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We acknowledge Jules Vidal for fruitful discussions on this topic during his stay in UCM. This work was supported by the Spanish Ministry of Economy and Competitiveness [RTI2018-095802-B-I00]., Ligands change the chemical and mechanical properties of polymers. In particular, single strand binding protein (SSB) non-specifically bounds to single-stranded DNA (ssDNA), modifying the ssDNA stiffness and the DNA replication rate, as recently measured with single-molecule techniques. SSB is a large ligand presenting cooperativity in some of its binding modes. We aim to develop an accurate kinetic model for the cooperative binding kinetics of large ligands. Cooperativity accounts for the changes in the affinity of a ligand to the polymer due to the presence of another bound ligand. Large ligands, attaching to several binding sites, require a detailed counting of the available binding possibilities. This counting has been done by McGhee and von Hippel to obtain the equilibrium state of the ligands-polymer complex. The same procedure allows to obtain the kinetic equations for the cooperative binding of ligands to long polymers, for all ligand sizes. Here, we also derive approximate cooperative kinetic equations in the large ligand limit, at the leading and next-to-leading orders. We found cooperativity is negligible at the leading-order, and appears at the next-to-leading order. Positive cooperativity (increased affinity) can be originated by increased binding affinity or by decreased release affinity, implying different kinetics. Nevertheless, the equilibrium state is independent of the origin of cooperativity and only depends on the overall increase in affinity. Next-to-leading approximation is found to be accurate, particularly for small cooperativity. These results allow to understand and characterize relevant ligand binding processes, as the binding kinetics of SSB to ssDNA, which has been reported to affect the DNA replication rate for several SSB-polymerase pairs. (C) 2022 The Author(s). Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology., Spanish Ministry of Economy and Competitiveness, Ministerio de Economía y Competitividad, Depto. de Estructura de la Materia, Física Térmica y Electrónica, Fac. de Ciencias Físicas, TRUE, pub

©2020 American Physical Society., Ligand binding to polymers modifies the physical and chemical properties of the polymers, leading to physical, chemical, and biological implications. McGhee and von Hippel obtained the equilibrium coverage as a function of the ligand affinity, through the computation of the possible binding sites for the ligand. Here, we complete this theory deriving the kinetic model for the ligand-binding dynamics and the associated equilibrium chemical potential, which turns out to be of the Tonks gas model type. At low coverage, the Tonks chemical potential becomes the Fermi chemical potential and even the ideal gas chemical potential. We also discuss kinetic models associated with these chemical potentials. These results clarify the kinetic models of ligand binding, their relations with the chemical potentials, and their range of validity. Our results highlight the inaccuracy of ideal and simplified kinetic approaches for medium and high coverages., Depto. de Estructura de la Materia, Física Térmica y Electrónica, Fac. de Ciencias Físicas, TRUE, pub

© 2017 Jarillo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. This work was supported by grants GR35/14-920911 (Banco Santander and Universidad Complutense de Madrid, Spain), and FIS2010-17440 and FIS2015-67765-R (MINECO, Spain) to JJ, EB, JPGV, and FJC, and by grant BFU2012-31825 and BFU2015-63714-R (MINECO, Spain) to JM and BI. Additionally, JJ and EB acknowledge financial support from FPU13/02934 and FPU13/02826 grants (Ministerio de Educación, Cultura y Deporte, Spain), respectively., Ligands binding to polymers regulate polymer functions by changing their physical and chemical properties. This ligand regulation plays a key role in many biological processes. We propose here a model to explain the mechanical, thermodynamic, and kinetic properties of the process of binding of small ligands to long biopolymers. These properties can now be measured at the single molecule level using force spectroscopy techniques. Our model performs an effective decomposition of the ligand-polymer system on its covered and uncovered regions, showing that the elastic properties of the ligand-polymer depend explicitly on the ligand coverage of the polymer (i.e., the fraction of the polymer covered by the ligand). The equilibrium coverage that minimizes the free energy of the ligand-polymer system is computed as a function of the applied force. We show how ligands tune the mechanical properties of a polymer, in particular its length and stiffness, in a force dependent manner. In addition, it is shown how ligand binding can be regulated applying mechanical tension on the polymer. Moreover, the binding kinetics study shows that, in the case where the ligand binds and organizes the polymer in different modes, the binding process can present transient shortening or lengthening of the polymer, caused by changes in the relative coverage by the different ligand modes. Our model will be useful to understand ligand-binding regulation of biological processes, such as the metabolism of nucleic acid. In particular, this model allows estimating the coverage fraction and the ligand mode characteristics from the force extension curves of a ligand-polymer system., Ministerio de Economía y Competitividad (MINECO), Universidad Complutense de Madrid/Banco Santander, Ministerio de Educación, Cultura y Deporte (MEC), Depto. de Estructura de la Materia, Física Térmica y Electrónica, Fac. de Ciencias Físicas, TRUE, pub

Systematic evolution of ligands by exponential enrichment (SELEX) is an established procedure for developing short single-stranded nucleic acid ligands called aptamers against a target of choice. This approach has also been used for developing aptamers specific to whole cells named Cell-SELEX. Aptamers selected by Cell-SELEX have the potential to act as cell specific therapeutics, cell specific markers or cell specific drug delivery and imaging agents. However, aptamer development is a laborious and time-consuming process which is often challenging due to the requirement of frequent optimization of various steps involved in Cell-SELEX procedures. This review provides an insight into various procedures for selection, aptamer enrichment, regeneration and aptamer-binding analysis, in addition to a very recent update on all aptamers selected by Cell-SELEX procedures.

In this work we highlight the potential of NEXAFS-near-edge X-ray absorption fine structure-analysis to perform refinements of hydrogen-bond structure in DNA. For this purpose we have carried out first-principle calculations of the N1s NEXAFS spectra of the guanine and cytosine nucleobases and their tautomers, nucleosides, and nucleotides in the gas phase, as well as for five crystal structures of guanine, cytosine, or guanosine. The spectra all clearly show imine (pi*(1)) and amine (pi*(2)) nitrogen absorption bands with a characteristic energy difference (Delta). Among all of the intramolecule covalent connections, the tautomerism of hydrogens makes the largest influence, around +/- 0.4-0.5 eV change of Delta, to the spectra due to a switch of single-double bonds. Deoxyribose and ribose sugars can cause at most 0.2 eV narrowing of Delta, while the phosphate groups have nearly negligible effects on the spectra. Two kinds of intermolecule interactions are analyzed, the hydrogen bonds and the stacking effect, by comparing "compressed" and "expanded" models. or by comparing models including or excluding the nearest stacking molecules. The shortening of hydrogen-bond length by 0.2-0.3 angstrom can result in the reduction of Delta by 0.2-0.8 eV. This is because the hydrogen bonds make the electrons more delocalized, and the amine and imine nitrogens become less distinguishable. Moreover, the hydrogen bond has a different ability to influence the spectra of different crystals, with guanine crystals as the largest (change by 0.8 eV) and the guanosine crystal as the smallest (change by 0.2 eV). The stacking has negligible effects on the spectra in all studied systems. A comparison of guanosine to guanine crystals shows that the sugars in the crystal could create "blocks" in the pi-and hydrogen bonds network of bases and thus makes the imine and amine nitrogens More distinguishable with a larger Delta. Our theoretical calculations offer a good match with experimental findin, QC 20101227

This work describes the synthesis and characterization of a new family of DNA amphiphiles containing modified nucleobases. The hydrophobicity was imparted by the introduction of a dodec-1-yne chain at the 5-position of the uracil base, which allowed precise and simple tuning of the hydrophobic properties through solid-phase DNA synthesis. The micelles formed from these modified DNA sequences were characterized by atomic force microscopy, dynamic light scattering, and polyacrylamide gel electrophoresis. These experiments revealed the role of the quantity and location of the hydrophobic units in determining the morphology and stability of the micelles. The effects of hybridization on the physical characteristics of the DNA micelles were also studied; these results showed potential for the sequence-specific noncovalent functionalization of the self-assembled aggregates.

The Neisseria gonorrhoeae type IV secretion system secretes chromosomal DNA that acts in natural transformation. To examine the mechanism of DNA processing for secretion, we made mutations in the putative relaxase gene traI and used nucleases to characterize the secreted DNA. The nuclease experiments demonstrated that the secreted DNA is single-stranded and blocked at the 5' end. Mutation of traI identified Tyr(93) as required for DNA secretion, while substitution of Tyr(201) resulted in intermediate levels of DNA secretion. TraI exhibits features of relaxases, but also has features that are absent in previously characterized relaxases, including an HD phosphohydrolase domain and an N-terminal hydrophobic region. The HD domain residue Asp(120) was required for wild-type levels of DNA secretion. Subcellular localization studies demonstrated that the TraI N-terminal region promotes membrane interaction. We propose that Tyr(93) initiates DNA processing and Tyr(201) is required for termination or acts in DNA binding. Disruption of an inverted-repeat sequence eliminated DNA secretion, suggesting that this sequence may serve as the origin of transfer for chromosomal DNA secretion. The TraI domain architecture, although not previously described, is present in 53 uncharacterized proteins, suggesting that the mechanism of TraI function is a widespread process for DNA donation.

Unlabeled helper oligonucleotides assisting a bead-based sandwich hybridization assay were tested for the optimal placement of the capture and detection probes. The target used was a full-length in vitro synthesized mRNA molecule. Helper probes complementary to regions adjacent to the binding site of the 5' end attached capture probe were found much more effective than helper probes targeting positions adjacent to the detection probe binding site. The difference is believed to be caused by a disruption of the RNA secondary structure in the area where the capture probe binds, thereby reducing structural interference from the bead. The use of additional helpers showed an additive effect. Using helpers, at both sides of the capture and detection probes showed a 15- to 40-fold increase in hybridization efficiency depending on the target, thereby increasing the sensitivity of the hybridization assays. Using an electrical chip linked to the detection probe for the detection of p-ominophenol, which is produced by alkaline phosphatase, a detection limit of 2 x 10(-13) M mRNA molecules was reached without the use of a nucleic acid amplification step., QC 20100525 QC 20111101

Mutation detection and single-nucleotide polymorphisin genotyping require screening of large samples of materials and therefore the importance of high-throughput DNA analysis techniques is significant. Pyrosequencing is a four-enzyme bioluminometric DNA sequencing technology based on the sequencing-by-synthesis principle. Currently, the technique is limited to simultaneous analysis of 96 or 384 samples. Earlier, attempts to increase the sample capacity were made using micromachined filter chamber arrays where parallel analyses of nanoliter samples could be monitored in real time. We have developed a strategy for specific immobilization of the light-producing enzyme luciferase to the DNA template within a reaction chamber. By this approach, luciferase is genetically fused to a DNA-binding protein (Klenow polymerase or Escherichia coli single-stranded DNA-binding (SSB) protein) and to a purification handle (Z(basic)). The proteins are produced in E. coli and purified using cation and anion exchange chromatography with removal of Z(basic). The produced proteins have been analyzed using an assay for complete primer extension of DNA templates immobilized on magnetic beads detected by pyrosequencing chemistry. Results from these experiments show that the proteins bind selectively to the immobilized DNA and that their enzymatic domains were active. Z(basic)-SSB-luciferase produced the highest signal in this assay and was further exploited as enzymatic reagent for DNA sequencing., QC 20100525 QC 20111026