Enhanced Recognition of Single-Base Mismatch Using Locked Nucleic Acid-Integrated Hairpin DNA Probes Revealed by Atomic Force Microscopy Nanolithography

2010 ◽  
Vol 82 (6) ◽  
pp. 2395-2400 ◽  
Author(s):  
Wen-Hsin Han ◽  
Jun-Min Liao ◽  
Kuan-Liang Chen ◽  
Shin-Mou Wu ◽  
Yi-Wen Chiang ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Nucleic Acid ◽  
Dna Probes ◽  
Locked Nucleic Acid ◽  
Hairpin Dna ◽  
Single Base ◽  
Force Microscopy ◽  
Atomic Force ◽  
Single Base Mismatch ◽  
Base Mismatch

scholarly journals Atomic Force Microscopy Investigation of the Interactions between the MCM Helicase and DNA

Materials ◽  
2021 ◽  
Vol 14 (3) ◽  
pp. 687
Author(s):  
Amna Abdalla Mohammed Khalid ◽  
Pietro Parisse ◽  
Barbara Medagli ◽  
Silvia Onesti ◽  
Loredana Casalis
Keyword(s):  
Atomic Force Microscopy ◽  
Nucleic Acid ◽  
Single Molecule ◽  
Protein Complex ◽  
The Other ◽  
Contour Length ◽  
Force Microscopy ◽  
Atomic Force ◽  
Mcm Complex ◽  
Dna Double Helix

The MCM (minichromosome maintenance) protein complex forms an hexameric ring and has a key role in the replication machinery of Eukaryotes and Archaea, where it functions as the replicative helicase opening up the DNA double helix ahead of the polymerases. Here, we present a study of the interaction between DNA and the archaeal MCM complex from Methanothermobacter thermautotrophicus by means of atomic force microscopy (AFM) single molecule imaging. We first optimized the protocol (surface treatment and buffer conditions) to obtain AFM images of surface-equilibrated DNA molecules before and after the interaction with the protein complex. We discriminated between two modes of interaction, one in which the protein induces a sharp bend in the DNA, and one where there is no bending. We found that the presence of the MCM complex also affects the DNA contour length. A possible interpretation of the observed behavior is that in one case the hexameric ring encircles the dsDNA, while in the other the nucleic acid wraps on the outside of the ring, undergoing a change of direction. We confirmed this topographical assignment by testing two mutants, one affecting the N-terminal β-hairpins projecting towards the central channel, and thus preventing DNA loading, the other lacking an external subdomain and thus preventing wrapping. The statistical analysis of the distribution of the protein complexes between the two modes, together with the dissection of the changes of DNA contour length and binding angle upon interaction, for the wild type and the two mutants, is consistent with the hypothesis. We discuss the results in view of the various modes of nucleic acid interactions that have been proposed for both archaeal and eukaryotic MCM complexes.

Atomic Force Microscopy and Anodic Porous Allumina of Nucleic Acid Programmable Protein Arrays

2013 ◽  
Vol 7 (2) ◽  
pp. 112-121 ◽  
Author(s):  
Claudio Nicolini ◽  
Tercio Correia ◽  
Enrico Stura ◽  
Claudio Larosa ◽  
Rosanna Spera ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Nucleic Acid ◽  
Protein Arrays ◽  
Force Microscopy ◽  
Atomic Force

scholarly journals Mismatch detection in DNA monolayers by atomic force microscopy and electrochemical impedance spectroscopy

2016 ◽  
Vol 7 ◽  
pp. 220-227 ◽  
Author(s):  
Maryse D Nkoua Ngavouka ◽  
Pietro Capaldo ◽  
Elena Ambrosetti ◽  
Giacinto Scoles ◽  
Loredana Casalis ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Electrochemical Impedance Spectroscopy ◽  
Impedance Spectroscopy ◽  
Electrochemical Impedance ◽  
Complementary Sequence ◽  
Single Base ◽  
Force Microscopy ◽  
Dna Monolayers ◽  
Atomic Force ◽  
The Difference

Background: DNA hybridization is at the basis of most current technologies for genotyping and sequencing, due to the unique properties of DNA base-pairing that guarantee a high grade of selectivity. Nonetheless the presence of single base mismatches or not perfectly matched sequences can affect the response of the devices and the major challenge is, nowadays, to distinguish a mismatch of a single base and, at the same time, unequivocally differentiate devices read-out of fully and partially matching sequences. Results: We present here two platforms based on different sensing strategies, to detect mismatched and/or perfectly matched complementary DNA strands hybridization into ssDNA oligonucleotide monolayers. The first platform exploits atomic force microscopy-based nanolithography to create ssDNA nano-arrays on gold surfaces. AFM topography measurements then monitor the variation of height of the nanostructures upon biorecognition and then follow annealing at different temperatures. This strategy allowed us to clearly detect the presence of mismatches. The second strategy exploits the change in capacitance at the interface between an ssDNA-functionalized gold electrode and the solution due to the hybridization process in a miniaturized electrochemical cell. Through electrochemical impedance spectroscopy measurements on extended ssDNA self-assembled monolayers we followed in real-time the variation of capacitance, being able to distinguish, through the difference in hybridization kinetics, not only the presence of single, double or triple mismatches in the complementary sequence, but also the position of the mismatched base pair with respect to the electrode surface. Conclusion: We demonstrate here two platforms based on different sensing strategies as sensitive and selective tools to discriminate mismatches. Our assays are ready for parallelization and can be used in the detection and quantification of single nucleotide mismatches in microRNAs or in genomic DNA.

scholarly journals Atomic Force Microscopy Investigation of Human Immunodeficiency Virus (HIV) and HIV-Infected Lymphocytes

2003 ◽  
Vol 77 (22) ◽  
pp. 11896-11909 ◽  
Author(s):  
Y. G. Kuznetsov ◽  
J. G. Victoria ◽  
W. E. Robinson ◽  
A. McPherson
Keyword(s):  
Human Immunodeficiency Virus ◽  
Atomic Force Microscopy ◽  
Nucleic Acid ◽  
Matrix Protein ◽  
Human Lymphocytes ◽  
Tween 20 ◽  
Force Microscopy ◽  
Virus Particles ◽  
Atomic Force ◽  
Immunodeficiency Virus

ABSTRACT Isolated human immunodeficiency virus (HIV) and HIV-infected human lymphocytes in culture have been imaged for the first time by atomic force microscopy (AFM). Purified virus particles spread on glass substrates are roughly spherical, reasonably uniform, though pleomorphic in appearance, and have diameters of about 120 nm. Similar particles are also seen on infected cell surfaces, but morphologies and sizes are considerably more varied, possibly a reflection of the budding process. The surfaces of HIV particles exhibit “tufts” of protein, presumably gp120, which do not physically resemble spikes. The protein tufts, which number about 100 per particle, have average diameters of about 200 Å, but with a large variance. They likely consist of arbitrary associations of small numbers of gp120 monomers on the surface. In examining several hundred virus particles, we found no evidence that the gp120 monomers form threefold symmetric trimers. Although >95% of HIV-infected H9 lymphocytic cells were producing HIV antigens by immunofluorescent assay, most lymphocytes displayed few or no virus on their surfaces, while others were almost covered by a hundred or more viruses, suggesting a dependence on cell cycle or physiology. HIV-infected cells treated with a viral protease inhibitor and their progeny viruses were also imaged by AFM and were indistinguishable from untreated virions. Isolated HIV virions were disrupted by exposure to mild neutral detergents (Tween 20 and CHAPS) at concentrations from 0.25 to 2.0%. Among the products observed were intact virions, the remnants of completely degraded virions, and partially disrupted particles that lacked sectors of surface proteins as well as virions that were split or broken open to reveal their empty interiors. Capsids containing nucleic acid were not seen, suggesting that the capsids were even more fragile than the envelope and were totally degraded and lost. From these images, a good estimate of the thickness of the envelope protein-membrane-matrix protein outer shell of the virion was obtained. Treatment with even low concentrations (<0.1%) of sodium dodecyl sulfate completely destroyed all virions but produced many interesting products, including aggregates of viral proteins with strands of nucleic acid.

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