RESUMEN
Pulmonary delivery of mRNA via inhalation is a very attractive approach for RNA-based therapy for treatment of lung diseases. In this work, we have demonstrated successful development of an mRNA-lipid nanoparticle (LNP) dry powder product (DPP), wherein the LNPs were spray dried using hydroalcoholic solvent along with mannitol and leucine as excipients. The desired critical attributes for the DPP were accomplished by varying the excipients, lipid composition, concentration of LNPs, and weight percentage of mRNA. Leucine alone or in combination with mannitol improved the formulation by increasing the mRNA yield as well as decreasing the particle size. Intratracheal administration of the DPP in mice resulted in luciferase expression in the trachea and lungs indicating successful delivery of functional mRNA. Our results show formulation optimization of mRNA LNPs administered in the form of DPP results in an efficacious functional delivery with great promise for future development of mRNA therapeutics for lung diseases.
Asunto(s)
Enfermedades Pulmonares , Nanopartículas , Ratones , Animales , Polvos/metabolismo , ARN Mensajero , Excipientes , Leucina , Pulmón/metabolismo , Manitol , Enfermedades Pulmonares/tratamiento farmacológico , Tamaño de la PartículaRESUMEN
Lipid nanoparticles (LNPs) have shown great promise as delivery vehicles to transport messenger ribonucleic acid (mRNA) into cells and act as vaccines for infectious diseases including COVID-19 and influenza. The ionizable lipid incorporated within the LNP is known to be one of the main driving factors for potency and tolerability. Herein, we describe a novel family of ionizable lipids synthesized with a piperazine core derived from the HEPES Good buffer. These ionizable lipids have unique asymmetric tails and two dissimilar degradable moieties incorporated within the structure. Lipids tails of varying lengths, degrees of unsaturation, branching, and the inclusion of additional ester moieties were evaluated for protein expression. We observed several key lipid structure activity relationships that correlated with improved protein production in vivo, including lipid tails of 12 carbons on the ester side and the effect of carbon spacing on the disulfide arm of the lipids. Differences in LNP physical characteristics were observed for lipids containing an extra ester moiety. The LNP structure and lipid bilayer packing, visualized through Cryo-TEM, affected the amount of protein produced in vivo. In non-human primates, the Good HEPES LNPs formulated with an mRNA encoding an influenza hemagglutinin (HA) antigen successfully generated functional HA inhibition (HAI) antibody titers comparable to the industry standards MC3 and SM-102 LNPs, demonstrating their promise as a potential vaccine.
Asunto(s)
COVID-19 , Vacunas contra la Influenza , Gripe Humana , Animales , Humanos , HEPES , Membrana Dobles de Lípidos , Carbono , Ésteres , Vacunas de ARNmRESUMEN
Three probe chemistries are evaluated with respect to thermal denaturation temperatures, UV-Vis and fluorescence characteristics, recognition of complementary and mismatched DNA hairpin targets, and recognition of chromosomal DNA targets in the context of non-denaturing fluorescence in situ hybridization (nd-FISH) experiments: (i) serine-γPNAs (SγPNAs), i.e., single-stranded peptide nucleic acid (PNA) probes that are modified at the γ-position with (R)-hydroxymethyl moieties, (ii) Invader probes, i.e., DNA duplexes modified with +1 interstrand zippers of 2'-O-(pyren-1-yl)methyl-RNA monomers, a molecular arrangement that results in a violation of the neighbor exclusion principle, and (iii) double-stranded chimeric SγPNAs:Invader probes, i.e., duplexes between complementary SγPNA and Invader strands, which are destabilized due to the poor compatibility between intercalators and PNA:DNA duplexes. Invader probes resulted in efficient, highly specific, albeit comparatively slow recognition of the model DNA hairpin targets. Recognition was equally efficient and faster with the single-stranded SγPNA probes but far less specific, whilst the double-stranded chimeric SγPNAs:Invader probes displayed recognition characteristics that were intermediate of the parent probes. All three probe chemistries demonstrated the capacity to target chromosomal DNA in nd-FISH experiments, with Invader probes resulting in the most favorable and consistent characteristics (signals in >90% of interphase nuclei against a low background and no signal in negative control experiments). These probe chemistries constitute valuable additions to the molecular toolbox needed for DNA-targeting applications.
Asunto(s)
Ácidos Nucleicos de Péptidos , Serina , Hibridación Fluorescente in Situ , ADN/química , Ácidos Nucleicos de Péptidos/química , ARN/química , Sondas de ADNRESUMEN
Major efforts have been devoted to the development of constructs that enable sequence-specific recognition of double-stranded (ds) DNA, fueled by the promise for enabling tools for applications in molecular biology, diagnostics, and medicine. Towards this end, we have previously introduced Invader probes, i.e., short DNA duplexes with +1 interstrand zipper arrangements of intercalator-functionalized nucleotides. The individual strands of these labile probes display high affinity towards complementary DNA (cDNA), which drives sequence-unrestricted dsDNA-recognition. However, recognition of long targets is challenging due to the high stability of the corresponding probes. To address this, we recently introduced toehold Invader probes, i.e., Invader probes with 5'-single-stranded overhangs. The toehold architecture allows for shorter double-stranded segments to be used, which facilitates probe dissociation and dsDNA-recognition. As an extension thereof, we here report the biophysical and dsDNA-targeting properties of nicked Invader probes. In this probe architecture, the single-stranded overhangs of toehold Invader probes are hybridized to short intercalator-modified auxiliary strands, leading to formation of additional labile segments. The extra binding potential from the auxiliary strands imparts nicked Invader probes with greater dsDNA-affinity than the corresponding toehold or blunt-ended probes. Recognition of chromosomal DNA targets, refractory to recognition by conventional Invader probes, is demonstrated for nicked Invader probes in the context of non-denaturing FISH experiments, which highlights their utility as dsDNA-targeting tools.
Asunto(s)
Sondas de ADN/química , ADN/análisis , Sustancias Intercalantes/química , Oligodesoxirribonucleótidos/química , Animales , Bovinos , Línea Celular , ADN/química , Sondas de ADN/síntesis química , Sustancias Intercalantes/síntesis química , Masculino , Estructura Molecular , Hibridación de Ácido Nucleico , Oligodesoxirribonucleótidos/síntesis química , Temperatura de TransiciónRESUMEN
The development of chemically modified oligonucleotides enabling robust, sequence-unrestricted recognition of complementary chromosomal DNA regions has been an aspirational goal for scientists for many decades. While several groove-binding or strand-invading probes have been developed towards this end, most enable recognition of DNA only under limited conditions (e.g., homopurine or short mixed-sequence targets, low ionic strength, fully modified probe strands). Invader probes, i.e., DNA duplexes modified with +1 interstrand zippers of intercalator-functionalized nucleotides, are predisposed to recognize DNA targets due to their labile nature and high affinity towards complementary DNA. Here, we set out to gain further insight into the design parameters that impact the thermal denaturation properties and binding affinities of Invader probes. Towards this end, ten Invader probes were designed, and their biophysical properties and binding to model DNA hairpins and chromosomal DNA targets were studied. A Spearman's rank-order correlation analysis of various parameters was then performed. Densely modified Invader probes were found to result in efficient recognition of chromosomal DNA targets with excellent binding specificity in the context of denaturing or non-denaturing fluorescence in situ hybridization (FISH) experiments. The insight gained from the initial phase of this study informed subsequent probe optimization, which yielded constructs displaying improved recognition of chromosomal DNA targets. The findings from this study will facilitate the design of efficient Invader probes for applications in the life sciences.
Asunto(s)
ADN , Oligonucleótidos , Hibridación Fluorescente in Situ , ADN/química , Oligonucleótidos/química , Nucleótidos , ADN Complementario , Sondas de ADNRESUMEN
Development of molecules capable of binding to specific sequences of double-stranded (ds) DNA continues to attract considerable interest, as this may yield useful tools for applications in life science, biotechnology, and medicine. We have previously demonstrated sequence-unrestricted of dsDNA using Invader probes, i.e., DNA duplexes that are energetically activated through incorporation of +1 interstrand zipper arrangements of O2'-intercalator-functionalized RNA monomers. Nonetheless, recognition of extended dsDNA target regions remains challenging due to the high stability of the corresponding probes. To address this, we introduce toehold Invader probes, i.e., Invader probes with 5'-single-stranded overhangs. This design provides access to probes with shortened double-stranded segments, which facilitates probe denaturation. The single-stranded overhangs can, furthermore, be modified with affinity-enhancing modifications like LNA (locked nucleic acid) monomers to additionally increase target affinity. Herein, we report the biophysical and dsDNA-targeting properties of different toehold Invader designs and compare them to conventional Invader probes. LNA-modified toehold Invader probes display promising recognition characteristics, including greatly improved affinity to dsDNA, excellent binding specificity, and fast recognition kinetics, which enabled recognition of chromosomal DNA targets that have proven refractory to recognition by conventional Invader probes. Thus, toehold Invader probes represent another step toward a robust, oligonucleotide-based approach for sequence-unrestricted dsDNA-recognition.
Asunto(s)
OligonucleótidosRESUMEN
Invader probes, i.e., DNA duplexes modified with +1 interstrand zippers of intercalator-functionalized nucleotides like 2'-O-(pyren-1-yl)methyl-RNA monomers, are energetically activated for sequence-unrestricted recognition of double-stranded DNA (dsDNA) as they are engineered to violate the neighbor exclusion principle, while displaying high affinity towards complementary DNA sequences. The impact on Invader-mediated dsDNA-recognition upon additional modification with different non-nucleotidic bulges is studied herein, based on the hypothesis that bulge-containing Invader probes will display additionally disrupted base-stacking, more extensive denaturation, and improved dsDNA-recognition efficiency. Indeed, Invader probes featuring a single central large bulge - e.g., a nonyl (C9) monomer - display improved recognition of model DNA hairpin targets vis-à-vis conventional Invader probes (C50 values â¼1.5 µM vs. â¼3.9 µM). In contrast, probes with two opposing central bulges display less favorable binding characteristics. Remarkably, C9-modified Invader probes display perfect discrimination between fully complementary dsDNA and dsDNA differing in only one of eighteen base-pairs, underscoring the high binding specificity of double-stranded probes. Cy3-labeled bulge-containing Invader probes are demonstrated to signal the presence of gender-specific DNA sequences in fluorescent in situ hybridization assays (FISH) performed under non-denaturing conditions, highlighting one potential application of dsDNA-targeting Invader probes.
Asunto(s)
ADN/química , Sondas Moleculares/química , Pirenos/química , Animales , Bovinos , Línea Celular , Hibridación Fluorescente in Situ , Masculino , Desnaturalización de Ácido Nucleico , Termodinámica , Cromosoma YRESUMEN
Four probe chemistries are characterized and compared with respect to thermal denaturation temperatures (Tms), thermodynamic parameters associated with duplex formation, and recognition of mixed-sequence double-stranded (ds) DNA targets: (i) oligodeoxyribonucleotides (ONs) modified with Locked Nucleic Acid (LNA) monomers, (ii) MPγPNAs, i.e., single-stranded peptide nucleic acid (PNA) probes that are functionalized at the γ-position with (R)-diethylene glycol (mini-PEG, MP) moieties, (iii) Invader probes, i.e., DNA duplexes modified with +1 interstrand zipper arrangements of 2'-O-(pyren-1-yl)methyl-RNA monomers, and (iv) intercalating nucleic acids (INAs), i.e., DNA duplexes with opposing insertions of 1-O-(1-pyrenylmethyl)glycerol bulges. Invader and INA probes, which are designed to violate the nearest-neighbor exclusion principle, denature readily, whereas the individual probe strands display exceptionally high affinity towards complementary DNA (cDNA) as indicated by increases in Tms of up to 8 °C per modification. Optimized Invader and INA probes enable efficient and highly specific recognition of mixed-sequence dsDNA targets with self-complementary regions (C50 = 30-50 nM), whereas recognition is less efficient with LNA-modified ONs and fully modified MPγPNAs due to lower cDNA affinity (LNA) and a proclivity for dimerization (LNA and MPγPNA). A Cy3-labeled Invader probe is shown to stain telomeric DNA of individual chromosomes in metaphasic spreads under non-denaturing conditions with excellent specificity.
Asunto(s)
ADN/química , Sondas Moleculares/química , Oligonucleótidos/química , Ácidos Nucleicos de Péptidos/química , Animales , Bovinos , Línea Celular , Núcleo Celular/química , Sondas Moleculares/síntesis química , Estructura MolecularRESUMEN
Double-stranded oligodeoxyribonucleotides with +1 interstrand zipper arrangements of 2'-O-(pyren-1-yl)methyl-RNA monomers are additionally activated for highly specific recognition of mixed-sequence DNA targets upon incorporation of non-nucleotidic spermine bulges.
Asunto(s)
Sondas de ADN/química , ADN/genética , Espermina/química , Secuencia de Bases , Estructura MolecularRESUMEN
Over the past three decades, a wide range of pyrene-functionalized oligonucleotides have been developed and explored for potential applications in material science and nucleic acid diagnostics. Our efforts have focused on their possible use as components of Invader probes, i.e., DNA duplexes with +1 interstrand zipper arrangements of intercalator-functionalized nucleotides. We have previously demonstrated that Invader probes based on 2'-O-(pyren-1-yl)methyl-RNA monomers are energetically activated for sequence-unrestricted recognition of chromosomal DNA targets under non-denaturing conditions. As part of ongoing efforts towards delineating structure-property relationships and optimizing Invader probes, we report the synthesis and biophysical characterization of oligodeoxyribonucleotides (ONs) modified with 2'-O-(7-neo-pentylpyren-1-yl)methyl-uridine monomer V and 2'-O-(7-tert-butyl-1-methoxypyren-5-yl)methyl-uridine monomer Y. ONs modified with monomer V display increased DNA affinity (ΔTm up to +10.5 °C), while Y-modified ONs display lower DNA affinity and up to 22-fold increases in fluorescence emission upon RNA binding. Although these monomers display limited potential as building blocks for Invader probes, their photophysical properties render them of interest for diagnostic RNA-targeting applications.
Asunto(s)
Pirenos/química , ARN/química , Alquilación , Relación Dosis-Respuesta a Droga , Estructura Molecular , Oligonucleótidos/síntesis química , Oligonucleótidos/química , Relación Estructura-Actividad , TermodinámicaRESUMEN
Development of hybridization-based probes that enable recognition of specific mixed-sequence double-stranded DNA (dsDNA) regions is of considerable interest due to their potential applications in molecular biology, biotechnology, and medicine. We have recently demonstrated that nucleic acid duplexes with +1 interstrand zipper arrangements of intercalator-functionalized nucleotides such as 2'-O-(pyren-1-yl)methyl RNA monomers are inherently activated for recognition of mixed-sequence dsDNA targets, including chromosomal DNA. In the present work, we follow up on our previous structure-activity relationship studies and explore if the dsDNA-recognition efficiency of these so-called Invader probes can be improved by using larger intercalators than pyrene. Oligodeoxyribonucleotides modified with 2'-O-(triphenylen-2-yl)methyl-uridine monomer X and 2'-O-(coronen-1-yl)methyl-uridine monomer Z form extraordinarily stabilized duplexes with complementary DNA (ΔTm's per modification of up to 13 °C and 20 °C, respectively). Invader probes based on X- and Z-monomers are shown to recognize model dsDNA targets with exceptional binding specificity, but are less efficient than reference probes modified with 2'-O-(pyren-1-yl)methyl-uridine monomer Y. The insight from this study will inform further optimization of Invader probes.
Asunto(s)
Crisenos/química , ADN/química , Compuestos Policíclicos/química , ARN/química , Secuencia de Bases , ADN/genética , Secuencias Invertidas Repetidas , Desnaturalización de Ácido Nucleico , Relación Estructura-Actividad , Temperatura , Uridina/químicaRESUMEN
Oligonucleotides (ONs) modified with 2'-O-(pyren-1-yl)methylribonucleotides have been explored for a range of applications in molecular biology, nucleic acid diagnostics, and materials science for more than 25 years. The first part of this review provides an overview of synthetic strategies toward 2'-O-(pyren-1-yl)methylribonucleotides and is followed by a summary of biophysical properties of nucleic acid duplexes modified with these building blocks. Insights from structural studies are then presented to rationalize the reported properties. In the second part, applications of ONs modified with 2'-O-(pyren-1-yl)methyl-RNA monomers are reviewed, which include detection of RNA targets, discrimination of single nucleotide polymorphisms, formation of self-assembled pyrene arrays on nucleic acid scaffolds, the study of charge transfer phenomena in nucleic acid duplexes, and sequence-unrestricted recognition of double-stranded DNA. The predictable binding mode of the pyrene moiety, coupled with the microenvironment-dependent properties and synthetic feasibility, render 2'-O-(pyren-1-yl)methyl-RNA monomers as a promising class of pyrene-functionalized nucleotide building blocks for new applications in molecular biology, nucleic acid diagnostics, and materials science.
Asunto(s)
Técnicas de Diagnóstico Molecular , Pirenos/química , Ribonucleótidos/química , Ciencia de los Materiales , Biología Molecular , Pirenos/síntesis química , Ribonucleótidos/síntesis químicaRESUMEN
Development of probes capable of recognizing specific regions of chromosomal DNA has been a long-standing goal for chemical biologists. Current strategies such as PNA, triplex-forming oligonucleotides, and polyamides are subject to target choice limitations and/or necessitate non-physiological conditions, leaving a need for alternative approaches. Toward this end, we have recently introduced double-stranded oligonucleotide probes that are energetically activated for DNA recognition through modification with +1 interstrand zippers of intercalator-functionalized nucleotide monomers. Here, probes with different chemistries and architectures - varying in the position, number, and distance between the intercalator zippers - are studied with respect to hybridization energetics and DNA-targeting properties. Experiments with model DNA targets demonstrate that optimized probes enable efficient (C50 < 1 µM), fast (t50 < 3h), kinetically stable (> 24h), and single nucleotide specific recognition of DNA targets at physiologically relevant ionic strengths. Optimized probes were used in non-denaturing fluorescence in situ hybridization experiments for detection of gender-specific mixed-sequence chromosomal DNA target regions. These probes present themselves as a promising strategy for recognition of chromosomal DNA, which will enable development of new tools for applications in molecular biology, genomic engineering and nanotechnology.
RESUMEN
Development of probes that allow for sequence-unrestricted recognition of double-stranded DNA (dsDNA) continues to attract much attention due to the prospect for molecular tools that enable detection, regulation, and manipulation of genes. We have recently introduced so-called Invader probes as alternatives to more established approaches such as triplex-forming oligonucleotides, peptide nucleic acids and polyamides. These short DNA duplexes are activated for dsDNA recognition by installment of +1 interstrand zippers of intercalator-functionalized nucleotides such as 2'-N-(pyren-1-yl)methyl-2'-N-methyl-2'-aminouridine and 2'-O-(pyren-1-yl)methyluridine, which results in violation of the nearest neighbor exclusion principle and duplex destabilization. The individual probes strands have high affinity toward complementary DNA strands, which generates the driving force for recognition of mixed-sequence dsDNA regions. In the present article, we characterize Invader probes that are based on phosphorothioate backbones (PS-DNA Invaders). The change from the regular phosphodiester backbone furnishes Invader probes that are much more stable to nucleolytic degradation, while displaying acceptable dsDNA-recognition efficiency. PS-DNA Invader probes therefore present themselves as interesting probes for dsDNA-targeting applications in cellular environments and living organisms.
Asunto(s)
Sondas de ADN , ADN/química , Oligonucleótidos Fosforotioatos , Sondas de ADN/síntesis química , Sondas de ADN/química , Oligonucleótidos Fosforotioatos/síntesis química , Oligonucleótidos Fosforotioatos/químicaRESUMEN
Double-stranded oligonucleotides with +1 interstrand zipper arrangements of intercalator-functionalized nucleotides are energetically activated for recognition of mixed-sequence double-stranded DNA. Incorporation of nonyl (C9) bulges at specific positions of these probes, results in more highly affine (>5-fold), faster (>4-fold) and more persistent dsDNA recognition relative to conventional Invader probes.
Asunto(s)
Sondas de ADN/química , Sondas de ADN/genética , ADN/análisis , ADN/genética , Termodinámica , Secuencia de Bases , ADN/química , Sondas de ADN/análisis , Cinética , Estructura MolecularRESUMEN
Formaldehyde is universally used to fix tissue specimens, where it forms hemiaminal and aminal adducts with biomolecules, hindering the ability to retrieve molecular information. Common methods for removing these adducts involve extended heating, which can cause extensive degradation of nucleic acids, particularly RNA. Here, we show that water-soluble bifunctional catalysts (anthranilates and phosphanilates) speed the reversal of formaldehyde adducts of mononucleotides over standard buffers. Studies with formaldehyde-treated RNA oligonucleotides show that the catalysts enhance adduct removal, restoring unmodified RNA at 37â °C even when extensively modified, while avoiding the high temperatures that promote RNA degradation. Experiments with formalin-fixed, paraffin-embedded cell samples show that the catalysis is compatible with common RNA extraction protocols, with detectable RNA yields increased by 1.5-2.4-fold using a catalyst under optimized conditions and by 7-25-fold compared with a commercial kit. Such catalytic strategies show promise for general use in reversing formaldehyde adducts in clinical specimens.
Asunto(s)
Aductos de ADN/química , ADN/química , Formaldehído/química , ARN/química , Compuestos de Anilina/química , Catálisis , ADN/metabolismo , Aductos de ADN/análisis , ARN/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Temperatura , ortoaminobenzoatos/químicaRESUMEN
The discovery of two new classes of catalysts for hydrazone and oxime formation in water at neutral pH, namely 2-aminophenols and 2-(aminomethyl)benzimidazoles, is reported. Kinetics studies in aqueous solutions at pH 7.4 revealed rate enhancements up to 7-fold greater than with classic aniline catalysis. 2-(Aminomethyl)benzimidazoles were found to be effective catalysts with otherwise challenging aryl ketone substrates.
Asunto(s)
Aminofenoles/química , Bencimidazoles/química , Hidrazonas/química , Oximas/química , Compuestos de Anilina/química , Catálisis , Concentración de Iones de Hidrógeno , Cetonas/química , Estructura MolecularRESUMEN
Despite advances with triplex-forming oligonucleotides, peptide nucleic acids, polyamides and--more recently--engineered proteins, there remains an urgent need for synthetic ligands that enable specific recognition of double-stranded (ds) DNA to accelerate studies aiming at detecting, regulating and modifying genes. Invaders, i.e., energetically activated DNA duplexes with interstrand zipper arrangements of intercalator-functionalized nucleotides, are emerging as an attractive approach toward this goal. Here, we characterize and compare Invaders based on 1-, 2- and 4-pyrenyl-functionalized O2'-alkylated uridine monomers X-Z by means of thermal denaturation experiments, optical spectroscopy, force-field simulations and recognition experiments using DNA hairpins as model targets. We demonstrate that Invaders with +1 interstrand zippers of X or Y monomers efficiently recognize mixed-sequence DNA hairpins with single nucleotide fidelity. Intercalator-mediated unwinding and activation of the double-stranded probe, coupled with extraordinary stabilization of probe-target duplexes (ΔT(m)/modification up to +14.0 °C), provides the driving force for dsDNA recognition. In contrast, Z-modified Invaders show much lower dsDNA recognition efficiency. Thus, even very conservative changes in the chemical makeup of the intercalator-functionalized nucleotides used to activate Invader duplexes, affects dsDNA-recognition efficiency of the probes, which highlights the importance of systematic structure-property studies. The insight from this study will guide future design of Invaders for applications in molecular biology and nucleic acid diagnostics.
Asunto(s)
ADN/química , Pirenos/química , ARN/química , Alquilación , Secuencias Invertidas Repetidas , Modelos Moleculares , Conformación de Ácido Nucleico , Compuestos Organofosforados/química , Estabilidad del ARN , TermodinámicaRESUMEN
Self-assembled monolayers (SAMs) of thiols of L-cysteine, 6-mercaptohexanol, 4-mercaptobenzoic acid, DL-thioctic acid and 11-(1-pyrenyl)-1-undecathiol, which have been selected for their propensity to interact with vaporized explosives, have been attached from solution onto gold decorated ZnO-coated nanosprings. X-ray and ultraviolet photoelectron spectroscopies (XPS and UPS) have been used to investigate the surface electronic structure of the SAMs coated nanosprings. On the basis of XPS analysis, it has been determined that the packing densities of L-cysteine, 6-mercaptohexanol, 4-mercaptobenzoic acid, DL-thioctic acid and 11-(1-pyrenyl)-1-undecathiol on gold (zinc oxide) are 5.42 × 10(14) (2.83 × 10(14)), 3.26 × 10(14) (2.54 × 10(14)), 9.50 × 10(13), 2.55 × 10(14) (1.12 × 10(14)), and 5.23 × 10(13) molecules/cm(2), respectively. A single S 2p core level doublet is observed for 4-mercaptobenzoic acid and 11-(1-pyrenyl)-1-undecathiol, which is assigned to the S-Au bond. The S 2p core level for L-cysteine, 6-mercaptohexanol, and DL-thioctic acid consist of two doublets, where one is S-Au bond and the other is the S-Zn bond. Analysis of the C/S ratios agrees well with the stoichiometry of the respective thiols. UPS analysis shows that the hybridization of S 3p states and Au d-bands produces antibonding and bonding states, above and below the Au d-bands, which is characteristic of molecular chemisorption on Au nanoparticles. Gas sensors were constructed with thiolated nanosprings and their responsiveness to ammonium nitrate at 100-150 °C was tested. Nanosprings sensors functionalized with 4-mercaptobenzoic acid and 6-mercaptohexanol showed the strongest responses by a factor of 4 to 5 relative to the less responsive thiols. The response to ammonium nitrate can be correlated to the packing density and ordering of the SAMs.