Surface-Area Enhanced Raman Spectroscopy of DNA in Porous Silica: A Quantitative and Reproducible Alternative to Plasmonic-Based SERS.

Despite the success of surface-enhanced Raman spectroscopy (SERS) for detecting DNA immobilized on plasmonic metal surfaces, its quantitative response is limited by the rapid falloff of enhancement with distance from the metal surface and variations in sensitivity that depend on orientation and proximity to plasmonic "hot spots". In this work, we assess an alternative approach for enhancing detection by immobilizing DNA on the interior surfaces of porous silica particles. These substrates provide over a 1000-fold greater surface area for detection compared to a planar support. The porous silica substrate is a purely dielectric material with randomly oriented internal surfaces, where scattering is independent of proximity and orientation of oligonucleotides relative to the silica surface. We characterize the quantitative response of Raman scattering from DNA in porous silica particles with sequences used in previous SERS investigations of DNA for comparison. The results show that Raman scattering of DNA in porous silica is independent of distance of nucleotides from the silica surface, allowing detection of longer DNA strands with constant sensitivity. The surface area enhancement within particles is reproducible (<4% particle-to-particle variation) owing to the uniform internal pore structure and surface chemistry of the silica support. DNA immobilization with a bis-thiosuccinimide linker provides a Raman-active internal standard for quantitative interpretation of Raman scattering results. Despite the high (30 mM) concentrations of immobilized DNA within porous silica particles, they can be used to measure nanomolar binding affinities of target molecules to DNA by equilibrating a very small number of particles with a sufficiently large volume of low-concentration solution of target molecules.

Simultaneous Multipass Resistive-Pulse Sensing and Fluorescence Imaging of Liposomes.

Simultaneous multipass resistive-pulse sensing and fluorescence imaging have been used to correlate the size and fluorescence intensity of individual E. coli lipid liposomes composed of E. coli polar lipid extracts labeled with membrane-bound 3,3-dioctadecyloxacarbocyanine (DiO) fluorescent molecules. Here, a nanopipet serves as a waveguide to direct excitation light to the resistive-pulse sensing zone at the end of the nanopipet tip. Individual DiO-labeled liposomes (>50 nm radius) were multipassed back and forth through the orifices of glass nanopipets' 110-150 nm radius via potential switching to obtain subnanometer sizing precision, while recording the fluorescence intensity of the membrane-bound DiO molecules. Fluorescence was measured as a function of liposome radius and found to be approximately proportional to the total membrane surface area. The observed relationship between liposome size and fluorescence intensity suggests that multivesicle liposomes emit greater fluorescence compared to unilamellar liposomes, consistent with all lipid membranes of the multivesicle liposomes containing DiO. Fluorescent and nonfluorescent liposomes are readily distinguished from each other in the same solution using simultaneous multipass resistive-pulse sensing and fluorescence imaging. A fluorescence "dead zone" of ∼1 µm thickness just outside of the nanopipet orifice was observed during resistive-pulse sensing, resulting in "on/off" fluorescent behavior during liposome multipassing.

Raman Scattering Reveals Ion-Dependent G-Quadruplex Formation in the 15-mer Thrombin-Binding Aptamer upon Association with α-Thrombin.

The discovery of DNA aptamers that bind biomolecular targets has enabled significant innovations in biosensing. Aptamers form secondary structures that exhibit selective high-affinity interactions with their binding partners. The binding of its target by an aptamer is often accompanied by conformational changes, and sensing by aptamers often relies on these changes to provide readout signals from extrinsic labels to detect target association. Many biosensing applications involve aptamers immobilized to surfaces, but methods to characterize conformations of immobilized aptamers and their in situ response have been lacking. To address this challenge, we have developed a structurally informative Raman spectroscopy method to determine conformations of the 15-mer thrombin-binding aptamer (TBA) immobilized on porous silica surfaces. The TBA is of interest because its binding of α-thrombin depends on the aptamer forming an antiparallel G-quadruplex, which is thought to drive signal changes that allow thrombin-binding to be detected. However, specific metal cations also stabilize the G-quadruplex conformation of the aptamer, even in the absence of its protein target. To develop a deeper understanding of the conformational response of the TBA, we utilize Raman spectroscopy to quantify the effects of the metal cations, K+ (stabilizing) and Li+ (nonstabilizing), on G-quadruplex versus unfolded populations of the TBA. In K+ or Li+ solutions, we then detect the association of α-thrombin with the immobilized aptamer, which can be observed in Raman scattering from the bound protein. The results show that the association of α-thrombin in K+ solutions produces no detectable change in aptamer conformation, which is found in the G-quadruplex form both before and after binding its target. In Li+ solutions, however, where the TBA is unfolded prior to α-thrombin association, protein binding occurs with the formation of a G-quadruplex by the aptamer.

Single-Molecule Electrical Currents Associated with Valinomycin Transport of K.

A quantitative description of ionophore-mediated ion transport is important in understanding ionophore activity in biological systems and developing ionophore applications. Herein, we describe the direct measurement of the electrical current resulting from K+ transport mediated by individual valinomycin (val) ionophores. Step fluctuations in current measured across a 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) bilayer suspended over a ∼400 nm radius glass nanopore result from dynamic partitioning of val between the bilayer and torus region, effectively increasing or decreasing the total number of val present in the membrane. In our studies, approximately 30 val are present in the membrane on average with a val entering or leaving the bilayer approximately every 50 s, allowing measurement of changes in electrical current associated with individual val. The single-molecule val(K+) transport current at 0.1 V applied potential is (1.3 ± 0.6) × 10-15 A, consistent with estimates of the transport kinetics based on large val ensembles. This methodology for analyzing single ionophore transport is general and can be applied to other carrier-type ionophores.

Nanomolar Binding of an Antibiotic Peptide to DNA Measured with Raman Spectroscopy.

Immobilization of DNA to surfaces offers a convenient means of screening the binding affinity and selectivity of potential small-molecule therapeutic candidates. Unfortunately, most surface-sensitive methods for detecting these binding interactions are not informative of the molecular structure, information that is valuable for understanding the non-covalent interactions that stabilize binding. In this work, we report a method to meet this challenge by employing confocal Raman microscopy to quantify the association of a minor-groove-binding antimicrobial peptide, netropsin, to duplex DNA hairpin sequences immobilized on the interior surfaces of porous silica particles. To assess binding selectivity, particles functionalized with different sequences of DNA were equilibrated with solutions of 100 nM netropsin, and selective association was detected based on the presence of netropsin Raman scattering in the particles. The selectivity study revealed that netropsin binds to sequences of duplex DNA having AT-rich recognition regions. To quantify binding affinities, these AT-rich DNA sequences were equilibrated with a range of netropsin solution concentrations (1 to 100 nM). Raman scattering intensities of netropsin versus solution concentration were well described by single-binding-site Langmuir isotherms with nanomolar dissociation constants, in agreement with previous isothermal calorimetry and surface plasmon resonance results. Target sequence binding was accompanied with changes in netropsin and DNA vibrational modes consistent with the hydrogen bonding between the amide groups of netropsin and adenine and thymine bases in the DNA minor groove. The binding of netropsin to a control sequence lacking the AT-rich recognition region exhibited an affinity nearly 4 orders of magnitude weaker than found for the target sequences. The Raman spectrum of netropsin interacting with this control sequence showed broad pyrrole and amide mode vibrations at frequencies similar to a free solution, revealing less constrained conformations compared with the specific binding interactions observed with AT-rich sequences.

Stable Immobilization of DNA to Silica Surfaces by Sequential Michael Addition Reactions Developed with Insights from Confocal Raman Microscopy.

The immobilization of DNA to surfaces is required for numerous biosensing applications related to the capture of target DNA sequences, proteins, or small-molecule analytes from solution. For these applications to be successful, the chemistry of DNA immobilization should be efficient, reproducible, and stable and should allow the immobilized DNA to adopt a secondary structure required for association with its respective target molecule. To develop and characterize surface immobilization chemistry to meet this challenge, it is invaluable to have a quantitative, surface-sensitive method that can report the interfacial chemistry at each step, while also being capable of determining the structure, stability, and activity of the tethered DNA product. In this work, we develop a method to immobilize DNA to silica, glass, or other oxide surfaces by carrying out the reactions in porous silica particles. Due to the high specific surface area of porous silica, the local concentrations of surface-immobilized molecules within the particle are sufficiently high that interfacial chemistry can be monitored at each step of the process with confocal Raman microscopy, providing a unique capability to assess the molecular composition, structure, yield, and surface coverage of these reactions. We employ this methodology to investigate the steps for immobilizing thiolated-DNA to thiol-modified silica surfaces through sequential Michael addition reactions with the cross-linker 1,4-phenylene-bismaleimide. A key advantage of employing a phenyl-bismaleimide over a comparable alkyl coupling reagent is the efficient conversion of the initial phenyl-thiosuccinimide to a more stable succinamic acid thioether linkage. This transformation was confirmed by in situ Raman spectroscopy measurements, and the resulting succinamic acid thioether product exhibited greater than 95% retention of surface-immobilized DNA after 12 days at room temperature in aqueous buffer. Confocal Raman microscopy was also used to assess the conformational freedom of surface-immobilized DNA by comparing the structure of a 23-mer DNA hairpin sequence under duplex-forming and unfolding conditions. We find that the immobilized DNA hairpin can undergo reversible intramolecular duplex formation based on the changes in frequencies and intensities of the phosphate backbone and base-specific vibrational modes that are informative of the hybridization state of DNA.

Inter-Leaflet Phospholipid Exchange Impacts the Ligand Density Available for Protein Binding at Supported Lipid Bilayers.

Phospholipid bilayers formed at solid-liquid interfaces have garnered interest as mimics of cell membranes to model association reactions of proteins with lipid bilayer-tethered ligands. Despite the importance of understanding how ligand density in a lipid bilayer impacts the protein-ligand association response, relating the ligand-modified lipid fraction to the absolute density of solution-accessible ligands in a lipid bilayer remains a challenge in interfacial quantitative analysis. In this work, confocal Raman microscopy is employed to quantify the association of anti-biotin IgG with a small fraction of biotinylated lipids dispersed in either gel-phase or liquid-crystalline supported lipid bilayers deposited on the interior surfaces of wide-pore silica surfaces. We examine the question of whether inter-leaflet lipid translocation contributes to the population of solution-accessible biotin ligands on the distal leaflet of a supported lipid bilayer by comparing their protein accumulation response with ligands dispersed in lipid monolayers on nitrile-derivatized silica surfaces. The binding of the antibody to biotin ligands dispersed in gel-phase bilayers exhibited an equivalent biotin coverage response as the accumulation of IgG onto gel-phase monolayers, indicating that gel-phase bilayer symmetry was preserved. This result contrasts with the ∼60% greater anti-biotin capture observed at fluid-phase bilayers compared to fluid-phase monolayers prepared at equivalent biotin fractions. This enhanced protein capture is attributed to biotin-capped lipids being transferred from the surface-associated proximal leaflet of the bilayer to the solution-exposed distal leaflet by the inter-leaflet exchange or lipid flip-flop, a facile process in fluid-phase supported lipid bilayers. The results suggest caution in interpreting the results of quantitative studies of protein binding to lipid-tethered ligands dispersed in fluid-phase phospholipid bilayers.

Raman Microscopy Investigation of GLP-1 Peptide Association with Supported Phospholipid Bilayers.

A wide range of important biological processes occur at phospholipid membranes including cell signaling, where a peptide or small molecule targets a membrane-localized receptor protein. In this work, we report the adaptation of confocal Raman microscopy to quantify populations of unlabeled glucagon-like peptide-1 (GLP-1), a membrane-active 30-residue incretin peptide, in supported phospholipid bilayers deposited on the interior surfaces of wide-pore porous silica particles. Quantification of lipid bilayer-associated peptide is achieved by measuring the Raman scattering intensity of the peptide relative to that of the supported lipid bilayer, which serves as an internal standard. The dependence of the bilayer-associated GLP-1 population on the solution concentration of GLP-1 produces an isotherm used to determine the equilibrium constant for peptide-bilayer association and the maximum peptide surface coverage. The maximum coverage of GLP-1 in the lipid bilayer was found to be only 1/5th of a full monolayer based on its hydrodynamic radius. The saturation coverage, therefore, is not limited by the size of GLP-1 but by the ability of the bilayer to accommodate the peptide at high concentrations within the bilayer. Raman spectra show that GLP-1 association with the supported bilayer is accompanied by structural changes consistent with the intercalation of the peptide into the bilayer, where the observed increase in acyl-chain order would increase the lipid density and provide free volume needed to accommodate the peptide. These results were compared with previous measurements of the association of fluorescently labeled GLP-1 with a planar-supported bilayer; the unlabeled peptide exhibits a 3-fold greater affinity for the lipid bilayer on the porous silica support, suggesting that the fluorescent label alters the GLP-1 lipid bilayer association.

Evaluating the effect of ionic strength on PNA:DNA duplex formation kinetics.

Peptide nucleic acid (PNA) is a unique synthetic nucleic acid analog that has been adopted for use in many biological applications. These applications rely upon the robust Franklin-Watson-Crick base pairing provided by PNA, particularly at lower ionic strengths. However, our understanding of the relationship between the kinetics of PNA:DNA hybridization and ionic strength is incomplete. Here we measured the kinetics of association and dissociation of PNA with DNA across a range of ionic strengths and temperatures at single-molecule resolution using total internal reflection fluorescence imaging. Unlike DNA:DNA duplexes, PNA:DNA duplexes are more stable at lower ionic strength, and we demonstrate that this is due to a higher association rate. While the dissociation rate of PNA:DNA duplexes is largely insensitive to ionic strength, it is significantly lower than that of DNA:DNA duplexes having the same number and sequence of base pairing interactions. The temperature dependence of PNA:DNA kinetic rate constants indicate a significant enthalpy barrier to duplex dissociation, and to a lesser extent, duplex formation. This investigation into the kinetics of PNA:DNA hybridization provides a framework towards better understanding and design of PNA sequences for future applications.

Confocal Raman Microscopy Enables Label-Free, Quantitative, and Structurally Informative Detection of DNA Hybridization at Porous Silica Surfaces.

Characterization of DNA at solid/liquid interfaces remains a challenge because most surface-sensitive techniques are unable to provide quantitative insight into the base content, length, or structure. Surface-enhanced Raman scattering measurements of DNA hybridization on plasmonic-metal substrates have been used to overcome small Raman-scattering cross-sections; however, surface-enhanced Raman spectroscopy measurements are not generally quantitative due to the fall-off in the scattering signal with the decay of the electric field enhancement from the surface, which also limits the length of oligonucleotides that can be investigated. In this work, we introduce an experimental methodology in which confocal Raman microscopy is used to characterize hybridization reactions of ssDNA immobilized at the solid/liquid interface of porous silica particles. By focusing the femtoliter confocal probe volume within a single porous particle, signal enhancement arises from the ∼1500-times greater surface area detected compared to a planar substrate. Because the porous support is a purely dielectric material, the scattering signal is independent of the proximity of the oligonucleotide to the silica surface. With this technique, we characterize a 19-mer capture strand and determine its hybridization efficiency with 9-mer and 16-mer target sequences from the scattering of a structurally insensitive phosphate-stretching mode. Changes in polarizability and frequency of scattering from DNA bases were observed, which are consistent with Watson-Crick base pairing. Quantification of base content from their duplex scattering intensities allows us to discriminate between hybridization of two target strands of equivalent length but with different recognition sequences. A duplex having a single-nucleotide polymorphism could be distinguished from hybridization of a fully complementary strand based on differences in base content and duplex conformation.

Hybrid-Lipid Bilayers Induce n-Alkyl-Chain Order in Reversed-Phase Chromatographic Surfaces, Impacting their Shape Selectivity for Aromatic Hydrocarbon Partitioning.

Shape selectivity is important in reversed-phase liquid chromatographic separations, where stationary phases are capable of separating geometric isomers, thereby resolving solutes based on their three-dimensional structure or shape rather than other chemical differences. Numerous chromatographic studies have been carried out using n-alkyl-chain-modified columns to understand how molecular shape affects retention. For polycyclic aromatic hydrocarbons (PAHs), it was found that planar compounds were selectively retained over nonplanar structures of comparable molecular weight on surfaces with longer n-alkyl chains, higher chain-density, or at lower temperatures, where selectivity likely arises with greater ordering of the n-alkyl chains. A limitation of these studies, however, is the small range of chain ordering that can be achieved and lack of a direct measure of the n-alkyl-chain order of the stationary phases. In this work, we employ a C18 stationary phase modified with a monolayer of phospholipid as a means of significantly varying the n-alkyl chain order. These hybrid-supported lipid bilayers, which have previously been employed as membrane-like stationary phases for measuring lipophilicity, provide a unique approach to control n-alkyl chain ordering by varying the acyl chain length and degree of unsaturation of the phospholipid modifier. The degree of alkyl-chain order of the resulting modified surfaces is determined from the ratio of trans- versus gauche-conformers, measured in situ within individual porous particles by confocal Raman microscopy. This methodology was also used to assess the affinity of these surfaces for planar versus nonplanar PAH molecules. The retention selectivity for the planar versus nonplanar compounds, thus determined, was found to vary significantly and systematically with the degree of order of the acyl/alkyl chains in the hybrid-supported lipid bilayers. The investigation also demonstrates the utility of confocal Raman microscopy for interrogating the impact of solute partitioning on stationary-phase structure within porous chromatographic particles.

Vibrational Spectroscopic Monitoring of the Gelation Transition in Nafion Ionomer Dispersions.

Infrared and Raman spectroscopy techniques were applied to investigate the drying and aggregation behavior of Nafion ionomer particles dispersed in aqueous solution. Gravimetric measurements aided the identification of gel-phase development within a series of time-resolved spectra that tracked transformations of a dispersion sample during solvent evaporation. A spectral band characteristic of ionomer sidechain end group vibration provided a quantitative probe of the dispersion-to-gel change. For sets of attenuated total reflection Fourier transform infrared (ATR FT-IR) spectra, adherence to Beer's law was attributed to the relatively constant refractive index in the frequency region of hydrated -SO3- group vibrations as fluorocarbon-rich ionomer regions aggregate in forming the structural framework of membranes and thin films. Although vibrational bands associated with ionomer backbone CF2 stretching vibrations were affected by distortion characteristic of wavelength-dependent refractive index change within a sample, the onset of band distortion signaled gel formation and coincided with ionomer mass % values just below the critical gelation point for Nafion aqueous dispersions. Similar temporal behavior was observed in confocal Raman microscopy experiments that monitored the formation of a thin ionomer film from an individual dispersion droplet. For the ATR FT-IR spectroscopy and confocal Raman microscopy techniques, intensity in the water H-O-H bending vibrational band dropped sharply at the ionomer critical gelation point and displayed a time dependence consistent with changes in water content derived from gravimetric measurements. The reported studies lay groundwork for examining the impact of dispersing solvents and above-ambient temperatures on fluorinated ionomer transformations that influence structural properties of dispersion-cast membranes and thin films.

Lacritin proteoforms prevent tear film collapse and maintain epithelial homeostasis.

Lipids in complex, protein-enriched films at air/liquid interfaces reduce surface tension. In the absence of this benefit, the light refracting and immunoprotective tear film on eyes would collapse. Premature collapse, coupled with chronic inflammation compromising visual acuity, is a hallmark of dry eye disease affecting 7 to 10% of individuals worldwide. Although collapse seems independent of mutation (unlike newborn lung alveoli), selective proteome and possible lipidome changes have been noted. These include elevated tissue transglutaminase and consequent inactivation through C-terminal cross-linking of the tear mitogen lacritin, leading to significant loss of lacritin monomer. Lacritin monomer restores homeostasis via autophagy and mitochondrial fusion and promotes basal tearing. Here, we discover that lacritin monomer C-terminal processing, inclusive of cysteine, serine, and metalloproteinase activity, generates cationic amphipathic α-helical proteoforms. Such proteoforms (using synthetic peptide surrogates) act like alveolar surfactant proteins to rapidly bind and stabilize the tear lipid layer. Immunodepletion of C- but not N-terminal proteoforms nor intact lacritin, from normal human tears promotes loss of stability akin to human dry eye tears. Stability of these and dry eye tears is rescuable with C- but not N-terminal proteoforms. Repeated topical application in rabbits reveals a proteoform turnover time of 7 to 33 h with gradual loss from human tear lipid that retains bioactivity without further processing. Thus, the processed C-terminus of lacritin that is deficient or absent in dry eye tears appears to play a key role in preventing tear film collapse and as a natural slow release mechanism that restores epithelial homeostasis.

Hybrid-Supported Bilayers Formed with Mixed-Charge Surfactants on C18-Functionalized Silica Surfaces.

Mixtures of cationic-anionic surfactants have been shown to spontaneously form ordered monolayers at hydrophobic-hydrophilic boundaries, including air-water and oil-water interfaces. In this work, confocal Raman microscopy is used to investigate the structure of hybrid-supported surfactant bilayers (HSSBs) formed by deposition of a distal leaflet of mixed cationic-anionic surfactants onto a proximal leaflet of n-alkane (C18) chains on the interior surfaces of chromatographic silica particles. The surface coverage of the two surfactants in a hybrid bilayer was determined from carbon analysis and the relative Raman scattering of their respective head-groups. Within the measurement uncertainty, the stoichiometric ratio of the two surfactants is one-to-one, equivalent to mixed-charge-surfactant monolayers at air-water and oil-water interfaces and consistent with the role of the head-group electrostatic interactions in their formation. When self-assembled on the hydrophobic surface, pairs of oppositely charged n-alkyl chain surfactants resemble a phospholipid (phosphatidylcholine) molecule, with its zwitterionic head-group and two hydrophobic acyl chain tails. Indeed, the structure of these hybrid-supported surfactant bilayers on C18-modified silica surfaces is similar to that of hybrid-supported lipid bilayers (HSLBs) on the same supports, but with denser and more-ordered n-alkyl chains. Hybrid-supported surfactant bilayers exhibit a melting phase transition (gel to liquid-crystalline phase) with structural and energetic characteristics similar to those of hybrid-supported bilayers prepared from a zwitterionic phospholipid of the same alkyl chain length. These mixed-charge surfactants on n-alkane-modified silica are stable in water over time (months), results that suggest the potential use of these hybrid bilayers for generating supported lipid-bilayer-like surfaces or for separation applications.

Probing the Mechanism of Structure-Switching Aptamer Assembly by Super-Resolution Localization of Individual DNA Molecules.

Oligonucleotide aptamers can be converted into structure-switching biosensors by incorporating a short, typically labeled oligonucleotide that is complementary to the analyte-binding region. Binding of a target analyte can disrupt the hybridization equilibrium between the aptamer and the labeled-complementary oligo producing a concentration-dependent signal for target-analyte sensing. Despite its importance in the performance of a biosensor, the mechanism of analyte-response of most structure-switching aptamers is not well understood. In this work, we employ single-molecule fluorescence imaging to investigate the competitive kinetics of association of a labeled complementary oligonucleotide and a target analyte, l-tyrosinamide (L-Tym), interacting with an L-Tym-binding aptamer. The complementary readout strand is fluorescently labeled, allowing us to measure its hybridization kinetics with individual aptamers immobilized on a surface and located with super-resolution techniques; the small-molecule L-Tym analyte is not labeled in order to avoid having an attached dye molecule impact its interactions with the aptamer. We measure the association kinetics of unlabeled L-Tym by detecting its influence on the hybridization of the labeled complementary strand. We find that L-Tym slows the association rate of the complementary strand with the aptamer but does not impact its dissociation rate, suggesting an SN1-like mechanism where the complementary strand must dissociate before L-Tym can bind. The competitive model revealed a slow association rate between L-Tym and the aptamer, producing a long-lived L-Tym-aptamer complex that blocks hybridization with the labeled complementary strand. These results provide insight about the kinetics and mechanism of analyte recognition in this structure-switching aptamer, and the methodology provides a general means of measuring the rates of unlabeled-analyte binding kinetics in aptamer-based biosensors.

Confocal Raman Microscopy Investigation of Phospholipid Monolayers Deposited on Nitrile-Modified Surfaces in Porous Silica Particles.

Phospholipid bilayers deposited on a variety of surfaces provide models for investigation of the lipid membrane structure and supports for biocompatible sensors. Hybrid-supported phospholipid bilayers (HSLBs) are stable membrane models for these investigations, typically prepared by self-assembly of a lipid monolayer over an n-alkane-modified surface. HSLBs have been prepared on n-alkyl chain-modified silica and used for lipophilicity-based chromatographic separations. The structure of these hybrid bilayers differs from vesicle membranes where the lipid head group spacing is greater due to interdigitation of the lipid acyl chains with the underlying n-alkyl chains bound to the silica surface. This interdigitated structure exhibits a broader melting transition at a higher temperature due to strong interactions between the lipid acyl chains and the immobile n-alkyl chains bound to silica. In the present work, we seek to reduce the interactions between a lipid monolayer and its supporting substrate by self-assembly of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) on porous silica functionalized with nitrile-terminated surface ligands. The frequency of Raman scattering of the surface -C≡N stretching mode at the lipid-nitrile interface is consistent with an n-alkane-like environment and insensitive to lipid head group charge, indicating that the lipid acyl chains are in contact with the surface nitrile groups. The head group area of this lipid monolayer was determined from the within-particle phospholipid concentration and silica specific surface area and found to be 54 ± 2 Å2, equivalent to the head group area of a DMPC vesicle bilayer. The structure of these nitrile-supported phospholipid monolayers was characterized below and above their melting transition by confocal Raman microscopy and found to be nearly identical to DMPC vesicle bilayers. Their narrow gel-to-fluid-phase melting transition is equivalent to dispersed DMPC vesicles, suggesting that the acyl chain structure on the nitrile support mimics the outer leaflet structure of a vesicle membrane.

Single-Molecule Kinetics Show DNA Pyrimidine Content Strongly Affects RNA:DNA and TNA:DNA Heteroduplex Dissociation Rates.

The heteroduplex hybridization thermodynamics of DNA with either RNA or TNA are greatly affected by DNA pyrimidine content, where increased DNA pyrimidine content leads to significantly increased duplex stability. Little is known, however, about the effect that purine or pyrimidine content has on the hybridization kinetics of these duplexes. In this work, single-molecule imaging is used to measure the hybridization kinetics of oligonucleotides having varying DNA pyrimidine content with complementary DNA, RNA, and TNA sequences. Results suggest that the change in duplex stability from DNA pyrimidine content (corresponding to purine content in the complementary TNA or RNA) is primarily due to changes in the dissociation rate, and not single-strand ordering or other structural changes that increase the association rate. Decreases in heteroduplex hybridization rates with pyrimidine content are similar for RNA and TNA, indicating that TNA behaves as a kinetic analogue for RNA.

Super-Resolution Imaging of Competitive Unlabeled DNA Hybridization Reveals the Influence of Fluorescent Labels on Duplex Formation and Dissociation Kinetics.

Single-molecule fluorescence imaging is a powerful method to measure reversible reaction kinetics, allowing one to monitor the bound state of individual probe molecules with fluorescently labeled targets. In the case of DNA hybridization, previous studies have shown that the presence of a fluorescent label on a target strand can exhibit significant influence on the stability of a DNA duplex that is formed. In this work, we have developed a super-resolution imaging method to measure the hybridization kinetics of unlabeled target DNA that compete with a fluorescently labeled tracer DNA strand to hybridize with an unlabeled probe DNA immobilized at a surface. The hybridization of an unlabeled DNA target cannot be detected directly, but its presence blocks the immobilized probe DNA, influencing the measured time intervals between labeled DNA hybridization events. We derive a model for competitive hybridization kinetics to extract the association and dissociation rate constants of the unlabeled species from the distribution of time intervals between hybridization events of the labeled tracer DNA at individual localized DNA probe sites. We use this methodology to determine the hybridization kinetics of a model 11-mer unlabeled target DNA strand and then determine how five different fluorescent labels attached to the same target DNA strand impact the hybridization kinetics. Compared to the unlabeled target, these labels can slow the association and dissociation rates by as much as a factor of 5. The super-resolution time-interval methodology provides a unique approach to determining fundamental (label-free) rates of DNA hybridization, revealing the significant influence of fluorescent labels on these kinetics. This measurement concept can be extended to studies of other reversible reaction systems, where kinetics of unlabeled species can be determined from their influence on the reaction of a labeled species with localized probe molecules on a surface.

Structural Elucidation of Bisulfite Adducts to Pseudouridine That Result in Deletion Signatures during Reverse Transcription of RNA.

The recent report of RBS-Seq to map simultaneously the epitranscriptomic modifications N1-methyladenosine, 5-methylcytosine, and pseudouridine (Ψ) via bisulfite treatment of RNA provides a key advance to locate these important modifications. The locations of Ψ were found by a deletion signature generated during cDNA synthesis after bisulfite treatment for which the chemical details of the reaction are poorly understood. In the present work, the bisulfite reaction with Ψ was explored to identify six isomers of bisulfite adducted to Ψ. We found four of these adducts involved the heterocyclic ring, similar to the reaction with other pyrimidines. The remaining two adducts were bonded to the 1' carbon, which resulted in opening of the ribose ring. The utilization of complementary 1D- and 2D-NMR, Raman, and electronic circular dichroism spectroscopies led to the assignment of the two ribose adducts being the constitutional isomers of an S- and an O-adduct of bisulfite to the ribose, and these are the final products after heating. A mechanistic proposal is provided to rationalize chemically the formation and stereochemistries of all six isomeric bisulfite adducts to Ψ; conversion of intermediate adducts to the two final products is proposed to involve E2, SN2', and [2,3]-sigmatropic shift reactions. Lastly, a synthetic RNA template with Ψ at a known location was treated with bisulfite, leading to a deletion signature after reverse transcription, supporting the RBS-Seq report. This classical bisulfite reaction used for epigenomic and epitranscriptomic sequencing diverges from the C nucleoside Ψ to form stable bisulfite end products that yield signatures for next-generation sequencing.

Confocal Raman Microscopy Investigation of Self-Assembly of Hybrid Phospholipid Bilayers within Individual Porous Silica Chromatographic Particles.

Hybrid-supported phospholipid bilayers are a model structure utilized for measurement of molecular interactions that typically occur at cell membranes. These membrane models are prepared by adsorption of a lipid monolayer onto a stable n-alkyl chain layer that is covalently bound to a support surface. Hybrid bilayers have been adapted to chromatographic retention measurements of lipophilicity through the assembly of a phospholipid monolayer onto n-alkane-modified silica surfaces in reversed-phase chromatographic particles. Recent Raman microscopy studies of these particles have shown that the acyl chains of the phospholipid interact with the C18-alkyl chains immobilized on the silica surface, where both lipid and C18 alkyl chains become ordered because of chain interdigitation. Confocal Raman microscopy has also been used to investigate the association of small molecules with hybrid-lipid bilayers in C18 chromatographic silica particles; the partitioning of model solutes compares favorably to that in lipid vesicle membranes with similar changes in acyl-chain structure (disordering) with solute partitioning. The present study seeks information about how these membrane-mimetic bilayers assemble onto the C18-derivatized silica surfaces of reversed-phase chromatographic silica particles. Confocal Raman microscopy is capable of interrogating the time-dependent internal composition and structure within individual silica particles. The Raman scattering data can be resolved into component Raman spectra and corresponding composition vectors that describe the time-dependent changes in intensity of the component spectra. This analysis provides insight into how the structures of both the lipid and C18 alkyl chains of hybrid lipid bilayers evolve during deposition and organization on the internal surfaces of reversed-phase chromatographic silica particles.