Small Volume Microrheology to Evaluate Viscoelastic Properties of Nucleic Acid-Based Supra-Assemblies.

Particle tracking (PT) microrheology is a passive microrheological approach that characterizes material properties of soft matter. Multicomponent materials with the ability to create extensive crosslinking, such as supra-assemblies, may exhibit a complex interplay of viscous and elastic properties with a substantial contribution of liquid phase still diffusing through the system. Microrheology analyzes the motion of microscopic beads immersed in a sample, making it possible to evaluate the rheological properties of biological supra-assemblies. This method requires only a small volume of the sample and a relatively simple, inexpensive experimental setup. The objective of this chapter is to describe the experimental procedures for the observation of particle motion, calibration of an optical setup for particle tracking, preparation of imaging chambers, and the use of image analysis software for particle tracking in viscoelastic nucleic acid-based supra-assemblies.

Characterization of RNA Nanoparticles and Their Dynamic Properties Using Atomic Force Microscopy.

The protocol described in this chapter allows for acquiring topography images of RNA-based nanoring structures and assessing their dynamic properties using atomic force microscopy (AFM) imaging. AFM is an indispensable tool for characterization of nucleic acid-based nanostructures with the exceptional capability of observing complexes in the range of a few nanometers. This method can visualize structural characteristics and evaluate differences between individual structurally different RNA nanorings. Due to the highly resolved AFM topography images, we introduce an approach that allows to distinguish the differences in the dynamic behavior of RNA nanoparticles not amenable to other experimental techniques. This protocol describes in detail the preparation procedures of RNA nanostructures, AFM imaging, and data analysis.

Optical, structural, and biological properties of silver nanoclusters formed within the loop of a C-12 hairpin sequence.

Silver nanoclusters (AgNCs) are the next-generation nanomaterials representing supra-atomic structures where silver atoms are organized in a particular geometry. DNA can effectively template and stabilize these novel fluorescent AgNCs. Only a few atoms in size - the properties of nanoclusters can be tuned using only single nucleobase replacement of C-rich templating DNA sequences. A high degree of control over the structure of AgNC could greatly contribute to the ability to fine-tune the properties of silver nanoclusters. In this study, we explore the properties of AgNCs formed on a short DNA sequence with a C12 hairpin loop structure (AgNC@hpC12). We identify three types of cytosines based on their involvement in the stabilization of AgNCs. Computational and experimental results suggest an elongated cluster shape with 10 silver atoms. We found that the properties of the AgNCs depend on the overall structure and relative position of the silver atoms. The emission pattern of the AgNCs depends strongly on the charge distribution, while all silver atoms and some DNA bases are involved in optical transitions based on molecular orbital (MO) visualization. We also characterize the antibacterial properties of silver nanoclusters and propose a possible mechanism of action based on the interactions of AgNCs with molecular oxygen.

DNA-Templated Silver Nanoclusters as Dual-Mode Sensitive Probes for Self-Powered Biosensor Fueled by Glucose.

Nanomaterials have been extensively explored in developing sensors due to their unique properties, contributing to the development of reliable sensor designs with improved sensitivity and specificity. Herein, we propose the construction of a fluorescent/electrochemical dual-mode self-powered biosensor for advanced biosensing using DNA-templated silver nanoclusters (AgNCs@DNA). AgNC@DNA, due to its small size, exhibits advantageous characteristics as an optical probe. We investigated the sensing efficacy of AgNCs@DNA as a fluorescent probe for glucose detection. Fluorescence emitted by AgNCs@DNA served as the readout signal as a response to more H2O2 being generated by glucose oxidase for increasing glucose levels. The second readout signal of this dual-mode biosensor was utilized via the electrochemical route, where AgNCs served as charge mediators between the glucose oxidase (GOx) enzyme and carbon working electrode during the oxidation process of glucose catalyzed by GOx. The developed biosensor features low-level limits of detection (LODs), ~23 µM for optical and ~29 µM for electrochemical readout, which are much lower than the typical glucose concentrations found in body fluids, including blood, urine, tears, and sweat. The low LODs, simultaneous utilization of different readout strategies, and self-powered design demonstrated in this study open new prospects for developing next-generation biosensor devices.

Detection of cancer-associated miRNA using a fluorescence switch of AgNC@NA and guanine-rich overhang sequences.

DNA-templated silver nanoclusters (AgNC@DNA) are a novel type of nanomaterial with advantageous optical properties. Only a few atoms in size, the fluorescence of nanoclusters can be tuned using DNA overhangs. In this study, we explored the properties of AgNCs manufactured on a short single-stranded (dC)12 when adjacent G-rich sequences (dGN , with N = 3-15) were added. The 'red' emission of AgNC@dC12 with λMAX = 660 nm dramatically changed upon the addition of a G-rich overhang with NG = 15. The pattern of the emission-excitation matrix (EEM) suggested the emergence of two new emissive states at λMAX = 575 nm and λMAX = 710 nm. The appearance of these peaks provides an effective way to design biosensors capable of detecting specific nucleic acid sequences with low fluorescence backgrounds. We used this property to construct an NA-based switch that brings AgNC and the G overhang near one another, turning 'ON' the new fluorescence peaks only when a specific miRNA sequence is present. Next, we tested this detection switch on miR-371, which is overexpressed in prostate cancer. The results presented provide evidence that this novel fluorescent switch is both sensitive and specific with a limit of detection close to 22 picomoles of the target miR-371 molecule.

Modular Micro Raman Reader Instrument for Fast SERS-Based Detection of Biomarkers.

Sensitive detection of biomarkers is very critical in the diagnosis, management, and monitoring of diseases. Recent efforts have suggested that bioassays using surface-enhanced Raman scattering as a signal read-out strategy possess certain unique beneficial features in terms of sensitivity and low limits of detection which set this method apart from its counterparts such as fluorescence, phosphorescence, and radiolabeling. Surface-enhanced Raman scattering (SERS) has also emerged as an ideal choice for the development of multiplexed bioassays. Such promising features have prompted the need for the development of SERS-based tools suitable for point-of-care applications. These tools must be easy to use, portable, and automated for the screening of many samples in clinical settings if diagnostic applications are considered. The availability of such tools will result in faster and more reliable detection of disease biomarkers, improving the accessibility of point-of-care diagnostics. In this paper, we describe a modular Raman reader instrument designed to create such a portable device suitable for screening a large number of samples with minimal operator assistance. The device's hardware is mostly built with commercially available components using our unique design. Dedicated software was created to automatically run sample screening and analyze the data measured. The mRR is an imaging system specifically created to automate measurements, eliminating human bias while enhancing the rate of data collection and analysis ~2000 times. This paper presents both the design and capabilities of the custom-built modular Raman reader system (mRR) capable of automated and fast measurements of sandwich immunoassay samples on gold substrates using modified gold nanoparticles as Raman tags. The limit of detection (LOD) of the tested MUC4-specific iSERS assay was measured to be 0.41 µg/mL.

Substituent Effects Impact Surface Charge and Aggregation of Thiophenol-Labeled Gold Nanoparticles for SERS Biosensors.

SERS immunoassay biosensors hold immense potential for clinical diagnostics due to their high sensitivity and growing interest in multi-marker panels. However, their development has been hindered by difficulties in designing compatible extrinsic Raman labels. Prior studies have largely focused on spectroscopic characteristics in selecting Raman reporter molecules (RRMs) for multiplexing since the presence of well-differentiated spectra is essential for simultaneous detection. However, these candidates often induce aggregation of the gold nanoparticles used as SERS nanotags despite their similarity to other effective RRMs. Thus, an improved understanding of factors affecting the aggregation of RRM-coated gold nanoparticles is needed. Substituent electronic effects on particle stability were investigated using various para-substituted thiophenols. The inductive and resonant effects of functional group modifications were strongly correlated with nanoparticle surface charge and hence their stability. Treatment with thiophenols diminished the negative surface charge of citrate-stabilized gold nanoparticles, but electron-withdrawing substituents limited the magnitude of this diminishment. It is proposed that this phenomenon arises by affecting the interplay of competing sulfur binding modes. This has wide-reaching implications for the design of biosensors using thiol-modified gold surfaces. A proof-of-concept multiplexed SERS biosensor was designed according to these findings using the two thiophenol compounds with the most electron-withdrawing substitutions: NO2 and CN.

DNA-Templated Fluorescent Silver Nanoclusters Inhibit Bacterial Growth While Being Non-Toxic to Mammalian Cells.

Silver has a long history of antibacterial effectiveness. The combination of atomically precise metal nanoclusters with the field of nucleic acid nanotechnology has given rise to DNA-templated silver nanoclusters (DNA-AgNCs) which can be engineered with reproducible and unique fluorescent properties and antibacterial activity. Furthermore, cytosine-rich single-stranded DNA oligonucleotides designed to fold into hairpin structures improve the stability of AgNCs and additionally modulate their antibacterial properties and the quality of observed fluorescent signals. In this work, we characterize the sequence-specific fluorescence and composition of four representative DNA-AgNCs, compare their corresponding antibacterial effectiveness at different pH, and assess cytotoxicity to several mammalian cell lines.

Micro RNA Sensing with Green Emitting Silver Nanoclusters.

Micro RNA (miR) are regulatory non-coding RNA molecules, which contain a small number of nucleotides ~18-28 nt. There are many various miR sequences found in plants and animals that perform important functions in developmental, metabolic, and disease processes. miRs can bind to complementary sequences within mRNA molecules thus silencing mRNA. Other functions include cardiovascular and neural development, stem cell differentiation, apoptosis, and tumors. In tumors, some miRs can function as oncogenes, others as tumor suppressors. Levels of certain miR molecules reflect cellular events, both normal and pathological. Therefore, miR molecules can be used as biomarkers for disease diagnosis and prognosis. One of these promising molecules is miR-21, which can serve as a biomarker with high potential for early diagnosis of various types of cancer. Here, we present a novel design of miR detection and demonstrate its efficacy on miR-21. The design employs emissive properties of DNA-silver nanoclusters (DNA/AgNC). The detection probe is designed as a hairpin DNA structure with one side of the stem complimentary to miR molecule. The binding of target miR-21 opens the hairpin structure, dramatically modulating emissive properties of AgNC hosted by the C12 loop of the hairpin. "Red" fluorescence of the DNA/AgNC probe is diminished in the presence of the target miR. At the same time, "green" fluorescence is activated and its intensity increases several-fold. The increase in intensity of "green" fluorescence is strong enough to detect the presence of miR-21. The intensity change follows the concentration dependence of the target miR present in a sample, which provides the basis of developing a new, simple probe for miR detection. The detection strategy is specific, as demonstrated using the response of the DNA/AgNC probe towards the scrambled miR-21 sequence and miR-25 molecule. Additionally, the design reported here is very sensitive with an estimated detection limit at ~1 picomole of miR-21.

Tuning properties of silver nanoclusters with RNA nanoring assemblies.

Combining atomically resolved DNA-templated silver nanoclusters (AgNCs) with nucleic acid nanotechnology opens new exciting possibilities for engineering bioinorganic nanomaterials with uniquely tunable properties. In this unforeseen cooperation, nucleic acids not only drive the formation of AgNCs but also promote their spatial organization in supra-assemblies. In this work, we confirm the feasibility of this approach using programmable RNA rings to control formation and optical properteis of six individual AgNCs. "Red" (λEXC/λEM = 565/623 nm) and "green" (λEXC/λEM = 440/523 nm) emitting AgNCs are templated on cytosine-rich DNA fragments embedded into the RNA rings. Optical properties of the AgNCs formed on the RNA rings are characterized in detail. While all "red" species passively transition to "green" emitters with time, the initial fluorescent properties and relative stabilities of "red" AgNCs can be regulated by altering the relative orientation of AgNCs within the RNA rings. As such, the oxidative stability increases dramatically for AgNC positioned towards the center of the RNA rings rather than facing outward. Overall, our findings expand the existing AgNC fluorescent toolkit while uncovering the complexity of the AgNC electronic structures with the abundance of possibilities for controlling de-excitation processes.

Post-ER Stress Biogenesis of Golgi Is Governed by Giantin.

BACKGROUND: The Golgi apparatus undergoes disorganization in response to stress, but it is able to restore compact and perinuclear structure under recovery. This self-organization mechanism is significant for cellular homeostasis, but remains mostly elusive, as does the role of giantin, the largest Golgi matrix dimeric protein. METHODS: In HeLa and different prostate cancer cells, we used the model of cellular stress induced by Brefeldin A (BFA). The conformational structure of giantin was assessed by proximity ligation assay and atomic force microscopy. The post-BFA distribution of Golgi resident enzymes was examined by 3D SIM high-resolution microscopy. RESULTS: We detected that giantin is rather flexible than an extended coiled-coil dimer and BFA-induced Golgi disassembly was associated with giantin monomerization. A fusion of the nascent Golgi membranes after BFA washout is forced by giantin re-dimerization via disulfide bond in its luminal domain and assisted by Rab6a GTPase. GM130-GRASP65-dependent enzymes are able to reach the nascent Golgi membranes, while giantin-sensitive enzymes appeared at the Golgi after its complete recovery via direct interaction of their cytoplasmic tail with N-terminus of giantin. CONCLUSION: Post-stress recovery of Golgi is conducted by giantin dimer and Golgi proteins refill membranes according to their docking affiliation rather than their intra-Golgi location.

First Step Towards Larger DNA-Based Assemblies of Fluorescent Silver Nanoclusters: Template Design and Detailed Characterization of Optical Properties.

Besides being a passive carrier of genetic information, DNA can also serve as an architecture template for the synthesis of novel fluorescent nanomaterials that are arranged in a highly organized network of functional entities such as fluorescent silver nanoclusters (AgNCs). Only a few atoms in size, the properties of AgNCs can be tuned using a variety of templating DNA sequences, overhangs, and neighboring duplex regions. In this study, we explore the properties of AgNCs manufactured on a short DNA sequence-an individual element designed for a construction of a larger DNA-based functional assembly. The effects of close proximity of the double-stranded DNA, the directionality of templating single-stranded sequence, and conformational heterogeneity of the template are presented. We observe differences between designs containing the same AgNC templating sequence-twelve consecutive cytosines, (dC)12. AgNCs synthesized on a single "basic" templating element, (dC)12, emit in "red". The addition of double-stranded DNA core, required for the larger assemblies, changes optical properties of the silver nanoclusters by adding a new population of clusters emitting in "green". A new population of "blue" emitting clusters forms only when ssDNA templating sequence is placed on the 5' end of the double-stranded core. We also compare properties of silver nanoclusters, which were incorporated into a dimeric structure-a first step towards a larger assembly.

Surface-Enhanced Raman Scattering-Based Immunoassay Technologies for Detection of Disease Biomarkers.

Detection of biomarkers is of vital importance in disease detection, management, and monitoring of therapeutic efficacy. Extensive efforts have been devoted to the development of novel diagnostic methods that detect and quantify biomarkers with higher sensitivity and reliability, contributing to better disease diagnosis and prognosis. When it comes to such devastating diseases as cancer, these novel powerful methods allow for disease staging as well as detection of cancer at very early stages. Over the past decade, there have been some advances in the development of platforms for biomarker detection of diseases. The main focus has recently shifted to the development of simple and reliable diagnostic tests that are inexpensive, accurate, and can follow a patient's disease progression and therapy response. The individualized approach in biomarker detection has been also emphasized with detection of multiple biomarkers in body fluids such as blood and urine. This review article covers the developments in Surface-Enhanced Raman Scattering (SERS) and related technologies with the primary focus on immunoassays. Limitations and advantages of the SERS-based immunoassay platform are discussed. The article thoroughly describes all components of the SERS immunoassay and highlights the superior capabilities of SERS readout strategy such as high sensitivity and simultaneous detection of a multitude of biomarkers. Finally, it introduces recently developed strategies for in vivo biomarker detection using SERS.

Effect of acidic pH on the stability of α-synuclein dimers.

Environmental factors, such as acidic pH, facilitate the assembly of α-synuclein (α-Syn) in aggregates, but the impact of pH on the very first step of α-Syn aggregation remains elusive. Recently, we developed a single-molecule approach that enabled us to measure directly the stability of α-Syn dimers. Unlabeled α-Syn monomers were immobilized on a substrate, and fluorophore-labeled monomers were added to the solution to allow them to form dimers with immobilized α-Syn monomers. The dimer lifetimes were measured directly from the fluorescence bursts on the time trajectories. Herein, we applied the single-molecule tethered approach for probing of intermolecular interaction to characterize the effect of acidic pH on the lifetimes of α-Syn dimers. The experiments were performed at pH 5 and 7 for wild-type α-Syn and for two mutants containing familial type mutations E46K and A53T. We demonstrate that a decrease of pH resulted in more than threefold increase in the α-Syn dimers lifetimes with some variability between the α-Syn species. We hypothesize that the stabilization effect is explained by neutralization of residues 96-140 of α-Syn and this electrostatic effect facilitates the association of the two monomers. Given that dimerization is the first step of α-Syn aggregation, we posit that the electrostatic effect thereby contributes to accelerating α-Syn aggregation at acidic pH. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 715-724, 2016.

Polymorphism of amyloid fibrils formed by a peptide from the yeast prion protein Sup35: AFM and Tip-Enhanced Raman Scattering studies.

Aggregation of prion proteins is the cause of various prion related diseases. The infectious form of prions, amyloid aggregates, exist as multiple strains. The strains are thought to represent structurally different prion protein molecules packed into amyloid aggregates, but the knowledge on the structure of different types of aggregates is limited. Here we report on the use of AFM (Atomic Force Microscopy) and TERS (Tip-Enhanced Raman Scattering) to study morphological heterogeneity and access underlying conformational features of individual amyloid aggregates. Using AFM we identified the morphology of amyloid fibrils formed by the peptide (CGNNQQNY) from the yeast prion protein Sup35 that is critically involved in the aggregation of the full protein. TERS results demonstrate that morphologically different amyloid fibrils are composed of a distinct set of conformations. Fibrils formed at pH 5.6 are composed of a mixture of peptide conformations (ß-sheets, random coil and α-helix) while fibrils formed in pH~2 solution primarily have ß-sheets. Additionally, peak positions in the amide III region of the TERS spectra suggested that peptides have parallel arrangement of ß-sheets for pH~2 fibrils and antiparallel arrangement for fibrils formed at pH 5.6. We also developed a methodology for detailed analysis of the peptide secondary structure by correlating intensity changes of Raman bands in different regions of TERS spectra. Such correlation established that structural composition of peptides is highly localized with large contribution of unordered secondary structures on a fibrillar surface.

A flexible nanoarray approach for the assembly and probing of molecular complexes.

Immobilization is a key step involved in probing molecular interactions using single-molecule force spectroscopy methods, including atomic force microscopy (AFM). To our knowledge, we describe a novel approach termed flexible nanoarray (FNA) in which the interaction between the two internally immobilized amyloid ß peptides is measured by pulling of the tether. The FNA tether was synthesized with nonnucleotide phosphoramidite monomers using the DNA synthesis chemistry. The two anchoring points for immobilization of the peptides inside the tether were incorporated at defined distances between them and from the ends of the polymer. Decamers of amyloid ß peptide capable of dimer formation were selected as a test system. The formation of the peptide dimers was verified by AFM force spectroscopy by pulling the tether at the ends. In these experiments, the thiolated end of the FNA tether was covalently immobilized on the AFM substrate functionalized with maleimide. The other end of the FNA tether was functionalized with biotin to form a noncovalent link with the streptavidin functionalized AFM tip during the approach stage. The dimers' rupture fingerprint was unambiguously identified on the force curves by its position and the force value. The FNA design allowed reversible experiments in which the monomers were allowed to associate after the rupture of the dimers by performing the approach stage before the rupture of the biotin-streptavidin link. This suggests that the FNA technique is capable of analyzing multiple intermolecular interactions in the same molecular complex. The computational analysis showed that the tethered peptides assemble into the same dimer structure as that formed by nontethered peptides, suggesting that the FNA tether has the necessary flexibility to enable assembly of the dimer even during the course of the force spectroscopy experiment.

Direct Detection of α-Synuclein Dimerization Dynamics: Single-Molecule Fluorescence Analysis.

The aggregation of α-synuclein (α-Syn) is linked to Parkinson's disease. The mechanism of early aggregation steps and the effect of pathogenic single-point mutations remain elusive. We report here a single-molecule fluorescence study of α-Syn dimerization and the effect of mutations. Specific interactions between tethered fluorophore-free α-Syn monomers on a substrate and fluorophore-labeled monomers diffusing freely in solution were observed using total internal reflection fluorescence microscopy. The results showed that wild-type (WT) α-Syn dimers adopt two types of dimers. The lifetimes of type 1 and type 2 dimers were determined to be 197 ± 3 ms and 3334 ± 145 ms, respectively. All three of the mutations used, A30P, E46K, and A53T, increased the lifetime of type 1 dimer and enhanced the relative population of type 2 dimer, with type 1 dimer constituting the major fraction. The kinetic stability of type 1 dimers (expressed in terms of lifetime) followed the order A30P (693 ± 14 ms) > E46K (292 ± 5 ms) > A53T (226 ± 6 ms) > WT (197 ± 3 ms). Type 2 dimers, which are more stable, had lifetimes in the range of several seconds. The strongest effect, observed for the A30P mutant, resulted in a lifetime 3.5 times higher than observed for the WT type 1 dimer. This mutation also doubled the relative fraction of type 2 dimer. These data show that single-point mutations promote dimerization, and they suggest that the structural heterogeneity of α-Syn dimers could lead to different aggregation pathways.

Nano-immunoassay with improved performance for detection of cancer biomarkers.

Nano-immunoassay utilizing surface-enhanced Raman scattering (SERS) effect is a promising analytical technique for early detection of cancer. In its current standing the assay is capable of discriminating samples of healthy individuals from samples of pancreatic cancer patients. Further improvements in sensitivity and reproducibility will extend practical applications of the SERS-based detection platforms to wider range of problems. In this report, we discuss several strategies designed to improve performance of the SERS-based detection system. We demonstrate that reproducibility of the platform is enhanced by using atomically smooth mica surface as a template for preparation of capture surface in SERS sandwich immunoassay. Furthermore, assay's stability and sensitivity can be further improved by using either polymer or graphene monolayer as a thin protective layer applied on top of the assay addresses. The protective layer renders signal to be more stable against photo-induced damage and carbonaceous contamination.

α-Synuclein misfolding assessed with single molecule AFM force spectroscopy: effect of pathogenic mutations.

Misfolding and subsequent aggregation of alpha-synuclein (α-Syn) protein are critically involved in the development of several neurodegenerative diseases, including Parkinson's disease (PD). Three familial single point mutations, A30P, E46K, and A53T, correlate with early onset PD; however, the molecular mechanism of the effects of these mutations on the structural properties of α-Syn and its propensity to misfold remains unclear. Here, we address this issue utilizing a single molecule AFM force spectroscopy approach in which structural details of dimers formed by all four variants of α-Syn are characterized. Analysis of the force spectroscopy data reflecting contour length distribution for α-Syn dimer dissociation suggests that multiple segments are involved in the assembly of the dimer. The interactions are not limited to the central nonamyloid-beta component (NAC) of the protein but rather expand beyond this segment. All three mutations alter the protein's folding and interaction patterns affecting interactions far beyond their immediate locations. Implementation of these findings to our understanding of α-Syn aggregation pathways is discussed.

Fibrillogenesis of huntingtin and other glutamine containing proteins.

This chapter focuses on the aggregation of glutamine containing peptides and proteins with an emphasis on huntingtin protein, whose aggregation leads to the development of Huntington's disease. The kinetics that leads to the formation of amyloids, the structure of aggregates of various types and the morphological mechanical properties of amyloid fibrils are described. The kinetics of amyloid fibril formation has been proposed to follow a nucleation dependent polymerization model, dependent upon the size of the nucleus. This model and the effect of the polyglutamine length on the nucleus size are reviewed. Aggregate structure is characterized at two different levels. The atomic-scale resolution structure of fibrillar and crystalline aggregates of polyglutamine containing proteins and peptides was determined by X-ray crystallography and solid-state nuclear magnetic resonance (NMR). The chapter outlines the results obtained by both these techniques. Atomic force microscopy (AFM) was instrumental in elucidating the morphology of fibrils, their organization and assembly. The chapter also discusses the high stability of amyloid fibrils, including their mechanical properties as revealed by AFM.