Sharp melting in DNA-linked nanostructure systems: thermodynamic models of DNA-linked polymers

Sharp melting that has been found for DNA-linked nanostructure systems such as DNA-linked gold nanoparticles enhances the resolution of DNA sequence detection enough to distinguish between a perfect match and single base pair mismatches. One intriguing explanation of the sharp melting involves the cooperative dehybridization of DNA strands between the nanostructures. However, in the DNA-linked gold nanoparticle system, strong optical absorption by the gold nanoparticles hinders the direct observation of cooperativity. Here, with a combination of theory and experiment, we investigate a DNA-linked polymer system in which we can show that the optical profile of the system at 260 nm is directly related to the individual DNA dehybridization profile, providing a clear distinction from other possible mechanisms. We find that cooperativity plays a crucial role in determining both the value of the melting temperature and the shape of the melting profile well away from the melting temperature. Our analysis suggests that the dehybridization properties of DNA strands in confined or dense structures differ from DNA in solution.     

Surface and bulk dissolution properties, and selectivity of DNA-linked nanoparticle assemblies

Using a simple mean-field model, we analyze the surface and bulk dissolution properties of DNA-linked nanoparticle assemblies. We find that the dissolution temperature and the sharpness of the dissolution profiles increase with the grafting density of the single-stranded DNA "probes" on the surface of colloids. The surface grafting density is controlled by the linker occupation number, in analogy with quantum particles obeying fractional statistics. The dissolution temperature increases logarithmically with the salt concentration. This is in agreement with the experimental findings [R. Jin, G. Wu, Z. Li, C. A. Mirkin, and G. C. Schatz, J. Am. Chem. Soc. 125, 1643 (2003)]. By exploiting the unique phase behavior of DNA-coated colloids, it should be possible to detect multiple "targets" in a single experiment by essentially mapping the DNA base-pair sequence onto the phase behavior of DNA-linked nanoparticle solution.     

Ranking targets in structure-based virtual screening of three-dimensional protein libraries: methods and problems

Structure-based virtual screening is a promising tool to identify putative targets for a specific ligand. Instead of docking multiple ligands into a single protein cavity, a single ligand is docked in a collection of binding sites. In inverse screening, hits are in fact targets which have been prioritized within the pool of best ranked proteins. The target rate depends on specificity and promiscuity in protein-ligand interactions and, to a considerable extent, on the effectiveness of the scoring function, which still is the Achilles' heel of molecular docking. In the present retrospective study, virtual screening of the sc-PDB target library by GOLD docking was carried out for four compounds (biotin, 4-hydroxy-tamoxifen, 6-hydroxy-1,6-dihydropurine ribonucleoside, and methotrexate) of known sc-PDB targets and, several ranking protocols based on GOLD fitness score and topological molecular interaction fingerprint (IFP) comparison were evaluated. For the four investigated ligands, the fusion of GOLD fitness and two IFP scores allowed the recovery of most targets, including the rare proteins which are not readily suitable for statistical analysis, while significantly filtering out most false positive entries. The current survey suggests that selecting a small number of targets (<20) for experimental evaluation is achievable with a pure structure-based approach.     

Diversity of phage-displayed libraries of peptides during panning and amplification

The amplification of phage-displayed libraries is an essential step in the selection of ligands from these libraries. The amplification of libraries, however, decreases their diversity and limits the number of binding clones that a screen can identify. While this decrease might not be a problem for screens against targets with a single binding site (e.g., proteins), it can severely hinder the identification of useful ligands for targets with multiple binding sites (e.g., cells). This review aims to characterize the loss in the diversity of libraries during amplification. Analysis of the peptide sequences obtained in several hundred screens of peptide libraries shows explicitly that there is a significant decrease in library diversity that occurs during the amplification of phage in bacteria. This loss during amplification is not unique to specific libraries: it is observed in many of the phage display systems we have surveyed. The loss in library diversity originates from competition among phage clones in a common pool of bacteria. Based on growth data from the literature and models of phage growth, we show that this competition originates from growth rate differences of only a few percent for different phage clones. We summarize the findings using a simple two-dimensional "phage phase diagram", which describes how the collapse of libraries, due to panning and amplification, leads to the identification of only a subset of the available ligands. This review also highlights techniques that allow elimination of amplification-induced losses of diversity, and how these techniques can be used to improve phage-display selection and enable the identification of novel ligands.     

Bioactive peptides from libraries

New ligands for a variety of biological targets can be selected from biological or synthetic combinatorial peptide libraries. The use of different libraries to select novel peptides with potential therapeutic applications is reviewed. The possible combination of molecular diversity provided by combinatorial libraries and a rational approach derived from computational modeling is also considered. Advantages and disadvantages of different approaches are compared. Possible strategies to bypass loss of peptide bioactivity in the transition from ligand selection to in vivo use are discussed.     

Recognition of single-base mismatch DNA by Au nanoparticle-assisted electroelution

A simple, convenient and effective method based on the surface plasmon resonance (SPR) technique was introduced for recognition of single-base mismatch DNA (smDNA) by Au nanoparticle (AuNPs)-assisted electroelution. In this method, target DNA, including perfectly matched DNA and smDNA, hybridized with the DNA probes immobilized on Au film and AuNPs, then the Au film was negatively charged. Owing to the difference in stability between single-base mismatch and perfect match DNA, effective distinction between complementary DNA (cDNA) and smDNA was achieved in the presence of an electric field: double-stranded DNA (dsDNA) formed between smDNA targets and DNA probes was denatured by the repulsion force acting on the negatively-charged DNA-linked AuNPs, while the perfectly matched duplex was not influenced. However, if the AuNPs were absent, the effects of cDNA and smDNA were not distinguishable. The effects of electric field intensity and mismatch sites were also investigated. All of the operations were performed under mild conditions. The results showed that AuNP-assisted electroelution may be exploited for the construction of biosensors with high selectivity.     

Molecular dynamics studies of ion distributions for DNA duplexes and DNA clusters: salt effects and connection to DNA melting

We present extensive molecular dynamics simulations of the ion distributions for DNA duplexes and DNA clusters using the Amber force field with implicit water. The distribution of ions and the electrostatic energy of ions around an isolated DNA duplex and clusters of DNA duplexes in different salt (NaCl) concentrations over the range 0.2-1.0 mol/L are determined on the basis of the simulation results. Using the electrostatic energy profile, we determine a local net charge fraction phi, which is found to increase with increasing of salt concentration. For DNA clusters containing two DNA duplexes (DNA pair) or four DNA duplexes, phi increases as the distance between the duplexes decreases. Combining this result with experimental results for the dependence of the DNA melting temperature on bulk salt concentration, we conclude that for a pair of DNA duplexes the melting temperature increases by 5-10 K for interaxis separations of 25-40 A. For a cluster of four DNA duplexes, an even larger melting temperature increase should occur. We argue that this melting temperature increase in dense DNA clusters is responsible for the cooperative melting mechanism in DNA-linked nanoparticle aggregates and DNA-linked polymer aggregates.     

Turbidimetric detection of ATP using polymeric micelles and DNA aptamers

Two types of turbidimetric detection of adenosine 5'-triphosphate (ATP) by the naked eye were achieved through a combination of non-cross-linking aggregation of DNA-linked polymeric micelles and molecular recognition of ATP by a DNA aptamer.     

Electrochemiluminescence induced photoelectrochemistry for sensing of the DNA based on DNA-linked CdS NPs superstructure with intercalator molecules

A novel detection protocol of DNA was developed using electrochemiluminescence (ECL) induced photoelectrochemistry (PEC) synthesis based on DNA-linked CdS NPs superstructure with methylene blue as the intercalator molecule.     

What controls the melting properties of DNA-linked gold nanoparticle assemblies?

We report a series of experiments and a theoretical model designed to systematically define and evaluate the relative importance of nanoparticle, oligonucleotide, and environmental variables that contribute to the observed sharp melting transitions associated with DNA-linked nanoparticle structures. These variables include the size of the nanoparticles, the surface density of the oligonucleotides on the nanoparticles, the dielectric constant of the surrounding medium, target concentration, and the position of the nanoparticles with respect to one another within the aggregate. The experimental data may be understood in terms of a thermodynamic model that attributes the sharp melting to a cooperative mechanism that results from two key factors: the presence of multiple DNA linkers between each pair of nanoparticles and a decrease in the melting temperature as DNA strands melt due to a concomitant reduction in local salt concentration. The cooperative melting effect, originating from short-range duplex-to-duplex interactions, is independent of DNA base sequences studied and should be universal for any type of nanostructured probe that is heavily functionalized with oligonucleotides. Understanding the fundamental origins of the melting properties of DNA-linked nanoparticle aggregates (or monolayers) is of paramount importance because these properties directly impact one's ability to formulate high sensitivity and selectivity DNA detection systems and construct materials from these novel nanoparticle materials.     

Ideal‐filter capillary electrophoresis: A highly efficient partitioning method for selection of protein binders from oligonucleotide libraries

Selection of affinity ligands for protein targets from oligonucleotide libraries currently involves multiple rounds of alternating steps of partitioning of protein‐bound oligonucleotides (binders) from protein‐unbound oligonucleotides (nonbinders). We have recently introduced ideal‐filter capillary electrophoresis (IFCE) for binder selection in a single step of partitioning. In IFCE, protein‐binder complexes and nonbinders move inside the capillary in the opposite directions, and the efficiency of their partitioning reaches 10⁹, i.e., only one of a billion molecules of nonbinders leaks through IFCE while all binders pass through. The condition of IFCE can be satisfied when the magnitude of the mobility of EOF is smaller than that of the protein‐binder complexes and larger than that of nonbinders. The efficiency of partitioning in IFCE is 10 million times higher than those of solid‐phase‐based methods of partitioning typically used in selection of affinity ligands for protein targets from oligonucleotide libraries. Here, we provide additional details on our justification for IFCE development. We elaborate on electrophoretic aspects of the method and define the theoretical range of EOF mobilities that support IFCE. Based on these theoretical results, we identify an experimental range of background electrolyte's ionic strength that supports IFCE. We also extend our interpretation of the results and discuss in‐depth IFCE's prospective in practical applications and fundamental studies.     

Detection of small-molecule enzyme inhibitors with peptides isolated from phage-displayed combinatorial peptide libraries

BACKGROUND: The rapidly expanding list of pharmacologically important targets has highlighted the need for ways to discover new inhibitors that are independent of functional assays. We have utilized peptides to detect inhibitors of protein function. We hypothesized that most peptide ligands identified by phage display would bind to regions of biological interaction in target proteins and that these peptides could be used as sensitive probes for detecting low molecular weight inhibitors that bind to these sites. RESULTS: We selected a broad range of enzymes as targets for phage display and isolated a series of peptides that bound specifically to each target. Peptide ligands for each target contained similar amino acid sequences and competition analysis indicated that they bound one or two sites per target. Of 17 peptides tested, 13 were found to be specific inhibitors of enzyme function. Finally, we used two peptides specific for Haemophilus influenzae tyrosyl-tRNA synthetase to show that a simple binding assay can be used to detect small-molecule inhibitors with potencies in the micromolar to nanomolar range. CONCLUSIONS: Peptidic surrogate ligands identified using phage display are preferentially targeted to a limited number of sites that inhibit enzyme function. These peptides can be utilized in a binding assay as a rapid and sensitive method to detect small-molecule inhibitors of target protein function. The binding assay can be used with a variety of detection systems and is readily adaptable to automation, making this platform ideal for high-throughput screening of compound libraries for drug discovery.     

Encoded peptide libraries and the discovery of new cell binding ligands

Cells over-expressing integrins or CCR6 were incubated on a DNA microarray, pre-hybridized with a 10,000 member PNA-encoded peptide library allowing novel cell specific ligands for integrins and CCR6 to be identified.     

20 years of DNA-encoded chemical libraries

The identification of specific binding molecules is a central problem in chemistry, biology and medicine. Therefore, technologies, which facilitate ligand discovery, may substantially contribute to a better understanding of biological processes and to drug discovery. DNA-encoded chemical libraries represent a new inexpensive tool for the fast and efficient identification of ligands to target proteins of choice. Such libraries consist of collections of organic molecules, covalently linked to a unique DNA tag serving as an amplifiable identification bar code. DNA-encoding enables the in vitro selection of ligands by affinity capture at sub-picomolar concentrations on virtually any target protein of interest, in analogy to established selection methodologies like antibody phage display. Multiple strategies have been investigated by several academic and industrial laboratories for the construction of DNA-encoded chemical libraries comprising up to millions of DNA-encoded compounds. The implementation of next generation high-throughput sequencing enabled the rapid identification of binding molecules from DNA-encoded libraries of unprecedented size. This article reviews the development of DNA-encoded library technology and its evolution into a novel drug discovery tool, commenting on challenges, perspectives and opportunities for the different experimental approaches.     

Synthesis and functional evaluation of DNA-assembled polyamidoamine dendrimer clusters for cancer cell-specific targeting

We sought to produce dendrimers conjugated to different biofunctional moieties (fluorescein [FITC] and folic acid [FA]), and then link them together using complementary DNA oligonucleotides to produce clustered molecules that target cancer cells that overexpress the high-affinity folate receptor. Amine-terminated, generation 5 polyamidoamine (G5 PAMAM) dendrimers are first partially acetylated and then conjugated with FITC or FA, followed by the covalent attachment of complementary, 5'-phosphate-modified 34-base-long oligonucleotides. Hybridization of these oligonucleotide conjugates led to the self-assembly of the FITC- and FA-conjugated dendrimers. In vitro studies of the DNA-linked dendrimer clusters indicated specific binding to KB cells expressing the folate receptor. Confocal microscopy also showed the internalization of the dendrimer cluster. These results demonstrate the ability to design and produce supramolecular arrays of dendrimers using oligonucleotide bridges. This will also allow for further development of DNA-linked dendrimer clusters as imaging agents and therapeutics.     

Subtractive Genomics Approach to Identify Putative Drug Targets and Identification of Drug-like Molecules for Beta Subunit of DNA Polymerase III in Streptococcus Species

The prolonged use of the antibiotics over the years has transformed many organisms resistant to multiple drugs. This has made the field of drug discovery of vital importance in curing various infections and diseases. The drugs act by binding to a specific target protein of prime importance for the cell??s survival. Streptococcus agalactiae, Streptococcus pneumoniae, and Streptococcus pyogenes are the few gram positive organisms that have developed resistance to drugs. It causes pneumonia, meningitis, pharyngitis, otitis media, sinusitis, bacteremia, pericarditis, and arthritis infections. The present study was carried out to identify potential drug targets and inhibitors for beta subunit of DNA polymerase III in these three Streptococcus species that might facilitate the discovery of novel drugs in near future. Various steps were adopted to find out novel drug targets. And finally 3D structure of DNA polymerase III subunit beta was modeled. The ligand library was generated from various databases to find the most suitable ligands. All the ligands were docked using Molegro Virtual Docker and the lead molecules were investigated for ADME and toxicity.     

The design and application of target-focused compound libraries

Target-focused compound libraries are collections of compounds which are designed to interact with an individual protein target or, frequently, a family of related targets (such as kinases, voltage-gated ion channels, serine/cysteine proteases). They are used for screening against therapeutic targets in order to find hit compounds that might be further developed into drugs. The design of such libraries generally utilizes structural information about the target or family of interest. In the absence of such structural information, a chemogenomic model that incorporates sequence and mutagenesis data to predict the properties of the binding site can be employed. A third option, usually pursued when no structural data are available, utilizes knowledge of the ligands of the target from which focused libraries can be developed via scaffold hopping. Consequently, the methods used for the design of target-focused libraries vary according to the quantity and quality of structural or ligand data that is available for each target family. This article describes examples of each of these design approaches and illustrates them with case studies, which highlight some of the issues and successes observed when screening target-focused libraries.     

Chemical libraries towards protein kinase inhibitors

Over 500 human protein kinases identified to date are susceptible to play crucial roles in the regulation of many signal transduction pathways, making them significant drug discovery targets. However, their active sites share a high level of similarity, which constitutes a major challenge in the finding of selective and safe inhibitors. In order to meet this challenge, whether via traditional or alternative approaches, the use of chemical libraries to find either unknown natural ligands or specific inhibitors of particular kinases is more important than ever. This review briefly summarizes the recent literature on such libraries of peptides, natural product analogues, and small molecules. Significant chemical scaffolds, some synthetic routes particularly on solid-phase support, and computational tools employed for the efficient design of both selective and bioavailable inhibitors are highlighted.     

Phase transitions in DNA-linked nanoparticle assemblies: a decorated-lattice model

We use decorated-lattice models to explore the phase behavior of two types of DNA-linked colloidal mixtures: systems with identical nanoparticles functionalized with two different DNA strands (mixture Aab) and mixtures involving two types of particles each one functionalized with a different DNA strand (mixture Aa-Ab). The model allows us to derive the properties of the mixtures from the well-known behavior of underlying spin-n Ising models with temperature and activity dependent effective interactions. The predicted evolution of the dissolution profiles for the colloidal assemblies as a function of temperature and number of single DNA strands on a nanoparticle M is in qualitative agreement with that observed in real systems. According to our model, the temperature at which the assemblies dissolve can be expected to increase with increasing M only for concentrations of colloids below a certain threshold. For more concentrated solutions, the dissolution temperature is a decreasing function of M. Linker-mediated interactions between Aa and Ab particles in the Aa-Ab mixture render the phase separation involving disordered aggregates metastable with respect to a phase transition between a solvent-rich and an ordered phase. The stability of the DNA-linked assembly is enhanced by the ordering of the colloidal network and the ordered aggregates dissolve at higher temperatures. Our results may explain the contrasting evolution of the dissolution temperatures with increasing probe size in Aab and Aa-Ab mixtures as observed experimentally.     

Identification of affinity ligands from peptide libraries and their applications

Summary Combinatorial peptide libraries may be screened to identify novel ligands for use in the affinity purification of proteins. Strategies for the screening of libraries are summarized and characterization of the interaction of a particular target protein (fibrinogen) with a ligand (FLLVPL) is described. Factors important for implementing an affinity resin in a production environment are highlighted. Presented at: Affinity Chromatography Conference, Cambridge, UK, July 1–3, 1997.