DNA bimodified gold nanoparticles

We report a general approach to bimodify gold nanoparticles (GNPs) with two different DNA strands via DNA template reaction. Two thioctic acid modified DNA strands, one at 5' end and one at 3' end, were attached to GNPs through bivalent thiol-gold bond. By sequence design, assemblies of 5 nm GNPs chains, 10 nm GNPs chains and alternative arrangement of 5 and 10 nm GNPs could be achieved. Gel electrophoresis, transmission electron microscope (TEM), UV-vis spectra were used to characterize the assemblies. It is believed that this new kind of bimodified GNPs with two different DNA strands at different ends would enrich the toolbox of DNA-GNP conjugates and provide diverse selectivity for further assembly.     

Enzymatic ligation creates discrete multinanoparticle building blocks for self-assembly

Enzymatic ligation of discrete nanoparticle-DNA conjugates creates nanoparticle dimer and trimer structures in which the nanoparticles are linked by single-stranded DNA, rather than by double-stranded DNA as in previous experiments. Ligation was verified by agarose gel and small-angle X-ray scattering. This capability was utilized in two ways: first, to create a new class of multiparticle building blocks for nanoscale self-assembly and, second, to develop a system that can amplify a population of discrete nanoparticle assemblies.     

Unlimited increase in the resolution of DNA ladders

Fractionation of DNA ladders by gel electrophoresis is limited by the progressive compressing of the long DNA end of a ladder. Improvement in the resolution of this DNA is achieved by use of the following two-step electrophoresis. Initially, the DNA ladder is fractionated by conventional constant field agarose gel electrophoresis. Subsequently, gel electrophoresis is performed in the reverse direction by pulsing the electrical field (PFGE). A newly developed type of pulsing is used, which causes inversion of a double-stranded DNA ladder: the distance migrated increases as the length of the DNA molecule increases. Thus, the resolution of DNA bands continues to increase during the PFGE. These two stages of electrophoresis are serially repeated. Eventually, both the short and the long DNA ends of the ladder migrate out of the gel while a selected region of the ladder undergoes progressive increase in resolution during back-and-forth migration. Improved resolution of DNA bands is achieved, without a known limit.     

DNA dissociation and degradation at gold nanoparticle surfaces

The instability of DNA-functionalized gold nanoparticles (Au-DNA conjugates) upon exposure to high temperatures is characterized using fluorescence spectroscopy, gel electrophoresis and ion-exchange chromatography. Above 70 °C, aqueous Au-DNA conjugates decompose within hours due to both desorption of thiol-terminated DNA from the gold nanoparticle surface and chemical degradation of DNA in the presence of colloidal gold. Although the chemical mechanism for DNA degradation was not identified in this study, the gold surface participates directly in the cleavage reaction. These results have important implications for the use of Au-DNA conjugates in biotechnological and clinical applications that require high temperatures, such as polymerase chain reaction (PCR).     

Sequence‐Specific DNA Alkylation by Tandem Py–Im Polyamide Conjugates

Tandem N‐methylpyrrole? N‐methylimidazole (Py? Im) polyamides with good sequence‐specific DNA‐alkylating activities have been designed and synthesized. Three alkylating tandem Py? Im polyamides with different linkers, which each contained the same moiety for the recognition of a 10 bp DNA sequence, were evaluated for their reactivity and selectivity by DNA alkylation, using high‐resolution denaturing gel electrophoresis. All three conjugates displayed high reactivities for the target sequence. In particular, polyamide 1 , which contained a β‐alanine linker, displayed the most‐selective sequence‐specific alkylation towards the target 10 bp DNA sequence. The tandem Py? Im polyamide conjugates displayed greater sequence‐specific DNA alkylation than conventional hairpin Py? Im polyamide conjugates ( 4 and 5 ). For further research, the design of tandem Py? Im polyamide conjugates could play an important role in targeting specific gene sequences.     

Rapid synthesis of stable and functional conjugates of DNA/gold nanoparticles mediated by Tween 80

Gold nanoparticles conjugated with DNA represent an attractive and alternative platform for broad applications in biosensors, medical diagnostic, and biological analysis. However, current methods to conjugate DNA to gold nanoparticles are time-consuming. In this study, we report a novel approach to rapidly conjugate DNA to gold nanoparticles (AuNPs) to form functional DNA/AuNPs in 2-3 h using Tween 80 as protective agent. With a fluorescence-based technique, we determine that the DNA density on the surface of AuNPs achieves about ～60 strands per particles, which is comparable to the loading density in the current methods. Moreover, the DNA/AuNPs synthesized by our approach exhibit an excellent stability as a function of temperature, pH, and freeze-thaw cycle, and the functionality of DNA/AuNPs conjugates is also verified. The work presented here has important implications to develop the fast and reproducible synthesis of stable DNA-functionalized gold nanoparticles.     

Monte Carlo simulation of DNA electrophoresis

This paper describes an attempt to study the electrophoresis mobility of a DNA molecule in a gel by means of a Monte Carlo simulation. We find that the electrophoresis mobility mu can be well described by the empirical equation mu v kappa 1/N + kappa 2E2 with N being the number of monomers of the model chain and E being the applied field. For small E the data can merge into the linear response result mu = kappa 1/N. The paper also discusses necessary extensions of the present approach.     

Gold‐Aryl nanoparticles coated with polyelectrolytes for adsorption and protection of DNA against nuclease degradation

Binding DNA on nanoparticles was pursued to form nanoplatform for formation of non‐viral gene system. Carboxyl derivatized gold‐aryl nanoparticles can bind with biodegradable cationic polyelectrolytes such as polydiallyldimethylammonium chloride (PDADMAC). In our study, we used gold‐aryl nanoparticles (AuNPs) treated with PDADMAC to form conjugates with non‐thiol or non‐disulfide modified oligonucleotide DNA. Both AuNPs‐DNA and PDADMAC‐AuNPs‐DNA biomaterials were characterized using UV–Vis, dynamic light scattering (DLS), atomic force microscopy (AFM), transmission electron microscopy (TEM) and agarose gel electrophoresis. UV–Vis showed a red shift in the plasmon peak as compared with unconjugated AuNPs. DLS measurements also showed difference in the size of AuNPs‐DNA and PDADMAC‐AuNPs‐DNA. AFM and TEM results showed proper conjugation of DNA with AuNPs. Gel electrophoresis proved the presence of interaction between PDADMAC‐AuNPs and negatively charged DNA. The binding of DNA in the described bioconjugate enhanced its protection against nuclease degradation and prolonged its presence in the digestive environment of DNase‐I. From the results we expect that these biomaterials can be used in nanomedicine with emphasis on non‐viral gene system.     

Toward reliable gold nanoparticle patterning on self-assembled DNA nanoscaffold

Assembly of gold nanoparticles (AuNP) into designer architectures with reliablity is important for nanophotonics and nanoelectronics applications. Toward this goal we present a new strategy to prepare AuNPs monofunctionalized with lipoic acid modified DNA oligos. This strategy offers increased bonding strength between DNA oligos and AuNP surface. These conjugates are further selectively mixed with other DNA strands and assembled into fixed sized DNA nanostructures carring a discrete number of AuNPs at desired positions. Atomic force microscopy imaging reveals a dramatically improved yield of the AuNPs on DNA tile structure compared to the ensembles using monothiolate AuNP-DNA conjugates.     

Four-way-branched DNA-porphyrin conjugates for construction of four double-helix-DNA assembled structures

[reaction: see text] DNA-porphyrin conjugates having four DNA strands were designed and synthesized. Four double helices were assembled using two DNA-porphyrin conjugates and their complementary strands, and the formation of the four double-helix assembled structures with the two DNA-porphyrin units was examined by gel electrophoresis and spectroscopic analysis. The interaction between two porphyrin chromophores in the complex was investigated by measurement of fluorescence lifetimes, and the singlet energy transfer between the two different phorphyrin units (Zn-porphyrin and H2-porphyrin) was observed. These results indicate that multiple and different porphyrin chromophores can be integrated into the DNA structures by programming the sequences of the DNA strands.     

Electrochemical genosensor based on three-dimensional DNA polymer brushes monolayers

A new approach to the three dimensional integration of short DNA strands at gold electrode surfaces via the in situ formation of DNA-acrylamide conjugates is presented. Surface initiated atom transfer radical polymerisation was employed to grow acrylamide brushes co-polymerised in the presence of acrylamide modified DNA probes. This strategy was demonstrated for the realisation of biofunctionalised thin polymer films capable of binding its complementary 105-base DNA amplicon. The synthesised brushes were characterised using atomic force microscopy, attenuated total reflectance spectroscopy and electrochemical impedance spectroscopy. Once characterised, the polymer brushes were applied to the quantitative detection of target DNA using an enzyme labelled reporter DNA probe in a sandwich-type format.     

End-labeled free-solution electrophoresis of DNA

DNA is a free-draining polymer. This subtle but "unfortunate" property of highly charged polyelectrolytes makes it impossible to separate nucleic acids by free-flow electrophoresis. This is why one must typically use a sieving matrix, such as a gel or an entangled polymer solution, in order to obtain some electrophoretic size separation. An alternative approach consists of breaking the charge to friction balance of free-draining DNA molecules. This can be achieved by labeling the DNA with a large, uncharged molecule (essentially a hydrodynamic parachute, which we also call a drag-tag) prior to electrophoresis; the resulting methodology is called end-labeled free-solution electrophoresis (ELFSE). In this article, we review the development of ELFSE over the last decade. In particular, we examine the theoretical concepts used to predict the ultimate performance of ELFSE for single-stranded (ssDNA) sequencing, the experimental results showing that ELFSE can indeed overcome the free-draining issue raised above, and the technological advances that are needed to speed the development of competitive ELFSE-based sequencing and separation technologies. Finally, we also review the reverse process, called free-solution conjugate electrophoresis (FSCE), wherein uncharged polymers of different sizes can be analyzed using a short DNA molecule as an electrophoretic engine.     

DNA-gold nanorod conjugates for remote control of localized gene expression by near infrared irradiation

Gold nanorods were attached to the gene of enhanced green fluorescence protein (EGFP) for the remote control of gene expression in living cells. The UV-vis spectroscopy, electrophoresis, and transmission electron microscopy (TEM) were used to study the optical and structural properties of the EGFP DNA and gold nanorod (EGFP-GNR) conjugates before and after femto-second near-infrared (NIR) laser irradiation. Upon NIR irradiation, the gold nanorods of EGFP-GNR conjugates underwent shape transformation that resulted in the release of EGFP DNA. When EGFP-GNR conjugates were delivered to cultured HeLa cells, induced GFP expression was specifically observed in cells that were locally exposed to NIR irradiation. Our results demonstrate the feasibility of using gold nanorods and NIR irradiation as means of remote control of gene expression in specific cells. This approach has potential applications in biological and medical studies.     

Cross-linking of a DNA conjugate tethering a cis-bifunctional platinated complex to a target DNA duplex

Tethering an ethylene diamine linker to the 5' terminus of an oligothymidine sequence provides a ligand for complexation with K2PtCl4. Post-synthetic reaction of the platinum reagent with the diamino oligothymidine generates the diamino dichloro platinum-DNA conjugate that can be used for DNA duplex targeting by oligodeoxyncleotide-mediated triplex formation. Cross-linking between the third strand and the duplex occurs exclusively with the duplex target strand directly involved in triplex formation. No examples of cross-linking to the complementary target strand or cases of cross-linking to both target strands are observed. Most efficient cross-linking occurs when the dinucleotide d(GpG) is present in the target strand and no cross-linking occurs with the corresponding 7-deazaG dinucleotide target. Cross-linking is also observed when dC or dA residues are present in the target strand, or even with a single dG residue, but it is not observed in any cases to dT residues. Triplex formation provides the ability to target specific sequences of double-stranded DNA; conjugates of the type described here offer the potential of delivering a platinum complex to a specific DNA site.     

Molecular design of a pyrrole-imidazole hairpin polyamides for effective DNA alkylation

New hairpin polyamide-CPI (CPI = cyclopropylpyrroloindole) conjugates, compounds 12-14, were synthesized and their DNA-alkylating activities compared with the previously prepared hairpin polyamide, compound 1, by high-resolution denaturing gel electrophoresis with 450 base pair (bp) DNA fragments and by HPLC product analysis of the synthetic decanucleotide. In accord with our previous results, alkylation by compound 1 occurred predominantly at the G moiety of the sequence 5'-AGTCAG-3' (site 3). However, compound 12, in which the structure of the alkylating moiety of compound 1 is replaced with segment A of duocarmycin A DU-86 (CPI), did not show any DNA alkylating activity. In clear contrast, the hairpin CPI conjugate 13, which differs from compound 1 in that it lacks one Py unit and possesses a vinyl linker, alkylated the A of 5'-AGTCAG-3' (site 3) efficiently at nanomolar concentrations. Alkylation by compound 14, which has a vinyl linker, occurred at the A of 5'-AGTCCA-3' (site 6) and at several minor alkylation sites, including mismatch alkylation at A of 5'-TCACAA-3' (site 2). The significantly different reactivity of the alkylating hairpin polyamides 1, 12, 13, and 14 was further confirmed by HPLC product analysis by using a synthetic decanucleotide. The results suggest that hairpin polyamide--CPI conjugate 13 alkylates effectively according to Dervan's pairing rule, and with a new mode of recognition in which the Im-vinyl linker (L) pair targets G-C base pairs. These results demonstrate that incorporation of the vinyl-linker pairing with Im dramatically improves the reactivity of hairpin polyamide--CPI conjugates.     

DNA constraints allow rational control of macromolecular conformation

We report that double-helical DNA constraints can be used to control the conformation of another molecule, RNA. When a covalently attached DNA constraint is structurally incompatible with the native Mg2+-dependent RNA conformation, RNA folding is disrupted, as revealed by nondenaturing gel electrophoresis and independently by chemical probing. Our approach is distinct from other efforts in DNA nanotechnology, which have prepared DNA objects by self-assembly, built static DNA lattices for assembly of other objects, and created nanomachines made solely of DNA. In contrast, our dynamic use of DNA to control the conformations of other macromolecules should have wide impact in nanotechnology applications ranging from materials science to biology.     

Hetero-assembly of gold nanoparticles on a DNA origami template

Hetero-assembling of spherical building blocks with well-defined spatial distribution holds great significance in developing chiral nanostructures. Herein, a strategy for hetero-assembling of gold nanoparticles(Au NPs) was demonstrated using rigid bifacial DNA origami as templates. By tuning the sizes and the fixed location of Au NPs on DNA origami, right-handed and left-handed Au NPs nanostructures were respectively constructed. Gel electrophoresis indicated the formation of the DNA origami-Au NPs complex and transmission electron microscopy(TEM) visually displayed the arrangement of Au NPs in these two chiral structures. The spatial configuration and 3D geometry of Au NPs were further illustrated by the stereographic TEM with tilting angles from ?30° to 30°. This strategy provides a universal approach to construct the asymmetrical 3D geometries, which may have potential applications in biomimicking and nanophotonics.     

Gel electrophoretic analysis of DNA branched junctions

Gel electrophoresis has provided much of the detailed information we have about the properties of DNA junctions, stable branched molecules formed from oligonucleotide or polynucleotide strands. Here we review these applications, and present the results of an electrophoretic investigation of conformationally restricted junctions formed by covalently connecting two different pairs of strands in a junction with four arms. Native gel electrophoresis is employed to establish the formation and stoichiometry of the multistrand complexes. Ferguson analysis of native gel mobility shows that junctions have retardation coefficients that are distinct from those of linear DNA duplexes. Denaturing gel electrophoresis is the primary tool for characterizing junctions that have been covalently linked together to form both linear and macrocyclic oligomers of junctions (oligojunctions). Radioactively labelled strands enable one to monitor the progress of the ligation reaction: both linear and closed cyclic molecules result, and these can be distinguished by applying Ferguson analysis to denaturing gels. Combinations of exonuclease III, restriction enzymes and sequencing reactions have been applied to oligojunction molecules, and the results are all analyzed on denaturing gels. Junctions containing intramolecular "tethers" that restrict the conformation freedom of the complex comprise a new system for analyzing the conformations of branched molecules. In these tethered junctions, the ability of arms to move relative to each other is restricted substantially by covalently connecting pairs of arms in the original complex with short, flexible loops. The two tethers used here constrain the helical domains of the structure to be roughly parallel or anti-parallel. In this article, we use Ferguson analysis to compare two tethered junctions with an untethered junction. At high gel concentrations, the mobility of the untethered complex is found to be closer to that of the molecule tethered anti-parallel than to the one tethered parallel. Curvature in the Ferguson plots for all three of these junctions is detected over a range of compositions. At low gel concentrations, differences in electrophoretic mobility persist, suggesting that the untethered junction differs in charge as well as conformational freedom from the tethered analogs. We expect that studies of this kind will be able to define the conformational repertoire of junctions of different kinds, and to explore the effects of electrophoresis on these states.     

DNA alkylation by pyrrole-imidazole seco-CBI conjugates with an indole linker: sequence-specific DNA alkylation with 10-base-pair recognition through heterodimer formation

The sequence-specific DNA alkylation by conjugates 4 and 5, which consist of N-methylpyrrole (Py)-N-methylimidazole (Im) polyamides and 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI) linked with an indole linker, was investigated in the absence or presence of partner Py-Im polyamide 6. High-resolution denaturing polyacrylamide gel electrophoresis revealed that conjugate 4 alkylates DNA at the sequences 5'-(A/T)GCCTA-3' through hairpin formation, and alkylates 5'-GGAAAGAAAA-3' through an extended binding mode. However, in the presence of partner Py-Im polyamide 6, conjugate 4 alkylates DNA at a completely different sequence, 5'-AGGTTGTCCA-3'. Alkylation of 4 in the presence of 6 was effectively inhibited by a competitor 7. Surface plasmon resonance (SPR) results indicated that conjugate 4 does not bind to 5'-AGGTTGTCCA-3', whereas 6 binds tightly to this sequence. The results suggest that alkylation proceeds through heterodimer formation, indicating that this is a general way to expand the recognition sequence for DNA alkylation by Py-Im seco-CBI conjugates.     

Highly efficient sequence-specific DNA interstrand cross-linking by pyrrole/imidazole CPI conjugates

We have developed a novel type of DNA interstrand cross-linking agent by synthesizing dimers of a pyrrole (Py)/imidazole (Im)-diamide-CPI conjugate, ImPyLDu86 (1), connected using seven different linkers. The tetramethylene linker compound, 7b, efficiently produces DNA interstrand cross-links at the nine-base-pair sequence, 5'-PyGGC(T/A)GCCPu-3', only in the presence of a partner triamide, ImImPy. For efficient cross-linking by 7b with ImImPy, one A.T base pair between two recognition sites was required to accommodate the linker region. Elimination of the A.T base pair and insertion of an additional A.T base pair and substitution with a G.C base pair significantly reduced the degree of cross-linking. The sequence specificity of the interstrand cross-linking by 7b was also examined in the presence of various triamides. The presence of ImImIm slightly reduced the formation of a cross-linked product compared to ImImPy. The mismatch partners, ImPyPy and PyImPy, did not produce an interstrand cross-link product with 7b, whereas ImPyPy and PyImPy induced efficient alkylation at their matching site with 7b. The interstrand cross-linking abilities of 7b were further examined using denaturing polyacrylamide gel electrophoresis with 5'-Texas Red-labeled 400- and 67-bp DNA fragments. The sequencing gel analysis of the 400-bp DNA fragment with ImImPy demonstrated that 7b alkylates several sites on the top and bottom strands, including one interstrand cross-linking match site, 5'-PyGGC(T/A)GCCPu-3'. To obtain direct evidence of interstrand cross-linkages on longer DNA fragments, a simple method using biotin-labeled complementary strands was developed, which produced a band corresponding to the interstrand cross-linked site on both top and bottom strands. Densitometric analysis indicated that the contribution of the interstrand cross-link in the observed alkylation bands was approximately 40%. This compound efficiently cross-linked both strands at the target sequence. The present system consisted of a 1:2 complex of the alkylating agent and its partner ImImPy and caused an interstrand cross-linking in a sequence-specific fashion according to the base-pair recognition rule of Py-Im polyamides.