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The non-canonical structures formed by G- or C-rich DNA regions, such as quadruplexes and i-motifs, as well as their associates, have recently been attracting increasing attention both because of the arguments in favor of their existence in vivo and their potential application in nanobiotechnology. When studying the structure and properties of non-canonical forms of DNA, as well as when controlling the artificially created architectures based on them, visualization plays an important role. This review analyzes the methods used to visualize quadruplexes, i-motifs, and their associates with high spatial resolution: fluorescence microscopy, transmission electron microscopy (TEM), and atomic force microscopy (AFM). The key approaches to preparing specimens for the visualization of this type of structures are presented. Examples of visualization of non-canonical DNA structures having various morphologies, such as G-wires, G-loops, as well as individual quadruplexes, i-motifs and their associates, are considered. The potential for using AFM for visualizing non-canonical DNA structures is demonstrated.

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We studied the ability of oligonucleotides CnT25 (n = 2, 5, 7, 9, 12, 25) to form an intermolecular i-motif using circular dichroism, ultra-violet spectroscopy, nuclear magnetic resonance, high-resolution atomic force microscopy, high-performance liquid chromatography, and molecular dynamics simulations. The arrangement of single-stranded oligonucleotides in multimer i-motifs was very unusual: C-tracts of different oligonucleotides followed each other consecutively in order to fold into a closed intermolecular i-motif core with minimal loops (one cytidine in a loop spanning over a minor groove, three cytidines in a loop over a major groove); intact T-tracts protruded from predefined loci allowing visualization of beetle-like nanostructures by atomic force microscopy. The same structures were formed from analogous biotinylated oligonucleotides demonstrating one of the potential applications of such structures as carriers of multiple functional groups. Our findings open up possibilities for the rational design of pH-sensitive DNA aggregates and evaluation of the efficiency of their assembly.

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In this paper, we report results of systematic studies of conformational polymorphism of G-rich DNA fragments from Alu repeats. Alu retrotransposones are primate-specific short interspersed elements. Using the Alu sequence from the prooncogen bcl2 intron and the consensus AluSx sequence as representative examples, we determined characteristic Alu sites that are capable of adopting G-quadruplex (GQ) conformations (i.e., potential quadruplex sites - PQSAlu), and demonstrated by bioinformatics methods that those sites are Alu-specific in the human genome. Genomic frequencies of PQSAlu were assessed (~1/10000 b.p.). The sites were found to be characteristic of young (active) Alu families (Alu-Y). A recombinant DNA sequence bearing the Alu element from the human bcl2 gene (304 b.p.) and its PQS-mutant (Alu-PQS) were constructed. The formation of noncanonical structures in Alubcl2 dsDNA and the absence of such structures in the case of Alu-PQS were shown using DMS-footprinting and AFM microscopy. Expression vectors bearing wild-type and mutant Alu insertions in the promoter regions were obtained, and the effects of these insertions on the expression of the reporter gene in НЕК293 and HeLa cell lines were compared. Our findings on the spatial organization of Alu repeats may provide insight into the mechanisms of genomic rearrangements which underlie many oncological and neurodegenerative diseases.

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BACKGROUND: Fibrinogen has been intensively studied with transmission electron microscopy and x-ray diffraction. But until now, a complete 3D structure of the molecule has not yet been available because the two highly flexible αC regions could not be resolved in fibrinogen crystals. This study was aimed at determining whether the αC regions can be visualized by high-resolution atomic force microscopy. METHODS: Atomic force microscopy with super high resolution was used to image single molecules of fibrinogen and fibrin associates. The key approach was to use a graphite surface modified with the monolayer of amphiphilic carbohydrate-glycine molecules and unique supersharp cantilevers with 1 nm tip diameter. RESULTS: Fibrinogen αC regions were visualized along with the complete domain structure of the protein. In almost all molecules at pH 7.4 the D domain regions had one or two protrusions of average height 0.4 ± 0.1 nm and length 21 ± 6 nm. The complex, formed between thrombin and fibrinogen, was also visualized. Images of growing fibrin fibers with clearly visible αC regions have been obtained. CONCLUSIONS: Fibrin αC regions were visible in protofibrils and large fibers; αC regions intertwined near a branchpoint and looked like a zipper. These results support the idea that αC regions are involved in the thickening of fibrin fibers. In addition, new details were revealed about the behavior of individual fibrin molecules during formation of the fibrin network. Under the diluted condition, the positioning of the αC regions could suggest their involvement in long-range interactions between fibrin but not fibrinogen molecules.

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