss discovery how of discovery i-motif ligands of i-motif serve ligands as both serve mo- as both lecularmolecular tools to tease tools out to teasethese out nuclear these functi nuclearons functions and potential and potential new therapeutics new therapeutics for drug for drug development.development. We also We highlight also highlight the different the different screening screening approaches approaches used usedto identify to identify i-mo- i-motif tif interactiveinteractive agents. agents. For Forbackground, background, we weinclude include a brief a brief discussion discussion on onthe therequirements requirements for for i-motifi-motif formation formation and and the the recent recent detection detection of of these these structures structures in in the the nuclei nuclei of of cells, cells, but but direct direct readersreaders to more comprehensive reviewsreviews forfor detaileddetailed descriptionsdescriptions of offactors factors affecting affecting i-motif i-motifstructure structure and and formation formation [ 19[19–23].–23]. Figure 1. Building blocks of the i-motif structure using c-MYC as a model. (A) A representation Figure of1. Building Hoogsteen blocks base of pairing the i-motif within structure an i-motif; using ( Bc-MYC) diagram as a model. of the c-MYC (A) A representationi-motif folded of structure; Hoogsteenand base (C) the pairing C-rich within sequence an i-motif; that gives (B) diagram rise to an of i-motif the c-MYC with i-motif the pairing folded pattern structure; of cytosine and tracts (C) the illustratedC-rich sequence in two that different gives shadesrise to an of bluei-moti [24f with]. the pairing pattern of cytosine tracts illus- trated in two different shades of blue [24]. 2. i-Motif Formation and Detection in Living Cells 2. i-Motif FormationEven though and initial Detection