Uncovering potential interspecies signaling factors in plant-derived mixed microbial culture.

Microbes use signaling factors for intraspecies and interspecies communications. While many intraspecies signaling factors have been found and characterized, discovery of factors for interspecies communication is lagging behind. To facilitate the discovery of such factors, we explored the potential of a mixed microbial culture (MMC) derived from wheatgrass, in which heterogeneity of this microbial community might elicit signaling factors for interspecies communication. The stability of Wheatgrass MMC in terms of community structure and metabolic output was first characterized by 16S ribosomal RNA amplicon sequencing and liquid chromatography/mass spectrometry (LC/MS), respectively. In addition, detailed MS analyses led to the identification of 12-hydroxystearic acid (12-HSA) as one of the major metabolites produced by Wheatgrass MMC. Stereochemical analysis revealed that Wheatgrass MMC produces mostly the (R)-isomer, although a small amount of the (S)-isomer was also observed. Furthermore, 12-HSA was found to modulate planktonic growth and biofilm formation of various marine bacterial strains. The current study suggests that naturally derived MMCs could serve as a simple and reproducible platform to discover potential signaling factors for interspecies communication. In addition, the study indicates that hydroxylated long-chain fatty acids, such as 12-HSA, may constitute a new class of interspecies signaling factors.

A general LC-MS-based RNA sequencing method for direct analysis of multiple-base modifications in RNA mixtures.

A complete understanding of the structural and functional potential of RNA requires understanding of chemical modifications and non-canonical bases; this in turn requires advances in current sequencing methods to be able to sequence not only canonical ribonucleotides, but at the same time directly sequence these non-standard moieties. Here, we present the first direct and modification type-independent RNA sequencing method via introduction of a 2-dimensional hydrophobic end-labeling strategy into traditional mass spectrometry-based sequencing (2D HELS MS Seq) to allow de novo sequencing of RNA mixtures and enhance sample usage efficiency. Our method can directly read out the complete sequence, while identifying, locating, and quantifying base modifications accurately in both single and mixed RNA samples containing multiple different modifications at single-base resolution. Our method can also quantify stoichiometry/percentage of modified RNA versus its canonical counterpart RNA, simulating a real biological sample where modifications exist but may not be 100% at a particular site in the RNA. This method is a critical step towards fully sequencing real complex cellular RNA samples of any type and containing any modification type and can also be used in the quality control of modified therapeutic RNAs.

Needle in a haystack: Antibacterial activity-guided fractionation of a potato wound tissue extract.

Erwinia carotovora is a major cause of potato tuber infection, which results in disastrous failures of this important food crop. There is currently no effective antibiotic treatment against E. carotovora. Recently we reported antibacterial assays of wound tissue extracts from four potato cultivars that exhibit a gradient of russeting character, finding the highest potency against this pathogen for a polar extract from the tissue formed immediately after wounding by an Atlantic cultivar. In the current investigation, antibacterial activity-guided fractions of this extract were analyzed by liquid chromatography-mass spectrometry (LC-MS) utilizing a quadrupole-time-of-flight (QTOF) mass spectrometer. The most active chemical compounds identified against E. carotovora were: 6-O-nonyl glucitol, Lyratol C, n-[2-(4-Hydroxyphenyl)] ethyldecanamide, α-chaconine and α-solanine. Interactions among the three compounds, ferulic acid, feruloyl putrescine, and α-chaconine, representing metabolite classes upregulated during initial stages of wound healing, were also evaluated, offering possible explanations for the burst in antibacterial activity after tuber wounding and a chemical rationale for the temporal resistance phenomenon.

Solvent Modulation of Aromatic Substituent Effects in Molecular Balances Controlled by CH-π Interactions.

CH-π aromatic interactions are ubiquitous in nature and are capable of regulating important chemical and biochemical processes. Solvation and aromatic substituent effects are known to perturb the CH-π aromatic interactions. However, the nature by which the two factors influence one another is relatively unexplored. Here we demonstrate experimentally that there is a quantitative correlation between substituent effects in CH-π interactions and the hydrogen-bond acceptor constants of the solvating molecule. The CH-π interaction energies were measured by the conformational study of a series of aryl-substituted molecular balances in which the conformational preferences depended on the relative strengths of the methyl and aryl CH-π interactions in the folded and unfolded states, respectively. Due to the favorable methyl-aromatic interactions, the balances were found to exist predominantly in the folded state. The observed substituent effect in the conformational preferences of the balances was controlled by the explicit solvation/desolvation of the aryl proton. The interpretation of the conformational free energy as a function of substituents and solvation using Hunter's solvation model revealed that a linear relationship exists between the sensitivity of aromatic substituent effects (i.e., the ρ values derived from Hammett plots) and the hydrogen-bond acceptor propensity (ßs) of the solvent molecule: ρ = 0.06ßs - 0.04.

How to Make a Mimic? Brood Parasitic Striped Cuckoo Eggs Match Host Shell Color but Not Pigment Concentrations.

Hosts of avian brood parasites often use visual cues to reject foreign eggs, and several lineages of brood parasites have evolved mimetic eggshell coloration and patterning to circumvent host recognition. What is the mechanism of parasitic egg color mimicry at the chemical level? Mimetic egg coloration by Common Cuckoos Cuculus canorus is achieved by depositing similar concentrations of colorful pigments into their shells as their hosts. The mechanism of parasitic egg color mimicry at the chemical level in other lineages of brood parasites remains unexplored. Here we report on the chemical basis of egg color mimicry in an evolutionarily independent, and poorly studied, host-parasite system: the Neotropical Striped Cuckoo Tapera naevia and one of its hosts, the Rufous-and-white Wren Thryophilus rufalbus. In most of South America, Striped Cuckoos lay white eggs that are identical to those of local host species. In Central America, however, Striped Cuckoos lay blue eggs that match those of the Rufous-and-white Wren, suggesting that blue egg color in these cuckoo populations is an adaptation to mimic host egg appearance. Here we confirm that Striped Cuckoo eggs are spectrally similar to those of their hosts and consistently contain the same major eggshell pigment, biliverdin. However, wren eggshells lacked protoporphyrin, which was present in the parasitic cuckoo eggshells. Furthermore, biliverdin concentrations were significantly lower in cuckoo eggshells than in host eggshells. Similarity of host-parasite eggshell appearance, therefore, need not always be paralleled by a quantitative chemical match to generate effective visual mimicry in birds.

Targetable Clinical Nanoparticles for Precision Cancer Therapy Based on Disease-Specific Molecular Inflection Points.

Novel translational approaches based on clinical modular nanoplatforms are needed in order to treat solid cancers according to their discrete molecular features. In the present study, we show that the clinical nanopharmaceutical Ferumoxytol, which consists of a glucose-based coat surrounding an iron oxide core, could identify molecular characteristics of prostate cancer, corresponding to unique phases of the disease continuum. By affixing a targeting probe for the prostate-specific membrane antigen on its surface, the nanopharmaceutical was able to assess the functional state of the androgen receptor pathway via MRI, guiding therapy and delivering it with the same clinical nanoparticle. In order to simultaneously inhibit signaling from key oncogenic pathways of more advanced forms of prostate cancer, a single-agent therapy for early stage disease to inhibit DNA replication, as well as combination therapy with two drugs co-retained within the nanopharmaceutical's polymeric coating, were tested and resulted in complete tumor ablation. Recalcitrant and terminal forms of the disease were effectively treated with a nanopharmaceutical delivering a combination that upregulates endoplasmic reticulum stress and inhibits metastasis, thereby showing that this multifunctional nanoplatform can be used in the clinic for patient stratification, as well as precision treatment based on the individual's unique disease features.

Cationic CHπ interactions as a function of solvation.

The energy (ΔG) of a cationic CHπ interaction was measured experimentally through the conformational studies of new molecular torsion balances using proton NMR spectroscopy. Each of the molecular balance adopted folded and unfolded conformations for which the ratio of the conformational equilibrium (i.e., folded/unfolded ratio) provided a quantitative measure of the ΔG as a function of solvation. An excellent linear solvation energy relationship between the ΔG values and the Hunter's solvent hydrogen-bond parameters (αs and ßs) revealed that electrostatic interaction is the physical origin of the observed conformational preferences in solution.

Ultrasmall dual-modality silica nanoparticle drug conjugates: Design, synthesis, and characterization.

The physicochemical design and synthesis of effective cancer-directed and particle-based nanotherapeutic imaging agents remains a challenging task. Of critical importance is the ability to demonstrate maximum delivery, retention, and treatment efficacy for platforms designed to deposit their cargo at sites of disease without attendant dose-limiting toxicity. In this work, we describe dual-modality nanoparticle drug conjugates (NDCs) which utilize protease sensitive linkers to attached drug compounds and imaging labels to a clinically translated class of ultrasmall silica nanoparticle (C' dots). We describe the synthesis and characterization of these linker-drug constructs. Linkers incorporating dipeptide enzyme substrates are attached to analogs of a prototypical epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), through a cleavable amide bond or para-aminobenzyloxycarbonyl (PABC) group. These constructs are conjugated onto C' dots leading to the desired NDCs. These NDCs exhibit fast and predictable release kinetics in the presence of model proteases, and are stable in various biological media. Finally, in vitro assays show NDCs to be highly active in reducing phosphorylated EGFR levels in H1650 cells, a human tumor-derived cell line. The data suggests that NDCs exhibit desirable properties that warrant further development toward oncological therapy.

De novo structure prediction and experimental characterization of folded peptoid oligomers.

Peptoid molecules are biomimetic oligomers that can fold into unique three-dimensional structures. As part of an effort to advance computational design of folded oligomers, we present blind-structure predictions for three peptoid sequences using a combination of Replica Exchange Molecular Dynamics (REMD) simulation and Quantum Mechanical refinement. We correctly predicted the structure of a N-aryl peptoid trimer to within 0.2 Å rmsd-backbone and a cyclic peptoid nonamer to an accuracy of 1.0 Å rmsd-backbone. X-ray crystallographic structures are presented for a linear N-alkyl peptoid trimer and for the cyclic peptoid nonamer. The peptoid macrocycle structure features a combination of cis and trans backbone amides, significant nonplanarity of the amide bonds, and a unique "basket" arrangement of (S)-N(1-phenylethyl) side chains encompassing a bound ethanol molecule. REMD simulations of the peptoid trimers reveal that well folded peptoids can exhibit funnel-like conformational free energy landscapes similar to those for ordered polypeptides. These results indicate that physical modeling can successfully perform de novo structure prediction for small peptoid molecules.

Evaluation of glycodendron and synthetically modified dextran clearing agents for multistep targeting of radioisotopes for molecular imaging and radioimmunotherapy.

A series of N-acetylgalactosamine-dendrons (NAG-dendrons) and dextrans bearing biotin moieties were compared for their ability to complex with and sequester circulating bispecific antitumor antibody streptavidin fusion protein (scFv4-SA) in vivo, to improve tumor-to-normal tissue concentration ratios for multistep targeted (MST) radioimmunotherapy and diagnosis. Specifically, a total of five NAG-dendrons employing a common synthetic scaffold structure containing 4, 8, 16, or 32 carbohydrate residues and a single biotin moiety were prepared (NAGB), and for comparative purposes, a biotinylated-dextran with an average molecular weight of 500 kD was synthesized from amino-dextran (DEXB). One of the NAGB compounds, CA16, has been investigated in humans; our aim was to determine if other NAGB analogues (e.g., CA8 or CA4) were bioequivalent to CA16 and/or better suited as MST reagents. In vivo studies included dynamic positron-emission tomography (PET) imaging of (124)I-labeled-scFv4-SA clearance and dual-label biodistribution studies following MST directed at subcutaneous (s.c.) human colon adenocarcinoma xenografts in mice. The MST protocol consists of three injections: first, a scFv4-SA specific for an antitumor-associated glycoprotein (TAG-72); second, CA16 or other clearing agent; and third, radiolabeled biotin. We observed using PET imaging of the (124)I-labeled-scFv4-SA clearance that the spatial arrangement of ligands conjugated to NAG (i.e., biotin linked with an extended spacer, referred to herein as long-chain (LC)) can impact the binding to the antibody in circulation and subsequent liver uptake of the NAG-antibody complex. Also, NAGB CA32-LC or CA16-LC can be utilized during MST to achieve comparable tumor-to-blood ratios and absolute tumor uptake seen previously with CA16. Finally, DEXB was equally effective as NAGB CA32-LC at lowering scFv4-SA in circulation, but at the expense of reducing absolute tumor uptake of radiolabeled biotin.

Quantifying macrocyclization-induced strain utilizing N-phenylimides as conformational reporters.

Incorporating an N-phenylimide unit into macrocycles enabled measurements of macrocyclization strains by comparing the N-phenylimide's conformational changes to similar units attached to a linear-chain control. Systems of larger macrocycles displayed negligible macrocyclization strain, while smaller macrocycles demonstrated proportionate effects, emphasizing the use of N-phenylimides as conformational reporters of macrocyclization strain.

On the mechanism of visible-light sensitized photosulfoxidation of toluidine blue O.

We report on the formation of toluidine blue O (TBO) sulfoxide by a self-sensitized photooxidation of TBO. Here, the photosulfoxidation process was studied by mass spectrometry (MS) and discussed in the context of photodemethylation processes which both contribute to TBO consumption over time. Analysis of solvent effects with D2O, H2O, and CH3CN along with product yields and MS fragmentation patterns provided mechanistic insight into TBO sulfoxide's formation. The formation of TBO sulfoxide is minor and detectable up to 12% after irradiation of 3 h. The photosulfoxidation process is dependent on oxygen wherein instead of a type II (singlet oxygen, 1O2) reaction, a type I reaction involving TBO to reach the TBO sulfoxide is consistent with the results. Density functional theory results point to the formation of the TBO sulfoxide by the oxidation of TBO via transiently formed peroxyl radical or thiadioxirane intermediates. We discover that the TBO photosulfoxidation arises competitively with TBO photodemethylation with the latter leading to formaldehyde formation.

N-acetylgalactosamino dendrons as clearing agents to enhance liver targeting of model antibody-fusion protein.

Dendrimer clearing agents represent a unique class of compounds for use in multistep targeting (MST) in radioimmunotherapy and imaging. These compounds were developed to facilitate the removal of excess tumor-targeting monoclonal antibody (mAb) prior to administration of the radionuclide to minimize exposure of normal tissue to radiation. Clearing agents are designed to capture the circulating mAb, and target it to the liver for metabolism. Glycodendrons are ideally suited for MST applications as these highly branched compounds are chemically well-defined, thus advantageous over heterogeneous macromolecules. Previous studies have described glycodendron 3 as a clearing agent for use in three-step MST protocols, and early in vivo assessment of 3 showed promise. However, synthetic challenges have hampered its availability for further development. In this report we describe a new sequence of chemical steps which enables the straightforward synthesis and analytical characterization of this class of dendrons. With accessibility and analytical identification solved, we sought to evaluate both lower and higher generation dendrons for hepatocyte targeting as well as clearance of a model protein. We prepared a series of clearing agents where a single biotin is connected to glycodendrons displaying four, eight, sixteen or thirty-two α-thio-N-acetylgalactosamine (α-SGalNAc) units, resulting in compounds with molecular weights ranging from 2 to 17 kDa, respectively. These compounds were fully characterized by LCMS and NMR. We then evaluated the capacity of these agents to clear a model (131)I-labeled single chain variable fragment antibody-streptavidin ((131)I-scFv-SAv) fusion protein from blood and tissue in mice, and compared their clearing efficiencies to that of a 500 kDa dextran-biotin conjugate. Glycodendrons and dextran-biotin exhibited enhanced blood clearance of the scFv-SAv construct. Biodistribution analysis showed liver targeting/uptake of the scFv-SAv construct to be 2-fold higher for compounds 1 to 4, as well as for the 500 kDa dextran, over saline. Additionally, the data suggest the glycodendrons clear through the liver, whereas the dextran through reticuloendothelial system (RES) metabolism.

Engineered Ultrasmall Nanoparticle Drug-Immune Conjugates with "Hit and Run" Tumor Delivery to Eradicate Gastric Cancer.

Despite advances by recently approved antibody-drug conjugates in treating advanced gastric cancer patients, substantial limitations remain. Here, several key obstacles are overcome by developing a first-in-class ultrasmall (sub-8-nanometer (nm)) anti-human epidermal growth factor receptor 2 (HER2)-targeting drug-immune conjugate nanoparticle therapy. This multivalent fluorescent core-shell silica nanoparticle bears multiple anti-HER2 single-chain variable fragments (scFv), topoisomerase inhibitors, and deferoxamine moieties. Most surprisingly, drawing upon its favorable physicochemical, pharmacokinetic, clearance, and target-specific dual-modality imaging properties in a "hit and run" approach, this conjugate eradicated HER2-expressing gastric tumors without any evidence of tumor regrowth, while exhibiting a wide therapeutic index. Therapeutic response mechanisms are accompanied by the activation of functional markers, as well as pathway-specific inhibition. Results highlight the potential clinical utility of this molecularly engineered particle drug-immune conjugate and underscore the versatility of the base platform as a carrier for conjugating an array of other immune products and payloads.

Mycobacterium abscessus Mutants with a Compromised Functional Link between the Type VII ESX-3 System and an Iron Uptake Mechanism Reliant on an Unusual Mycobactin Siderophore.

The opportunistic pathogen Mycobacterium abscessus subsp. abscessus (Mab) has become an emerging public health threat due to the increasing number of Mab-associated chronic pulmonary disease cases. Treatment requires multiple drug courses and is often combined with surgical resection. Cure rates are only ~50% due to treatment failure and comorbidities. Deeper understanding of the biology of Mab is required to illuminate potential avenues for the development of better therapeutics against Mab infections. The ESX-3 type VII protein secretion system of Mab has an important role in host inflammatory and pathological responses during infection. In this work, we demonstrate a functional link between ESX-3 and an iron uptake system based on an unusual mycobactin-type siderophore (designated MBT Ab) and exploit this link to implement a large screen for transposon mutants with an impaired ESX-3. Most mutants we identified carry insertions in genes encoding predicted ESX-3 secretion machinery components or potential ESX-3 substrates. The mutants overproduce MBT Ab, a trait consistent with an iron uptake defect. Our characterization of MBT Ab revealed structural features reminiscent of nocardial mycobactin-like compounds with cytotoxicity. This finding raises the possibility that MBT Ab may play roles in pathogenesis unlinked to iron homeostasis. The mutants generated herein will facilitate research to better understand the role of ESX-3 and its interplay with the siderophore system.

Ultrasmall Nanoparticle Delivery of Doxorubicin Improves Therapeutic Index for High-Grade Glioma.

PURPOSE: Despite dramatic growth in the number of small-molecule drugs developed to treat solid tumors, durable therapeutic options to control primary central nervous system malignancies are relatively scarce. Chemotherapeutic agents that appear biologically potent in model systems have often been found to be marginally effective at best when given systemically in clinical trials. This work presents for the first time an ultrasmall (<8 nm) multimodal core-shell silica nanoparticle, Cornell prime dots (or C' dots), for the efficacious treatment of high-grade gliomas. EXPERIMENTAL DESIGN: This work presents first-in-kind renally clearable ultrasmall (<8 nm) multimodal C' dots with surface-conjugated doxorubicin (DOX) via pH-sensitive linkers for the efficacious treatment in two different clinically relevant high-grade glioma models. RESULTS: Optimal drug-per-particle ratios of as-developed nanoparticle-drug conjugates were established and used to obtain favorable pharmacokinetic profiles. The in vivo efficacy results showed significantly improved biological, therapeutic, and toxicological properties over the native drug after intravenous administration in platelet-derived growth factor-driven genetically engineered mouse model, and an EGF-expressing patient-derived xenograft (EGFR PDX) model. CONCLUSIONS: Ultrasmall C' dot-drug conjugates showed great translational potential over DOX for improving the therapeutic outcome of patients with high-grade gliomas, even without a cancer-targeting moiety.

A General LC-MS-Based Method for Direct and De Novo Sequencing of RNA Mixtures Containing both Canonical and Modified Nucleotides.

Mass spectrometry (MS)-based sequencing has advantages in direct sequencing of RNA, compared to cDNA-based RNA sequencing methods, as it is completely independent of enzymes and base complementarity errors in sample preparation. In addition, it allows for sequencing of different RNA modifications in a single study, rather than just one specific modification type per study. However, many technical challenges remain in de novo MS sequencing of RNA, making it difficult to MS sequence mixed RNAs or to differentiate isomeric modifications such as pseudouridine (Ψ) from uridine (U). Our recent study incorporates a two-dimensional hydrophobic end labeling strategy into MS-based sequencing (2D-HELS MS Seq) to systematically address the current challenges in MS sequencing of RNA, making it possible to directly and de novo sequence purified single RNA and mixed RNA containing both canonical and modified nucleotides. Here, we describe the method to sequence representative single-RNA and mixed-RNA oligonucleotides, each with a different sequence and/or containing modified nucleotides such as Ψ and 5-methylcytosine (m5C), using 2D-HELS MS Seq.

A chemical window into the impact of RNAi silencing of the StNAC103 gene in potato tuber periderms: Soluble metabolites, suberized cell walls, and antibacterial defense.

The growth and survival of terrestrial plants require control of their interactions with the environment, e.g., to defend against desiccation and microbial invasion. For major food crops, the protection conferred by the outer skins (periderm in potato) is essential to cultivation, storage, and marketing of the edible tubers and fruits. Potatoes are particularly vulnerable to bacterial infections due to their high content of water and susceptibility to mechanical wounding. Recently, both specific and conserved gene silencing (StNAC103-RNAi and StNAC103-RNAi-c, respectively) were found to increase the load of wax and aliphatic suberin depolymerization products in tuber periderm, implicating this NAC gene as a repressor of the wax and suberin biosynthetic pathways. However, an important gap in our understanding of StNAC103 silencing concerns the metabolites produced in periderm cells as antimicrobial defense agents and potential building blocks of the deposited suberin biopolymer. In the current work, we have expanded prior studies on StNAC103 silenced lines by conducting comprehensive parallel analyses to profile changes in chemical constituents and antibacterial activity. Compositional analysis of the intact suberized cell walls using solid-state 13C NMR (ssNMR) showed that NAC silencing produced an increase in the long-chain aliphatic groups deposited within the periderm cell walls. LC-MS of polar extracts revealed up-regulation of glycoalkaloids in both StNAC103-RNAi and StNAC103-RNAi-c native periderms but down-regulation of a phenolic amine in StNAC103-RNAi-c and a phenolic acid in StNAC103-RNAi native periderms. The nonpolar soluble metabolites identified using GC-MS included notably abundant long-chain alkane metabolites in both silenced samples. By coordinating the differentially accumulated soluble metabolites and the suberin depolymerization products with the ssNMR-based profiles for the periderm polymers, it was possible to obtain a holistic view of the chemical changes that result from StNAC103 gene silencing. Correspondingly, the chemical composition trends served as a backdrop to interpret trends in the chemical barrier defense function of native tuber periderms, which was found to be more robust for the nonpolar extracts.

Peptoid macrocycles: making the rounds with peptidomimetic oligomers.

Macrocyclic constraints are often employed to rigidify the conformation of flexible oligomeric systems. This approach has recently been used to organize the structure of peptoid oligomers, which are peptidomimetics composed of chemically diverse N-substituted glycine monomer units. In this review, we describe advances in the synthesis and characterization of cyclic peptoids. We evaluate how the installation of covalent constraints between the oligomer termini or side chains has been effective in defining peptoid conformations. We also discuss the potential applications for this promising family of macrocyclic peptidomimetics.

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications.

Mass spectrometry (MS)-based sequencing approaches have been shown to be useful in direct sequencing RNA without the need for a complementary DNA (cDNA) intermediate. However, such approaches are rarely applied as a de novo RNA sequencing method, but used mainly as a tool that can assist in quality assurance for confirming known sequences of purified single-stranded RNA samples. Recently, we developed a direct RNA sequencing method by integrating a 2-dimensional mass-retention time hydrophobic end-labeling strategy into MS-based sequencing (2D-HELS MS Seq). This method is capable of accurately sequencing single RNA sequences as well as mixtures containing up to 12 distinct RNA sequences. In addition to the four canonical ribonucleotides (A, C, G, and U), the method has the capacity to sequence RNA oligonucleotides containing modified nucleotides. This is possible because the modified nucleobase either has an intrinsically unique mass that can help in its identification and its location in the RNA sequence, or can be converted into a product with a unique mass. In this study, we have used RNA, incorporating two representative modified nucleotides (pseudouridine (Ψ) and 5-methylcytosine (m5C)), to illustrate the application of the method for the de novo sequencing of a single RNA oligonucleotide as well as a mixture of RNA oligonucleotides, each with a different sequence and/or modified nucleotides. The procedures and protocols described here to sequence these model RNAs will be applicable to other short RNA samples (<35 nt) when using a standard high-resolution LC-MS system, and can also be used for sequence verification of modified therapeutic RNA oligonucleotides. In the future, with the development of more robust algorithms and with better instruments, this method could allow sequencing of more complex biological samples.