Dr. Olga Yu Limanskaya Profile

Dr. Olga Yu Limanskaya

at Mechnikov Institute M&I UAMS

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Publications (1)

Proceedings Article | 18 April 2003 Paper

Inverted repeats: computer analysis of microorganism genome and imaging of cruciform structure in DNA by atomic force microscopy

Alex Limansky, Olga Limanskaya

Proceedings Volume 5119, (2003) https://doi.org/10.1117/12.501637

KEYWORDS: Atomic force microscopy, Mica, Visualization, Microorganisms, Leukemia, Argon, Molecules, Viruses, In vivo imaging, Glasses

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Inverted repeats may regulate genetic procceses by formation of hairpin secondary structures that block DNA polymerases. Two different DNA conformations may cor-respond to inverted repeats: either a linear double stranded helix or a cruciform struc-ture consisting of two symmetrical hairpins. Theoretical and experimental studies have shown that cruciform structures can exist in negatively supercoiled DNA, cont-rary to relaxed molecules. Cruciform formation depends on many factors, firstly, on temperature and supercoils density. Recently application of the scanning probe mic-roscopy has allowed for significant progress in cruciform structure studies. The goal of present work is computer analysis of inverted repeats in viruses, bac-teria and plasmid DNA (human immunodeficiency virus (HIV), bovine immunode-ficiency virus (BIV), bovine leukemia virus (BLV), mycobacterium tuberculosis (MTB), plasmid pUC8) and direct visualization of the cruciform structure in super-coiled DNA by atomic force microscopy (AFM). The cruciform dimensions were determined. Analysis and modeling of the most thermodynamically stable cruciform formations in viral and bacterial DNA were carried out. The complete genome sequence of HIV, BIV, BLV is ~9000 base pairs (bp), my-cobacterium tuberculosis - over 4000000 bp, pUC8 DNA - 2665 bp. Computer ana-lysis showed that two different isolates of MTB with complete genome contain 45 and 50 inverted repeats; HIV, BIV, BLV and plasmid pUC8 contain only one palin-drome which can form cruciform structure in buffer solution. Cruciform in plasmid pUC8 supercoiled DNA, was directly visualized by atomic force microscopy. Cruciform is seen as clear-cut extrusions on the DNA filaments with the lengths of the arms fully consistent with the size of the hairpins expected from a 26 bp inverted repeat in pUC8 plasmid DNA. Application of the aminomodi-fied mica allowed to obtain stable DNA images. DNA molecules on aminomica are not stretched and their contours are smooth. The geometry of cruciform depends on ionic coditions. At low ionic strength cruciform can adopt an extended conformation with the angle of ca. 180° between the hairpins arms. AFM image shows that hairpin of cruciform structure is formed by 13-14 bp. Results of search for self-complementary regions of pUC8 DNA sequence confirmed that hairpin is formed by the double 11bp sequences and a loop of 4 nucleotides.

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