We report a universal strategy for functionalizing near-infrared light-responsive nanocarriers with both a peptide “cargo” and an orthogonal cell-penetrating peptide. Modularity of both the cargo and the internalization peptide attachment is an important feature of these materials relying on the robust affinity of polyhistidine tags to nitrilotriacetic acid in the presence of nickel as well as the affinity of biotin labeled peptides to streptavidin. Attachment to the gold surface uses thiol-labeled scaffolds terminated with the affinity partner. These materials allow for unprecedented spatiotemporal control over the release of the toxic α-helical amphipathic peptide (KLAKLAK)2 which disrupts mitochondrial membranes and initiates apoptotic cell death. Laser treatment at benign near-infrared wavelengths releases peptide from the gold surface as well as breaches the endosome barrier for cytosolic activity (with 105-fold improved response to peptide activity over the free peptide) and can be monitored in real time.

Caulobacter crescentus relies on DNA methylation by the cell cycle-regulated methyltransferase (CcrM) in addition to key transcription factors to control the cell cycle and direct cellular differentiation. CcrM is shown here to efficiently methylate its cognate recognition site 5′-GANTC-3′ in single-stranded and hemimethylated double-stranded DNA. We report the Km, kcat, kmethylation, and Kd for single-stranded and hemimethylated substrates, revealing discrimination of 107-fold for noncognate sequences. The enzyme also shows a similar discrimination against single-stranded RNA. Two independent assays clearly show that CcrM is highly processive with single-stranded and hemimethylated DNA. Collectively, the data provide evidence that CcrM and other DNA-modifying enzymes may use a new mechanism to recognize DNA in a key epigenetic process.

Water plays important but poorly understood roles in the functions of most biomolecules. We are interested in understanding how proteins use diverse search mechanisms to locate specific sites on DNA; here we present a study of the role of closely associated waters in diverse translocation mechanisms. The bacterial DNA adenine methyltransferase, Dam, moves across large segments of DNA using an intersegmental hopping mechanism, relying in part on movement through bulk water. In contrast, other proteins, such as the bacterial restriction endonuclease EcoRI, rely on a sliding mechanism, requiring the protein to stay closely associated with DNA. Here we probed how these two mechanistically distinct proteins respond to well-characterized osmolytes, dimethyl sulfoxide (DMSO), and glycerol. The ability of Dam to move over large segments of DNA is not impacted by either osmolyte, consistent with its minimal reliance on a sliding mechanism. In contrast, EcoRI endonuclease translocation is significantly enhanced by DMSO and inhibited by glycerol, providing further corroboration that these proteins rely on distinct translocation mechanisms. The well-established similar effects of these osmolytes on bulk water, and their differential effects on macromolecule-associated waters, support our results and provide further evidence of the importance of water in interactions between macromolecules and their ligands.

We demonstrate modulation of nitric oxide release in solution and in human prostate cancer cells from a thiol functionalized cupferron (TCF) absorbed on hollow gold nanoshells (HGNs) using near-infrared (NIR) light. NO release from the TCF–HGN conjugates occurs through localized surface heating due to NIR excitation of the surface plasmon. Specific HGN targeting is achieved through cell surface directed peptides, and excitation with tissue penetrating NIR light provides unprecedented spatio-temporal control of NO delivery to biological targets.

While a host of methods exist to deliver genetic materials or small molecules to cells, very few are available for protein delivery to the cytosol. We describe a modular, light-activated nanocarrier that transports proteins into cells by receptor-mediated endocytosis and delivers the cargo to the cytosol by light triggered endosomal escape. The platform is based on hollow gold nanoshells (HGN) with polyhistidine tagged proteins attached through an avidity-enhanced, nickel chelation linking layer; here, we used green fluorescent protein (GFP) as a model deliverable cargo. Endosomal uptake of the GFP loaded nanocarrier was mediated by a C-end Rule (CendR) internalizing peptide fused to the GFP. Focused femtosecond pulsed-laser excitation triggered protein release from the nanocarrier and endosome disruption, and the released protein was capable of targeting the nucleoli, a model intracellular organelle. We further demonstrate the generality of the approach by loading and releasing Sox2 and p53. This method for targeting of individual cells, with resolution similar to microinjection, provides spatial and temporal control over protein delivery.

The genomes of all cells are intimately associated with proteins, which are important for compaction, scaffolding, and gene regulation. Here we show that pre-existing protein–DNA complexes (roadblocks) diminish and―interestingly―enhance the ability of particular sequence-specific proteins to move along DNA to locate their binding sites. We challenge the bacterial DNA adenine methyltransferase (Dam, recognizes 5′-GATC-3′) with tightly bound EcoRV ENase–DNA complexes, which bend DNA. A single EcoRV roadblock does not alter processive (multiple modifications) methylation by Dam. This result disfavors a reliance on heavily touted mechanisms involving sliding or short hops for Dam. Specific conformations of two EcoRV roadblocks cause an increase in processivity. The histone-like leucine-responsive regulatory protein (Lrp) binds DNA nonspecifically as an octamer, and also increases Dam’s processivity. These results can be explained by our prior demonstration that Dam moves over large regions (>300 bp) within a single DNA molecule using an “intersegmental hopping” mechanism. This mechanism involves the protein hopping between looped DNA segments. Both roadblock systems can cause the DNA to loop and therefore facilitate intersegmental hopping. For Lrp, this only occurs when the Dam sites are separated (by >134bp) such that they can be looped around the protein. Intersegmental hopping may well be a general mechanism for proteins that navigate long distances along compacted DNA. Unlike Dam, EcoRI ENase (recognizes 5′-GAATTC-3′) relies extensively on a sliding mechanism, and as expected, Lrp decreases its processivity. Our systematic use of protein roadblocks provides a powerful strategy to differentiate between site location mechanisms.

We have combined a versatile and powerful route to deliver nucleic acids with peptide-based cell-specific targeting. siRNA targeting the polo-like kinase gene is in clinical trials for cancer treatment, and here we deliver this RNA selectively to cancer cells displaying the neuropilin-1 epitope using gold nanoshells. Release of the siRNA from the nanoparticles results from irradiation with a pulsed near-infrared laser, which also provides efficient endosomal escape within the cell. As a result, our approach requires 10-fold less material than standard nucleic acid transduction materials and is significantly more efficient than other particle-based methods. We also describe a particle–nucleic acid design that does not rely on modified RNA, thereby making the preparation of these materials more efficient and much less expensive. These improvements, when combined with control over when and where the siRNA is released, could provide the basis for diverse cell biological studies.

The passive search by proteins for particular DNA sequences involving nonspecific DNA is essential for gene regulation, DNA repair, phage defense, and diverse epigenetic processes. Distinct mechanisms contribute to these searches, and it remains unresolved as to which mechanism or blend of mechanisms best suits a particular protein and, more importantly, its biological role. To address this, we compare the translocation properties of two well-studied bacterial restriction endonucleases (ENases), EcoRI and EcoRV. These dimeric, magnesium-dependent enzymes hydrolyze related sites (EcoRI ENase, 5′-GAATTC-3′; EcoRV ENase, 5′-GATATC-3′), leaving overhangs and blunt DNA segments, respectively. Here, we demonstrate that the extensive sliding by EcoRI ENase, involving sliding up to ∼600 bp prior to dissociating from the DNA, contrasts with a larger reliance on hopping mechanism(s) by EcoRV ENase. The mechanism displayed by EcoRI ENase results in a highly thorough search of DNA, whereas the EcoRV ENase mechanism results in an extended, yet less rigorous, interrogation of DNA sequence space. We describe how these mechanistic distinctions are complemented by other aspects of these endonucleases, such as the 10-fold higher in vivo concentrations of EcoRI ENase compared to that of EcoRV ENase. Further, we hypothesize that the highly diverse enzyme arsenal that bacteria employ against foreign DNA involves seemingly similar enzymes that rely on distinct but complementary search mechanisms. Our comparative approach reveals how different proteins utilize distinct site-locating strategies.

A quantitative understanding of how conformational transitions contribute to enzyme catalysis and specificity remains a fundamental challenge. A suite of biophysical approaches was used to reveal several transient states of the enzyme–substrate complexes of the model DNA cytosine methyltransferase M.HhaI. Multidimensional, transverse relaxation-optimized nuclear magnetic resonance (NMR) experiments show that M.HhaI has the same conformation with noncognate and cognate DNA sequences. The high-affinity cognatelike mode requires the formation of a subset of protein–DNA interactions that drive the flipping of the target base from the helix to the active site. Noncognate substrates lacking these interactions undergo slow base flipping, and fluorescence tracking of the catalytic loop corroborates the NMR evidence of a loose, nonspecific binding mode prior to base flipping and subsequent closure of the catalytic loop. This slow flipping transition defines the rate-limiting step for the methylation of noncognate sequences. Additionally, we present spectroscopic evidence of an intermediate along the base flipping pathway that has been predicted but never previously observed. These findings provide important details of how conformational rearrangements are used to balance specificity with catalytic efficiency.

One of the most dramatic illustrations of enzymatic promotion of a high-energy intermediate is observed in DNA modification and repair enzymes where an individual base is rotated (flipped) 180° around the deoxyribose–phosphate backbone and into the active site. While the end states have been extensively characterized, experimental techniques have yet to yield a full description of the base flipping process and the role played by the enzyme. The C5 cytosine methyltransferase M.HhaI coordinates an ensemble of reciprocal DNA and enzyme rearrangements to efficiently flip the target cytosine from the DNA helix. We sought to understand the role of individual amino acids during base flipping. Our results demonstrate that M.HhaI initiates base flipping before closure of the catalytic loop and utilizes the conserved serine 85 in the catalytic loop to accelerate flipping and maintain distortion of the DNA backbone. Serine 87, which forms specific contacts within the DNA helix after base flipping, is not involved in the flipping process or in maintaining the catalytically competent complex. At the base of the catalytic loop, glycine 98 acts as a hinge to allow conformational dynamism of the loop and mutation to alanine inhibits stabilization of the closed loop. Our results illustrate how an enzyme utilizes numerous, distal residues in concert to transform substrate recognition into catalysis.

Despite intense academic and industrial efforts and innumerable in vitro and cell studies, no small-molecule telomerase inhibitors have emerged as drugs. Insufficient understanding of enzyme structure and mechanisms of interdiction coupled with the substantial complexities presented by its dimeric composition have stalled all progress toward small-molecule therapeutics. Here we challenge the assumption that human telomerase provides the best platform for inhibitor development by probing a monomeric Tetrahymena telomerase with six tool compounds. We find BIBR-1532 (2) and MST-312 (5) inhibit only human telomerase, whereas β-R (1), THyF (3), TMPyP4 (6), and EGCG (4) inhibit both enzymes. Our study demonstrates that some small-molecule scaffolds can be easily surveyed with in vitro studies using Tetrahymena telomerase, a finding that could lead to more tractable inhibitors with a greater potential for development given the more precise insights that can be gleaned from this more easily expressed and assayed monomeric enzyme.

The DNA methyltransferase M.HhaI is an excellent model for understanding how recognition of a nucleic acid substrate is translated into site-specific modification. In this study, we utilize direct, real-time monitoring of the catalytic loop position via engineered tryptophan fluorescence reporters to dissect the conformational transitions that occur in both enzyme and DNA substrate prior to methylation of the target cytosine. Using nucleobase analogues in place of the target and orphan bases, the kinetics of the base flipping and catalytic loop closure rates were determined, revealing that base flipping precedes loop closure as the rate-determining step prior to methyl transfer. To determine the mechanism by which individual specific hydrogen bond contacts at the enzyme−DNA interface mediate these conformational transitions, nucleobase analogues lacking hydrogen bonding groups were incorporated into the recognition sequence to disrupt the major groove recognition elements. The consequences of binding, loop closure, and catalysis were determined for four contacts, revealing large differences in the contribution of individual hydrogen bonds to DNA recognition and conformational transitions on the path to catalysis. Our results describe how M.HhaI utilizes direct readout contacts to accelerate extrication of the target base that offer new insights into the evolutionary history of this important class of enzymes.

We present two strategies for attaching self-assembled DNA arrays to the surfaces of cells. Our first approach uses biotin−streptavidin interactions to bind DNA architectures to biotinylated cells. The second approach takes advantage of specific antibody−cell surface interactions, conjugated arrays and the subsequent binding to native epidermal growth factor receptors expressed on cancer cells. DNA array−cell surface interactions were visualized by fluorescence, confocal microscopy, and scanning electron microscopy. This novel application of DNA nanoarrays provides strategies to specifically label cell surfaces with micrometer-sized patches, bind cells onto a designed functionalized DNA scaffold, engineer cell/cell networks into microtissues, and deliver materials to cell surfaces.

Methylation of GATC sites in Escherichia coli by DNA adenine methyltransferase (EcoDam) is essential for proper DNA replication timing, gene regulation, and mismatch repair. The low cellular concentration of EcoDam and the high number of GATC sites in the genome (∼20000) support the reliance on methylation efficiency-enhancing strategies such as extensive intersite processivity. Here, we present evidence that EcoDam has evolved other unique mechanisms of activation not commonly observed with restriction−modification methyltransferases. EcoDam dimerizes on short, synthetic DNA, resulting in enhanced catalysis; however, dimerization is not observed on large genomic DNA where the potential for intersite processive methylation precludes any dimerization-dependent activation. An activated form of the enzyme is apparent on large genomic DNA and can also be achieved with high concentrations of short, synthetic substrates. We suggest that this activation is inherent on polymeric DNA where either multiple GATC sites are available for methylation or the partitioning of the enzyme onto nonspecific DNA is favored. Unlike other restriction−modification methyltransferases, EcoDam carries out intrasite processive catalysis whereby the enzyme−DNA complex methylates both strands of an unmethylated GATC site prior to dissociation from the DNA. This occurs with short 21 bp oligonucleotides and is highly dependent upon salt concentrations. Kinetic modeling which invokes enzyme activation by both dimerization and excess substrate provides mechanistic insights into key regulatory checkpoints for an enzyme involved in multiple, diverse biological pathways.

Enzymatic sequence-specific DNA modification involves multiple poorly understood intermediates. DNA methyltransferases like M.HhaI initially bind nonspecific DNA and then selectively bind and modify a unique sequence. High-resolution NMR was used to map conformational changes occurring in M.HhaI upon binding nonspecific DNA, a one base pair altered noncognate DNA sequence, and both hemimethylated and unmethylated cognate DNA sequences. Comparisons with previous NMR studies of the apoenzyme and enzyme−cofactor complex provide snapshots of the pathway to sequence-specific complex formation. Dramatic chemical shift perturbations reaching many distal sites within the protein are detected with cognate DNA, while much smaller changes are observed upon nonspecific and noncognate DNA binding. A cooperative rather than stepwise transition from a nonspecific to a cognate complex is revealed. Furthermore, switching from unmethylated to hemimethylated cognate DNA involves detectable protein conformational changes 20−30 Å away from the methyl group, indicating high protein sensitivity and plasticity to DNA modification.

We describe the self-assembly of multilayer hexagonal DNA arrays through highly regular interlayer packing. Slow cooling of a mixture of three single-stranded DNA sequences with various Mg2+ concentrations leads to the self-assembly of diverse multilayer architectures. The self-assembled aggregates were deposited onto mica surfaces and examined with atomic force microscopy. The size of the two-dimensional arrays and subsequent stacking to form multilayer structures are highly dependent on Mg2+ concentration. DNA bilayers and multilayers of defined shape are favored in 2−5 mM Mg2+ with an average lateral size of 700 nm. Arrays are much larger (up to 20 μm across) in 10−15 mM Mg2+, although multiple layers still make up 20−60% of the observed structures. Domains within single layer architectures were identified using Moiré pattern analysis. Distinct structural phases within the multilayer assemblies include two layers translated by 17.5 nm and interlayer rotations of 20° and 30°. Three layer assemblies have cubic close packing and taller multilayer architectures of 2D DNA sheets were also identified.

The temporal and spatial control over the delivery of materials such as siRNA into cells remains a significant technical challenge. We demonstrate the pulsed near-infrared (NIR) laser-dependent release of siRNA from coated 40 nm gold nanoshells. Tat-lipid coating mediates the cellular uptake of the nanomaterial at picomolar concentration, while spatiotemporal silencing of a reporter gene (green fluorescence protein) was studied using photomasking. The NIR laser-induced release of siRNA from the nanoshells is found to be power- and time-dependent, through surface-linker bond cleavage, while the escape of the siRNA from endosomes occurs above a critical pulse energy attributed to local heating and cavitation. NIR laser-controlled drug release from functional nanomaterials should facilitate more sophisticated developmental biology and therapeutic studies.Keywords: gold; laser; nanoparticle; nanoshell; RNA; silencing; TAT

Over the past decade the emphasis on single-molecule sensitivity of surface-enhanced Raman spectroscopy (SERS) has brought to prominence the special role played by so-called SERS hot spots, oftentimes nanometer-scale junctions between nanoparticles (NPs). In this report, molecular linkers are used to mediate the assembly of NPs to dimers and small clusters. When the SERS enhancement is optimized, the aggregation process is quenched by polymer and protein stabilizers that subsequently act as encapsulants resulting in SERS substates with unprecedented enhancement uniformity, reproducibility, and stability. The polymer-stabilized NP junctions were then imprinted with a variety of small molecules that permeated the polymer coat and displaced the linker from the hot spot. The average SERS enhancement of these SERS “nanocapsules” was found to be at least 300× greater than for single NPs, while the Raman/Rayleigh scattering ratio was 104 higher for linked NPs versus nonoptimized aggregates. Single-particle statistics showed that almost every nanocapsule produced intense SERS, suggesting that they are NP dimers and small clusters with the probe molecule resident in a hot spot. Nanocapsules were functionalized and shown to compete successfully with fluorescence imaging in multiplexed identification of cancer cell epitopes at the single-cell and single-nanotag level.

A method to position nanoparticles onto DNA with high resolution using an enzyme-based approach is described. This provides a convenient route to assemble multiple nanoparticles (e.g., Au and CdSe) to specific positions with a high level of control and expandability to more complex assemblies. Atomic force microscopy is used to analyze the nanostructures, which have potential interest for biosensor, optical waveguide, molecular electronics, and energy transfer studies.

Biomolecular self-assembly provides a basis for the bottom-up construction of useful and diverse nanoscale architectures. DNA is commonly used to create these assemblies and is often exploited as a lattice or an array. Although geometrically rigid and highly predictable, these sheets of repetitive constructs often lack the ability to be enzymatically manipulated or elongated by standard biochemical techniques. Here, we describe two approaches for the construction of position-controlled, molecular-scale, discrete, three- and four-way DNA junctions. The first approach for constructing these junctions relies on the use of nonmigrating cruciforms generated from synthetic oligonucleotides to which large, biologically generated, double-stranded DNA segments are enzymatically ligated. The second approach utilitizes the DNA methyltransferase-based SMILing (sequence-specific methyltransferase-induced labeling of DNA) method to site-specifically incorporate a biotin within biologically derived DNA. Streptavidin is then used to form junctions between unique DNA strands. The resultant assemblies have precise and predetermined connections with lengths that can be varied by enzymatic or hybridization techniques, or geometrically controlled with standard DNA functionalization methods. These junctions are positioned with single nucleotide resolution on large, micrometer-length templates. Both approaches generate DNA assemblies which are fully compatible with standard recombinant methods and thus provide a novel basis for nanoengineering applications.

Gold nanoparticles were modified with RNA and utilized to detect specific DNA sequences and various RNA nucleases.

Molecular dynamics (MD) simulations of HhaI DNA methyltransferase and statistical coupling analysis (SCA) data on the DNA cytosine methyltransferase family were combined to identify residues that are coupled by coevolution and motion. The highest ranking correlated pairs from the data matrix product (SCA·MD) are colocalized and form stabilizing interactions; the anticorrelated pairs are separated on average by 30 Å and form a clear focal point centered near the active site. We suggest that these distal anti-correlated pairs are involved in mediating active-site compressions that may be important for catalysis. Mutants that disrupt the implicated interactions support the validity of our combined SCA·MD approach.

The role of Glu119 in S-adenosyl-l-methionine-dependent DNA methyltransferase M.HhaI-catalyzed DNA methylation was studied. Glu119 belongs to the highly conserved Glu/Asn/Val motif found in all DNA C5-cytosine methyltransferases, and its importance for M.HhaI function remains untested. We show that formation of the covalent intermediate between Cys81 and the target cytosine requires Glu119, since conversion to Ala, Asp or Gln lowers the rate of methyl transfer 102–106 fold. Further, unlike the wild-type M.HhaI, these mutants are not trapped by the substrate in which the target cytosine is replaced with the mechanism-based inhibitor 5-fluorocytosine. The DNA binding affinity for the Glu119Asp mutant is decreased 103-fold. Thus, the ability of the enzyme to stabilize the extrahelical cytosine is coupled directly to tight DNA binding. The structures of the ternary protein/DNA/AdoHcy complexes for both the Glu119Ala and Glu119Gln mutants (2.70 Å and 2.75 Å, respectively) show that the flipped base is positioned nearly identically with that observed in the wild-type M.HhaI complex. A single water molecule in the Glu119Ala structure between Ala119 and the extrahelical cytosine N3 is lacking in the Glu119Gln and wild-type M.HhaI structures, and most likely accounts for this mutant's partial activity. Glu119 has essential roles in activating the target cytosine for nucleophilic attack and contributes to tight DNA binding.

Escherichia coli DNA adenine methyltransferase (Dam) and Leucine-responsive regulatory protein (Lrp) are key regulators of the pap operon, which codes for the pilus proteins necessary for uropathogenic E. coli cellular adhesion. The pap operon is regulated by a phase variation mechanism in which the methylation states of two GATC sites in the pap regulatory region and the binding position of Lrp determine whether the pilus genes are expressed. The post-replicative reassembly of Dam, Lrp, and the local regulator PapI onto a hemimethylated pap intermediate is a critical step of the phase variation switching mechanism and is not well understood. We show that Lrp, in the presence and in the absence of PapI and nonspecific DNA, specifically protects pap regulatory GATC sites from Dam methylation when allowed to compete with Dam for assembly on unmethylated and hemimethylated pap DNA. The methylation protection is dependent upon the concentration of Lrp and does not occur with non-regulatory GATC sites. Our data suggest that only at low Lrp concentrations will Dam compete effectively for binding and methylation of the proximal GATC site, leading to a phase switch resulting in the expression of pili.

•Sliding and hopping models cannot adequately explain long-range protein movements.•DNA adenine methyltransferase (Dam) methylates ~ 20,000 sites along the genome.•Dam travels between its sites most efficiently when they are separated by ~ 500 bp.•Passive DNA looping facilitates these movements.•Other proteins possibly utilize this “intersegmental hopping” mechanism as well.Studies on how transcription factors and DNA modifying enzymes passively locate specific sites on DNA have yet to be reconciled with a sufficient set of mechanisms that can adequately account for the efficiency and speed of this process. This is especially true when considering that these DNA binding/modifying proteins have diverse levels of both cellular copy numbers and genomic recognition site densities. The monomeric bacterial DNA adenine methyltransferase (Dam) is responsible for the rapid methylation of the entire chromosome (with only ~ 100 Dam copies per cell) and the regulated methylation of closely spaced sites that controls the expression of virulence genes in several human pathogens. Provocatively, we find that Dam travels between its recognition sites most efficiently when those sites are ~ 500 bp apart. We propose that this is manifested by Dam moving between distal regions on the same DNA molecule, which is mediated by DNA looping, a phenomenon we designate as intersegmental hopping. Importantly, an intermediate found in other systems including two simultaneously bound, looped DNA strands is not involved here. Our results suggest that intersegmental hopping contributes to enzymatic processivity (multiple modifications), which invoke recent reports demonstrating that DNA looping can assist in site finding. Intersegmental hopping is possibly used by other sequence-specific DNA binding proteins, such as transcription factors and regulatory proteins, given certain biological context. While a general form of this mechanism is proposed by many research groups, our consideration of DNA looping in the context of processive catalysis provides new mechanistic insights and distinctions.Download high-res image (75KB)Download full-size image

Transcriptional regulatory proteins typically bind specific DNA sequences with ∼103–107-fold higher affinity than non-specific DNA and this discrimination is essential for their in vivo function. Here we show that the bacterial leucine-responsive regulatory protein (Lrp) does not follow this trend and has a ∼20–400-fold binding discrimination between specific and non-specific DNA sequences. We suggest that the dual function of Lrp to regulate genes and to organize DNA utilizes this unique property. A ∼20-fold decrease in binding affinity from specific DNA is dependent upon cryptic binding sites, including the sequence GN2-3TTT and A-tracts. Removal of these sites still results in high binding affinity, only ∼70-fold weaker than that of specific sites. Similar to Lrp's binding of specific sites in the pap and ilvIH promoters, Lrp binds cooperatively to non-specific DNA; thus, protein/protein interactions are important for both specific and non-specific DNA binding. When considering this cooperativity of Lrp binding, the binding selectivity to specific sites may increase to a maximum of ∼400-fold. Neither leucine nor the pap-specific local regulator PapI alter Lrp's non-specific binding affinity or cooperative binding of non-specific DNA. We hypothesize that Lrp combines low sequence discrimination and relatively high intracellular protein concentrations to ensure its ability to regulate the transcription of specific genes while also functioning as a nucleoid-associated protein. Modeling of Lrp binding data and comparison to other proteins with regulatory and nucleoid-associated properties suggests similar mechanisms.

We demonstrate modulation of nitric oxide release in solution and in human prostate cancer cells from a thiol functionalized cupferron (TCF) absorbed on hollow gold nanoshells (HGNs) using near-infrared (NIR) light. NO release from the TCF–HGN conjugates occurs through localized surface heating due to NIR excitation of the surface plasmon. Specific HGN targeting is achieved through cell surface directed peptides, and excitation with tissue penetrating NIR light provides unprecedented spatio-temporal control of NO delivery to biological targets.

Gold nanoparticles were modified with RNA and utilized to detect specific DNA sequences and various RNA nucleases.