Human genome function is underpinned by the primary storage of genetic information in canonical B-form DNA, with a second layer of DNA structure providing regulatory control. I-motif structures are thought to form in cytosine-rich regions of the genome and to have regulatory functions; however, in vivo evidence for the existence of such structures has so far remained elusive. Here we report the generation and characterization of an antibody fragment (iMab) that recognizes i-motif structures with high selectivity and affinity, enabling the detection of i-motifs in the nuclei of human cells. We demonstrate that the in vivo formation of such structures is cell-cycle and pH dependent. Furthermore, we provide evidence that i-motif structures are formed in regulatory regions of the human genome, including promoters and telomeric regions. Our results support the notion that i-motif structures provide key regulatory roles in the genome.

I-motif DNA structures are formed in the nuclei of human cells

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.

The i-Motif as a Molecular Target: More Than a Complementary DNA Secondary Structure.

The solution secondary structures and nucleotide conformations in the telomeric DNA of Oxytricha nova have been determined by Raman spectroscopy. Structural polymorphism is demonstrated for both single-stranded [d(T4G4)4] and double-stranded [d(G4T4G4).d(C4A4C4)] telomere models. In the case of d(T4G4)4, which is a prototype for the single-stranded telomeric tail, an interquadruplex equilibrium involving parallel and antiparallel strand configurations is shown to be governed by the solution concentrations of both Na+ and K+. In both the parallel and antiparallel quadruplexes of d(T4G4)4, the local geometry of the phosphodiester backbone is similar to that of canonical B DNA, and associations between bases of the guanine quartet involve Hoogsteen hydrogen bonding. However, the deoxyguanosine (dG) sugar conformations are significantly different in the two quadruplexes. In the extended parallel form, dG residues assume only the C2'-endo/anti conformation with respect to deoxyribose pucker and glycosyl orientation. In the foldback antiparallel form, dG residues are distributed equally between C2'-endo/anti and C2'-endo/syn conformations. In the case of d(G4T4G4).d(C4A4C4), which serves as a model for the double-stranded B DNA regions of Oxytricha telomeres, we show that disproportionation of the B form duplex to yield stable parallel quadruplexes of the guanine-rich strand occurs reversibly at high solution concentrations of either Na+ or K+. The present study reveals that both interquadruplex and duplex-quadruplex conversions of Oxytricha telomeric DNA are under the control of an alkali cation switch.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural Polymorphism of Telomere DNA: Interquadruplex and Duplex-Quadruplex Conversions Probed by Raman Spectroscopy. [Erratum to document cited in CA121:75118]

Nucleic acids have been among the first targets for antitumor drugs and antibiotics. With the unveiling of new biological roles in regulation of gene expression, specific DNA and RNA structures have become very attractive targets, especially when the corresponding proteins are undruggable. Biophysical assays to assess target structure as well as ligand binding stoichiometry, affinity, specificity, and binding modes are part of the drug development process. Mass spectrometry offers unique advantages as a biophysical method owing to its ability to distinguish each stoichiometry present in a mixture. In addition, advanced mass spectrometry approaches (reactive probing, fragmentation techniques, ion mobility spectrometry, ion spectroscopy) provide more detailed information on the complexes. Here, we review the fundamentals of mass spectrometry and all its particularities when studying noncovalent nucleic acid structures, and then review what has been learned thanks to mass spectrometry on nucleic acid structures, self-assemblies (e.g., duplexes or G-quadruplexes), and their complexes with ligands.

Mass Spectrometry of Nucleic Acid Noncovalent Complexes.

A free solution method was developed for evaluating the specific binding affinity and stoichiometry of small molecules with oligo DNA subsequent to cation-induced G-quadruplex formation. A nonlinear curve fitting equation capable of extracting specific binding constants in the presence of nonspecific binding without the need for reference compounds was proposed and tested. Electrospray ionization mass spectrometry was first used to rapidly screen the small molecule candidates; then, the stoichiometry and affinity constants of the native state binding pair in solution were obtained with capillary electrophoresis frontal analysis (CE-FA). The B cell lymphoma 2 (Bcl-2) oncogene is directly responsible for the expression of Bcl-2 protein, which plays a significant role in cell apoptosis. The binding of a G-quadruplex formed in the promoter region of the Bcl-2 oncogene with a small molecule could stabilize the quadruplex structure and potentially regulate the transcription of Bcl-2. Four natural product drug c...

Specific Binding Constant and Stoichiometry Determination in Free Solution by Mass Spectrometry and Capillary Electrophoresis Frontal Analysis

C-myb proto-oncogene is a potential therapeutic target for some human solid tumors and leukemias. A long cytosine-rich sequence, which locates the downstream of the transcription initiation site, is demonstrated to fold into an intramolecular i-motif DNA using electrospray ionization mass spectrometry (ESI-MS) and circular dichroism (CD) spectroscopy. Effects of factors, including the pH value, the number of C:C+ dimers, the concentration of buffer, the molecular crowding condition, and the coexistence of the complementary DNA, on the formation and the structural stability of the i-motif DNA are systematically studied. We have demonstrated that the i-motif folding in the c-myb promoter could be accelerated upon synergistic physiological stimuli including intracellular molecular crowding and low pH values, as well as the large number of the i-motif C:C+ dimers. Meanwhile, various inputs, such as acids/bases and metal ions, have exhibited their abilities in controlling the conformational switch of the c-myb GC-rich DNA. Acidic pH values and the presence of K+ ions can induce the dissociation of the double helix. Our present strategy can greatly extend the potential usages of i-motif DNA molecules with specific sequences as conformational switch-controlled devices. Moreover, this work demonstrates the superiority of CD spectroscopy associated with ESI-MS as a rapid, more cost-effective and sensitive structural change responsive method in the research of DNA conformational switching.

The formation and characteristics of the i-motif structure within the promoter of the c-myb proto-oncogene

RATIONALE The c-myb gene is a potential therapeutic target for human tumors and leukemias. Active ingredients from natural products may be used as drugs in chemotherapy for human cancers. Here, electrospray ionization mass spectrometry (ESI-MS) was used to probe the formation and recognition of the G-quadruplex structure from the G-rich sequence that is found in the c-myb gene promoter, 5'-GGGCTGGGCTGGGCGGGG-3'. The aim of our study is to evaluate a potential binder for the c-myb gene from natural products, and thereby to modulate c-myb gene expression. METHODS ESI-MS, as an effective method, was utilized not only to characterize the formation of the G-quadruplex in the c-myb oncogene, but also as a tool to probe the binding characteristics of alkaloid molecules with the target G-quadruplex DNA. RESULTS ESI-MS results with the support of circular dichroism (CD) spectra demonstrated the formation of an intramolecular parallel-stranded G-quadruplex in the c-myb oncogene promoter. A screening of six alkaloid molecules showed that brucine (P1) had a strong binding affinity to the c-myb G-quadruplex DNA. It is notable that P1 can bind selectively to the c-myb G-quadruplex with respect to duplex DNAs, as well as to G-quadruplexes in other types of gene sequences. According to ESI-MS results, in which the stability was tested by capillary heating and collision-induced dissociation, the binding of P1 could thermally stabilize the c-myb G-quadruplex DNA. CONCLUSIONS In this work, brucine (P1), an alkaloid molecule, has been found to bind to the intramolecular parallel G-quadruplex in the c-myb oncogene promoter with high affinity and selectivity, and could thermally stabilize the c-myb G-quadruplex DNA, indicating that the binding of P1 has the potential to modulate c-myb gene expression. Copyright © 2015 John Wiley & Sons, Ltd.

Jinhui Hai

Papers

The formation and characteristics of the i-motif structure within the promoter of the c-myb proto-oncogene

Exploration of binding affinity and selectivity of brucine with G-quadruplex in the c-myb proto-oncogene by electrospray ionization mass spectrometry.