Transfer Ribonucleic Acid Research Articles

Mitochondrial Dysfunctions Contribute to Hypertrophic Cardiomyopathy in Patient iPSC-Derived Cardiomyocytes with MT-RNR2 Mutation.

SummaryHypertrophic cardiomyopathy (HCM) is the most common cause of sudden cardiac death in young individuals. A potential role of mtDNA mutations in HCM is known. However, the underlying molecular mechanisms linking mtDNA mutations to HCM remain poorly understood due to lack of cell and animal models. Here, we generated induced pluripotent stem cell-derived cardiomyocytes (HCM-iPSC-CMs) from human patients in a maternally inherited HCM family who carry the m.2336T>C mutation in the mitochondrial 16S rRNA gene (MT-RNR2). The results showed that the m.2336T>C mutation resulted in mitochondrial dysfunctions and ultrastructure defects by decreasing the stability of 16S rRNA, which led to reduced levels of mitochondrial proteins. The ATP/ADP ratio and mitochondrial membrane potential were also reduced, thereby elevating the intracellular Ca2+ concentration, which was associated with numerous HCM-specific electrophysiological abnormalities. Our findings therefore provide an innovative insight into the pathogenesis of maternally inherited HCM.

Complete mitochondrial genome of Bactrocera ritsemai (Insecta: Tephritidae) and phylogenetic relationship with its congeners and related tephritid taxa

Bactrocera ritsemai is a dacine fruit fly found in Indonesia. We report here the complete mitogenome of this fruit fly from Lombok, Indonesia determined by Illumina MiSeq sequencing and its phylogenetic relationship with its congeners and related tephritid taxa. The whole mitogenome of B. ritsemai had a total length of 15,927bp, comprising 37 genes – 13 protein-coding genes (PCGs), 2 ribosomal ribonucleic acid (rRNA) and 22 transfer ribonucleic acid (tRNA) genes – and a control region (D-loop). Of the PCGs, 6 (atp6, cob, cox2, cox3, nad4, nad4l) had ATG start codon, 4 (nad2, nad3, nad5, nad6) had ATT, and one each had ATA (nad1), GTG (atp8) and TCG (cox1). Seven PCGs (atp6, atp8, cox2, cox3, nad2, nad4l, nad6) had TAA stop codon, 3 (cob, nad3, nad4) had TAG, and 3 had incomplete stop codon (cox1 – TA; nad1, nad5 – T). The TΨC-loop of tRNA was absent in trnF while trnS1 lacked the DHU-loop. Phylogenetic analysis based on 15 mt-genes (13 PCGs+2 rRNA genes) indicated B. ritsemai forming a sister group with B. umbrosa and the subgenus Bactrocera was monophyletic. The genera Bactrocera and Zeugodacus were monophyletic while the subfamilies Dacinae and Tephritinae were paraphyletic. A broader taxa sampling of the Tephritidae is needed to better elucidate the phylogenetics and systematics of the tribes and subfamilies of tephritid fruit flies.

Current Research on Non-Coding Ribonucleic Acid (RNA).

Non-coding ribonucleic acid (RNA) has without a doubt captured the interest of biomedical researchers. The ability to screen the entire human genome with high-throughput sequencing technology has greatly enhanced the identification, annotation and prediction of the functionality of non-coding RNAs. In this review, we discuss the current landscape of non-coding RNA research and quantitative analysis. Non-coding RNA will be categorized into two major groups by size: long non-coding RNAs and small RNAs. In long non-coding RNA, we discuss regular long non-coding RNA, pseudogenes and circular RNA. In small RNA, we discuss miRNA, transfer RNA, piwi-interacting RNA, small nucleolar RNA, small nuclear RNA, Y RNA, single recognition particle RNA, and 7SK RNA. We elaborate on the origin, detection method, and potential association with disease, putative functional mechanisms, and public resources for these non-coding RNAs. We aim to provide readers with a complete overview of non-coding RNAs and incite additional interest in non-coding RNA research.

%MinMax: A versatile tool for calculating and comparing synonymous codon usage and its impact on protein folding.

Most amino acids can be encoded by more than one synonymous codon, but these are rarely used with equal frequency. In many coding sequences the usage patterns of rare versus common synonymous codons is nonrandom and under selection. Moreover, synonymous substitutions that alter these patterns can have a substantial impact on the folding efficiency of the encoded protein. This has ignited broad interest in exploring synonymous codon usage patterns. For many protein chemists, biophysicists and structural biologists, the primary motivation for codon analysis is identifying and preserving usage patterns most likely to impact high-yield production of functional proteins. Here we describe the core functions and new features of %MinMax, a codon usage calculator freely available as a web-based portal and downloadable script (http://www.codons.org). %MinMax evaluates the relative usage frequencies of the synonymous codons used to encode a protein sequence of interest and compares these results to a rigorous null model. Crucially, for analyzing codon usage in common host organisms %MinMax requires only the coding sequence as input; with a user-input codon frequency table, %MinMax can be used to evaluate synonymous codon usage patterns for any coding sequence from any fully sequenced genome. %MinMax makes no assumptions regarding the impact of transfer ribonucleic acid concentrations or other molecular-level interactions on translation rates, yet its output is sufficient to predict the effects of synonymous codon substitutions on cotranslational folding mechanisms. A simple calculation included within %MinMax can be used to harmonize codon usage frequencies for heterologous gene expression.

SMORE: Synteny Modulator of Repetitive Elements.

Several families of multicopy genes, such as transfer ribonucleic acids (tRNAs) and ribosomal RNAs (rRNAs), are subject to concerted evolution, an effect that keeps sequences of paralogous genes effectively identical. Under these circumstances, it is impossible to distinguish orthologs from paralogs on the basis of sequence similarity alone. Synteny, the preservation of relative genomic locations, however, also remains informative for the disambiguation of evolutionary relationships in this situation. In this contribution, we describe an automatic pipeline for the evolutionary analysis of such cases that use genome-wide alignments as a starting point to assign orthology relationships determined by synteny. The evolution of tRNAs in primates as well as the history of the Y RNA family in vertebrates and nematodes are used to showcase the method. The pipeline is freely available.

Molecular recognition of pre-tRNA by Arabidopsis protein-only Ribonuclease P.

Protein-only ribonuclease P (PRORP) is an enzyme responsible for catalyzing the 5′ end maturation of precursor transfer ribonucleic acids (pre-tRNAs) encoded by various cellular compartments in many eukaryotes. PRORPs from plants act as single-subunit enzymes and have been used as a model system for analyzing the function of the metazoan PRORP nuclease subunit, which requires two additional proteins for efficient catalysis. There are currently few molecular details known about the PRORP–pre-tRNA complex. Here, we characterize the determinants of substrate recognition by the single subunit Arabidopsis thaliana PRORP1 and PRORP2 using kinetic and thermodynamic experiments. The salt dependence of binding affinity suggests 4–5 contacts with backbone phosphodiester bonds on substrates, including a single phosphodiester contact with the pre-tRNA 5′ leader, consistent with prior reports of short leader requirements. PRORPs contain an N-terminal pentatricopeptide repeat (PPR) domain, truncation of which results in a >30-fold decrease in substrate affinity. While most PPR-containing proteins have been implicated in single-stranded sequence-specific RNA recognition, we find that the PPR motifs of PRORPs recognize pre-tRNA substrates differently. Notably, the PPR domain residues most important for substrate binding in PRORPs do not correspond to positions involved in base recognition in other PPR proteins. Several of these residues are highly conserved in PRORPs from algae, plants, and metazoans, suggesting a conserved strategy for substrate recognition by the PRORP PPR domain. Furthermore, there is no evidence for sequence-specific interactions. This work clarifies molecular determinants of PRORP–substrate recognition and provides a new predictive model for the PRORP–substrate complex.

Abstract 1992: Changes in gene expression of stromal and epithelial cells during prostate cancer progression

Abstract Prostatic intraepithelial neoplasia (PIN) is a histologic abnormality which arises within the secretory epithelium of prostate glands, without disrupting the layer of basal cells which separates the epithelium from the surrounding stroma. PINs are generally considered to be a step towards development of prostatic adenocarcinoma (PCA). Molecular steps in the transition from normal glands to PIN to PCA are poorly understood, in part due to the difficulty of accessing and characterizing individual PINs without contamination from surrounding tissue. Furthermore, stromal cells surrounding pre-cancerous glands may play an active role in cancer development and progression, but are difficult to profile. We utilized the ultra-sensitive and quantitative TempO-Seq gene expression assay of the whole transcriptome to profile microdisections of stromal vs. epithelial cells in archival prostatectomy FFPE tissue. TempO-Seq is highly insensitive to RNA degradation (measurements of intact RNA RIN=9.1 to degraded RNA RIN=3.0 correlate with R2=0.97) and does not require RNA extraction, allowing measurement of both soluble and insoluble cross-linked RNA. This allowed deparaffinization and H&E staining of prostate sections prior to the assay, so that morphology was clearly visible and subsections could be readily extracted and profiled. Microdissections were performed on individual 5 µm FFPE sections of prostatectomies from patients with Gleason sum scores of 6, containing PIN alongside PCA. We removed an ~300 µm diameter region of stroma surrounding individual normal, PIN, and PCA glands, followed by targeted dissection of the gland epithelium itself which was lysed and assayed separately. Stroma and epithelial samples from 3 adjacent sections of the same glands were pooled together (adding up to ~0.005 mm3 tissue/replicate), and then profiled. We compared the whole transcriptome expression profile of stroma vs. epithelium at every stage of progression. Our results indicate that stroma and epithelium undergo separate and distinct changes in gene expression patterns throughout the development of PCA, most notably in genes involved in cell-cell signaling. Specifically, we found that changes in cAMP signaling in stromal cells accompanied the shift from normal glands to PIN. Furthermore, changes in androgen signaling pathways were present in stromal cells as well as in epithelium during the transition from PIN to invasive carcinoma. These results shed light on the molecular progression to PCA and demonstrate the feasibility of profiling gene expression at the level of tissue microstructure to elucidate changes within the tissue microenvironment. Since archival FFPE can be used, studies of progression can be carried out and the findings translated to diagnosis and prognosis as well as to the investigation of new therapeutic approaches. Citation Format: Milos Babic, Elliot Imler, Peter Shepard, Joanne Yeakley, Raymond Nagle, Bruce Seligmann. Changes in gene expression of stromal and epithelial cells during prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1992. doi:10.1158/1538-7445.AM2017-1992

Diverse Mechanisms of Sulfur Decoration in Bacterial tRNA and Their Cellular Functions.

Sulfur-containing transfer ribonucleic acids (tRNAs) are ubiquitous biomolecules found in all organisms that possess a variety of functions. For decades, their roles in processes such as translation, structural stability, and cellular protection have been elucidated and appreciated. These thionucleosides are found in all types of bacteria; however, their biosynthetic pathways are distinct among different groups of bacteria. Considering that many of the thio-tRNA biosynthetic enzymes are absent in Gram-positive bacteria, recent studies have addressed how sulfur trafficking is regulated in these prokaryotic species. Interestingly, a novel proposal has been given for interplay among thionucleosides and the biosynthesis of other thiocofactors, through participation of shared-enzyme intermediates, the functions of which are impacted by the availability of substrate as well as metabolic demand of thiocofactors. This review describes the occurrence of thio-modifications in bacterial tRNA and current methods for detection of these modifications that have enabled studies on the biosynthesis and functions of S-containing tRNA across bacteria. It provides insight into potential modes of regulation and potential evolutionary events responsible for divergence in sulfur metabolism among prokaryotes.

MappingPost-TranscriptionalModificationsonto TransferRibonucleicAcidSequencesbyLiquid ChromatographyTandemMassSpectrometry.

Liquid chromatography, coupled with tandem mass spectrometry, has become one of the most popular methods for the analysis of post-transcriptionally modified transfer ribonucleic acids (tRNAs). Given that the information collected using this platform is entirely determined by the mass of the analyte, it has proven to be the gold standard for accurately assigning nucleobases to the sequence. For the past few decades many labs have worked to improve the analysis, contiguous to instrumentation manufacturers developing faster and more sensitive instruments. With biological discoveries relating to ribonucleic acid happening more frequently, mass spectrometry has been invaluable in helping to understand what is happening at the molecular level. Here we present a brief overview of the methods that have been developed and refined for the analysis of modified tRNAs by liquid chromatography tandem mass spectrometry.

Stable Isotope Labeling for Improved Comparative Analysis of RNA Digests by Mass Spectrometry

Even with the advent of high throughput methods to detect modified ribonucleic acids (RNAs), mass spectrometry remains a reliable method to detect, characterize, and place post-transcriptional modifications within an RNA sequence. Here we have developed a stable isotope labeling comparative analysis of RNA digests (SIL-CARD) approach, which improves upon the original 18O/16O labeling CARD method. Like the original, SIL-CARD allows sequence or modification information from a previously uncharacterized in vivo RNA sample to be obtained by direct comparison with a reference RNA, the sequence of which is known. This reference is in vitro transcribed using a 13C/15N isotopically enriched nucleoside triphosphate (NTP). The two RNAs are digested with an endonuclease, the specificity of which matches the labeled NTP used for transcription. As proof of concept, several transfer RNAs (tRNAs) were characterized by SIL-CARD, where labeled guanosine triphosphate was used for the reference in vitro transcription. RNase T1 digestion products from the in vitro transcript will be 15Da higher in mass than the same digestion products from the in vivo tRNA that are unmodified, leading to a doublet in the mass spectrum. Singlets, rather than doublets, arise if a sequence variation or a post-transcriptional modification is present that results in a relative mass shift different from 15Da. Moreover, the use of the in vitro synthesized tRNA transcript allows for quantitative measurement of RNA abundance. Overall, SIL-CARD simplifies data analysis and enhances quantitative RNA modification mapping by mass spectrometry. Graphical Abstract ᅟ.

A new integrated symmetrical table for genetic codes

Degeneracy is a salient feature of genetic codes, because there are more codons than amino acids. The conventional table for genetic codes suffers from an inability of illustrating a symmetrical nature among genetic base codes. In fact, because the conventional wisdom avoids the question, there is little agreement as to whether the symmetrical nature actually even exists. A better understanding of symmetry and an appreciation for its essential role in the genetic code formation can improve our understanding of nature’s coding processes. Thus, it is worth formulating a new integrated symmetrical table for genetic codes, which is presented in this paper. It could be very useful to understand the Nobel laureate Crick’s wobble hypothesis — how one transfer ribonucleic acid can recognize two or more synonymous codons, which is an unsolved fundamental question in biological science.

Pseudouridine in the Anticodon of Escherichia coli tRNATyr(QΨA) Is Catalyzed by the Dual Specificity Enzyme RluF

Pseudouridine is found in almost all cellular ribonucleic acids (RNAs). Of the multiple characteristics attributed to pseudouridine, making messenger RNAs (mRNAs) highly translatable and non-immunogenic is one such feature that directly implicates this modification in protein synthesis. We report the existence of pseudouridine in the anticodon of Escherichia coli tyrosine transfer RNAs (tRNAs) at position 35. Pseudouridine was verified by multiple detection methods, which include pseudouridine-specific chemical derivatization and gas phase dissociation of RNA during liquid chromatography tandem mass spectrometry (LC-MS/MS). Analysis of total tRNA isolated from E. coli pseudouridine synthase knock-out mutants identified RluF as the enzyme responsible for this modification. Furthermore, the absence of this modification compromises the translational ability of a luciferase reporter gene coding sequence when it is preceded by multiple tyrosine codons. This effect has implications for the translation of mRNAs that are rich in tyrosine codons in bacterial expression systems.

Optical Kerr effect of tRNA solution induced by femtosecond laser pulses

The optical Kerr effect (OKE) in a transfer ribonucleic acid (tRNA) solution induced by femtosecond pulses of linearly polarized pump light (λi=800nm) and sounded by probe light (λp=800nm) was studied. The measurements were performed to find nonlinear optical parameters describing a single molecule (molecular Kerr constant K, mean nonlinear third order optical polarizability cpi) and to compare them with our previous OKE results obtained in ns and ps time range. The OKE experiment has proven to be an efficient method to obtain the nonlinear parameters of single molecules in solution, which reflects dynamic structure changes.

RNA therapeutics: RNAi and antisense mechanisms and clinical applications.

RNA therapeutics refers to the use of oligonucleotides to target primarily ribonucleic acids (RNA) for therapeutic efforts or in research studies to elucidate functions of genes. Oligonucleotides are distinct from other pharmacological modalities, such as small molecules and antibodies that target mainly proteins, due to their mechanisms of action and chemical properties. Nucleic acids come in two forms: deoxyribonucleic acids (DNA) and ribonucleic acids (RNA). Although DNA is more stable, RNA offers more structural variety ranging from messenger RNA (mRNA) that codes for protein to non-coding RNAs, microRNA (miRNA), transfer RNA (tRNA), short interfering RNAs (siRNAs), ribosomal RNA (rRNA), and long-noncoding RNAs (lncRNAs). As our understanding of the wide variety of RNAs deepens, researchers have sought to target RNA since >80% of the genome is estimated to be transcribed. These transcripts include non-coding RNAs such as miRNAs and siRNAs that function in gene regulation by playing key roles in the transfer of genetic information from DNA to protein, the final product of the central dogma in biology1. Currently there are two main approaches used to target RNA: double stranded RNA-mediated interference (RNAi) and antisense oligonucleotides (ASO). Both approaches are currently in clinical trials for targeting of RNAs involved in various diseases, such as cancer and neurodegeneration. In fact, ASOs targeting spinal muscular atrophy and amyotrophic lateral sclerosis have shown positive results in clinical trials2. Advantages of ASOs include higher affinity due to the development of chemical modifications that increase affinity, selectivity while decreasing toxicity due to off-target effects. This review will highlight the major therapeutic approaches of RNA medicine currently being applied with a focus on RNAi and ASOs.

SIMS of transfer ribonucleic acid molecules encapsulated between free-standing graphene sheets.

In this study, the authors used cluster-secondary ion mass spectrometry method to investigate the preserved transfer ribonucleic acid (tRNA) encapsulated between two free-standing graphene sheets. Single impacts of 50 keV C60 (2+) projectiles generated the emission of tRNA fragment ions in the transmission direction for mass selection and detection in a time-of-flight mass spectrometer. Ribonucleic acid (RNA) is extremely unstable and prone to rapid enzymatic degradation by ribonucleases. Employing graphene to isolate RNA from the environment, the authors prevent the aforementioned process. Encapsulation was achieved by drop casting a solution of tRNA, prepared using deuterated water, onto one graphene sheet and covering it with another. The event-by-event bombardment/detection mode allowed us to use colocalization analysis method to characterize the tRNA and its immediate environment. The authors found that upon drying, tRNA agglomerated into nanostructures ∼60 nm in diameter via formation and subsequent drying of aqua cells. The tRNA nanoagglomerates had a density of ∼42 structures per μm(2) with coverage of ∼12% of the surface area. In addition, trace amounts of water remained mostly around the tRNA nanoagglomerates, probably in the form of hydration.

Single TRAM domain RNA-binding proteins in Archaea: functional insight from Ctr3 from the Antarctic methanogen Methanococcoides burtonii.

TRAM domain proteins present in Archaea and Bacteria have a β-barrel shape with anti-parallel β-sheets that form a nucleic acid binding surface; a structure also present in cold shock proteins (Csps). Aside from protein structures, experimental data defining the function of TRAM domains is lacking. Here, we explore the possible functional properties of a single TRAM domain protein, Ctr3 (cold-responsive TRAM domain protein 3) from the Antarctic archaeon Methanococcoides burtonii that has increased abundance during low temperature growth. Ribonucleic acid (RNA) bound by Ctr3 in vitro was determined using RNA-seq. Ctr3-bound M. burtonii RNA with a preference for transfer (t)RNA and 5S ribosomal RNA, and a potential binding motif was identified. In tRNA, the motif represented the C loop; a region that is conserved in tRNA from all domains of life and appears to be solvent exposed, potentially providing access for Ctr3 to bind. Ctr3 and Csps are structurally similar and are both inferred to function in low temperature translation. The broad representation of single TRAM domain proteins within Archaea compared with their apparent absence in Bacteria, and scarcity of Csps in Archaea but prevalence in Bacteria, suggests they represent distinct evolutionary lineages of functionally equivalent RNA-binding proteins.

Long Noncoding RNAs in Metabolic Syndrome Related Disorders.

Ribonucleic acids (RNAs) are very complex and their all functions have yet to be fully clarified. Noncoding genes (noncoding RNA, sequences, and pseudogenes) comprise 67% of all genes and they are represented by housekeeping noncoding RNAs (transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), and small nucleolar RNA (snoRNA)) that are engaged in basic cellular processes and by regulatory noncoding RNA (short and long noncoding RNA (ncRNA)) that are important for gene expression/transcript stability. In this review, we summarize data concerning the significance of long noncoding RNAs (lncRNAs) in metabolic syndrome related disorders, focusing on adipose tissue and pancreatic islands.

Fatigue in Primary Sjögren's Syndrome: Clinical, Laboratory, Psychometric, and Biologic Associations.

To identify independent contributors of fatigue in primary Sjögren's syndrome (SS) patients, taking into account clinical, laboratory, and psychological features, and to explore the potential role of interferon (IFN)-induced gene indoleamine 2,3-dioxygenase (IDO-1), anti-21-hydroxylase (anti-21[OH]) antibodies, and soluble BAFF. Detailed clinical and laboratory characteristics were recorded for 106 primary SS patients. The Functional Assessment of Chronic Illness Therapy-Fatigue, Zung Depression Scale, State-Trait Anxiety Inventory, Eysenck Personality Questionnaire Scale, and Athens Insomnia Scale were adopted to assess fatigue, depression, anxiety, and sleep disturbances, respectively. Peripheral whole blood expression levels of IDO-1, as well as type I and II IFN-induced genes were calculated using quantitative reverse transcriptase-polymerase chain reaction. Serum anti-21(OH) antibodies and soluble BAFF levels were determined by a radioimmunoassay and an enzyme-linked immunosorbent assay, respectively. Univariate and multivariate models were performed to identify determinants of fatigue. Fatigue was detected in 32 of 106 (30.2%) primary SS patients. In univariate analysis, fatigue was associated with arthralgias/myalgias, fibromyalgia hydroxychloroquine therapy, both state and trait anxiety scores, depression, and neuroticism, as well as impaired sleep patterns. Multivariate analysis revealed neuroticism (odds ratio [OR] 6.9, [95% confidence interval (95% CI) 1.7-28.0]), depression (OR 3.0 [95% CI 0.8-11.0]), and fibromyalgia (OR 5.5 [95% CI 1.1-27.7]) as independent fatigue contributors. Soluble BAFF levels, anti-21(OH) autoantibodies, and IDO-1 messenger RNA expression did not significantly differ between fatigued and nonfatigued primary SS patients. Depression, neuroticism, and fibromyalgia play a major role in primary SS-associated fatigue and should be addressed in clinical practice, with active collaboration between rheumatologists and mental health professionals. Further studies are warranted in order to explore underlying pathophysiologic pathways that might explain fatigue in the setting of primary SS.

Genome-wide screen uncovers novel pathways for tRNA processing and nuclear-cytoplasmic dynamics.

Transfer ribonucleic acids (tRNAs) are essential for protein synthesis. However, key gene products involved in tRNA biogenesis and subcellular movement remain to be discovered. We conducted the first comprehensive unbiased analysis of the role of nearly an entire proteome in tRNA biology and describe 162 novel and 12 previously known Saccharomyces cerevisiae gene products that function in tRNA processing, turnover, and subcellular movement. tRNA nuclear export is of particular interest because it is essential, but the known tRNA exporters (Los1 [exportin-t] and Msn5 [exportin-5]) are unessential. We report that mutations of CRM1 (Exportin-1), MEX67/MTR2 (TAP/p15), and five nucleoporins cause accumulation of unspliced tRNA, a hallmark of defective tRNA nuclear export. CRM1 mutation genetically interacts with los1Δ and causes altered tRNA nuclear-cytoplasmic distribution. The data implicate roles for the protein and mRNA nuclear export machineries in tRNA nuclear export. Mutations of genes encoding actin cytoskeleton components and mitochondrial outer membrane proteins also cause accumulation of unspliced tRNA, likely due to defective splicing on mitochondria. Additional gene products, such as chromatin modification enzymes, have unanticipated effects on pre-tRNA end processing. Thus, this genome-wide screen uncovered putative novel pathways for tRNA nuclear export and extensive links between tRNA biology and other aspects of cell physiology.

Impact of size, secondary structure, and counterions on the binding of small ribonucleic acids to layered double hydroxide nanoparticles.

Use of ribonucleic acid (RNA) interference to regulate protein expression has become an important research topic and gene therapy tool, and therefore, finding suitable vehicles for delivery of small RNAs into cells is of crucial importance. Layered double metal hydroxides such as hydrotalcite (HT) have shown great promise as nonviral vectors for transport of deoxyribose nucleic acid (DNA), proteins, and drugs into cells, but the adsorption of RNAs to these materials has been little explored. In this study, the binding of small RNAs with different lengths and levels of secondary structure to HT nanoparticles has been analyzed and compared to results obtained with small DNAs in concurrent experiments. Initial experiments established the spectrophotometric properties of HT in aqueous solutions and determined that HT particles could be readily sedimented with near 100% efficiencies. Use of RNA+HT cosedimentation experiments as well as electrophoretic mobility shift assays demonstrated strong adsorption of RNA 25mers to HT, with twofold greater binding of single-stranded RNAs relative to double-stranded molecules. Strong affinities were also observed with ssRNA and dsRNA 54mers and with more complex transfer RNA molecules. Competition binding and RNA displacement experiments indicated that RNA-HT associations were strong and were only modestly affected by the presence of high concentrations of inorganic anions.