Programmable siRNA pro-drugs that activate RNAi activity in response to specific cellular RNA biomarkers.

Since Paul Ehrlich's introduction of the "magic bullet" concept in 1908, drug developers have been seeking new ways to target drug activity to diseased cells while limiting effects on normal tissues. In recent years, it has been proposed that coupling riboswitches capable of detecting RNA biomarkers to small interfering RNAs (siRNAs) to create siRNA pro-drugs could selectively activate RNA interference (RNAi) activity in specific cells. However, this concept has not been achieved previously. We report here that we have accomplished this goal, validating a simple and programmable new design that functions reliably in mammalian cells. We show that these conditionally activated siRNAs (Cond-siRNAs) can switch RNAi activity against different targets between clearly distinguished OFF and ON states in response to different cellular RNA biomarkers. Notably, in a rat cardiomyocyte cell line (H9C2), one version of our construct demonstrated biologically meaningful inhibition of a heart-disease-related target gene protein phosphatase 3 catalytic subunit alpha (PPP3CA) in response to increased expression of the pathological marker atrial natriuretic peptide (NPPA) messenger RNA (mRNA). Our results demonstrate the ability of synthetic riboswitches to regulate gene expression in mammalian cells, opening a new path for development of programmable siRNA pro-drugs.

Publisher Correction: The current state and future directions of RNAi-based therapeutics.

The use of the names for patisiran has been made consistent throughout the article in line with the journal style and typographical errors have been corrected.

Author Correction: The current state and future directions of RNAi-based therapeutics.

Errors in the alignment and structure of the siRNN and in the structure of the sisiRNA in the original version of Fig. 3 have been corrected.

The current state and future directions of RNAi-based therapeutics.

The RNA interference (RNAi) pathway regulates mRNA stability and translation in nearly all human cells. Small double-stranded RNA molecules can efficiently trigger RNAi silencing of specific genes, but their therapeutic use has faced numerous challenges involving safety and potency. However, August 2018 marked a new era for the field, with the US Food and Drug Administration approving patisiran, the first RNAi-based drug. In this Review, we discuss key advances in the design and development of RNAi drugs leading up to this landmark achievement, the state of the current clinical pipeline and prospects for future advances, including novel RNAi pathway agents utilizing mechanisms beyond post-translational RNAi silencing.

The PX Motif of DNA Binds Specifically to Escherichia coli DNA Polymerase I.

The PX motif of DNA is a four-stranded structure in which two parallel juxtaposed double-helical domains are fused by crossovers at every point where the strands approach each other. Consequently, its twist and writhe are approximately half of those of conventional DNA. This property has been shown to relax supercoiled plasmid DNA under circumstances in which head-to-head homology exists within the plasmid; the homology can be either complete homology or every-other-half-turn homology, known as PX homology. It is clearly of interest to establish whether the cell contains proteins that interact with this unusual and possibly functional motif. We have examined Escherichia coli extracts to seek such a protein. We find by gel mobility studies that the PX motif is apparently bound by a cellular component. Fractionation of this binding activity reveals that the component is DNA polymerase I (Pol I). Although the PX motif binds to Pol I, we find that PX-DNA is not able to serve as a substrate for the extension of a shortened strand. We cannot say at this time whether the binding is a coincidence or whether it represents an activity of Pol I that is currently unknown. We have modeled the interaction of Pol I and PX-DNA using symmetry considerations and molecular dynamics.

Prevalence and risk factors for cardiovascular disease among chronic kidney disease patients: results from the Chinese cohort study of chronic kidney disease (C-STRIDE).

BACKGROUND: Although a high incidence of cardiovascular disease (CVD) is observed among chronic kidney disease (CKD) patients in developed countries, limited information is available about CVD prevalence and risk factors in the Chinese CKD population. The Chinese Cohort of Chronic Kidney Disease (C-STRIDE) was established to investigate the prevalence and risk factors of CVD among Chinese CKD patients. METHODS: Participants with stage 1-4 CKD (18-74 years of age) were recruited at 39 clinical centers located in 28 cities from 22 provinces of China. At entry, the socio-demographic status, medical history, anthropometric measurements and lifestyle behaviors were documented, and blood and urine samples were collected. Estimated glomerular filtration rate (eGFR) was calculated by the CKD-EPI creatinine equation. CVD diagnosis was based on patient self-report and review of medical records by trained staff. A multivariable logistic regression model was used to estimate the association between risk factors and CVD. RESULTS: Three thousand four hundred fifty-nine Chinese patients with pre-stage 5 CKD were enrolled, and 3168 finished all required examinations and were included in the study. In total, 40.8% of the cohort was female, with a mean age of 48.21 ± 13.70 years. The prevalence of CVD was 9.8%, and in 69.1% of the CVD cases cerebrovascular disease was observed. Multivariable analysis showed that increasing age, lower eGFR, presence of hypertension, abdominal aorta calcification and diabetes were associated with comorbid CVD among CKD patients. The odds ratios and 95% confidence intervals for these risk factors were 3.78 (2.55-5.59) for age 45-64 years and 6.07 (3.89-9.47) for age ≥65 years compared with age <45 years; 2.07 (1.28-3.34) for CKD stage 3a, 1.66 (1.00-2.62) for stage 3b, and 2.74 (1.72-4.36) for stage 4 compared with stages 1 and 2; 2.57 (1.50-4.41) for hypertension, 1.82 (1.23-2.70) for abdominal aorta calcification, and 1.70 (1.30-2.23) for diabetes, respectively. CONCLUSIONS: We reported the CVD prevalence among a CKD patient cohort and found age, hypertension, diabetes, abdominal aorta calcification and lower eGFR were independently associated with higher CVD prevalence. Prospective follow-up and longitudinal evaluations of CVD risk among CKD patients are warranted.

DNA-Directed Self-Assembly of Highly Ordered and Dense Single-Walled Carbon Nanotube Arrays.

Single-walled carbon nanotubes (SWNT or CNT) have unique and well-known high-performance material properties that can enable revolutionary increases in the performance of electronic devices and architectures. However, fabrication of large-scale SWNT-based ICs is an enormously challenging, unsolved problem, and self-assembly is likely needed for critical steps. Over the past several years, methods have been introduced to created ordered carbon nanotube structures using DNA guided self-assembly. In this chapter, we briefly review the challenges involved in using DNA to assemble SWNT nanostructures, and then give detailed methods to assemble dense, aligned SWNT arrays. In particular, we discuss the preparation of DNA wrapped single-walled nanotubes (DNA-CNTs) using commercial carbon nanotube products that are suitable for electronics applications. Then, we discuss methods to characterize DNA-CNTs using fluid mode atomic force microscopy (AFM). Finally, we give detailed procedures for assembly of DNA-CNTs into dense parallel arrays via linker induced surface assembly (LISA).

Early transcriptomic adaptation to Na2CO3 stress altered the expression of a quarter of the total genes in the maize genome and exhibited shared and distinctive profiles with NaCl and high pH stresses.

Sodium carbonate (Na2CO3) presents a huge challenge to plants by the combined damaging effects of Na⁺, high pH, and CO3²â». Little is known about the cellular responses to Na2CO3 stress. In this study, the transcriptome of maize (Zea mays L. cv. B73) roots exposed to Na2CO3 stress for 5 h was compared with those of NaCl and NaOH stresses. The expression of 8,319 genes, representing over a quarter of the total number of genes in the maize genome, was altered by Na2CO3 stress, and the downregulated genes (5,232) outnumbered the upregulated genes (3,087). The effects of Na2CO3 differed from those of NaCl and NaOH, primarily by downregulating different categories of genes. Pathways commonly altered by Na2CO3, NaCl, and NaOH were enriched in phenylpropanoid biosynthesis, oxidation of unsaturated fatty acids, ATP-binding cassette (ABC) transporters, as well as the metabolism of secondary metabolites. Genes for brassinosteroid biosynthesis were specifically upregulated by Na2CO3, while genes involved in ascorbate and aldarate metabolism, protein processing in the endoplasmic reticulum and by N-glycosylation, fatty acid biosynthesis, and the circadian rhythm were downregulated. This work provides the first holistic picture of early transcriptomic adaptation to Na2CO3 stress, and highlights potential molecular pathways that could be manipulated to improve tolerance in maize.

DNA-linker-induced surface assembly of ultra dense parallel single walled carbon nanotube arrays.

Ultrathin film preparations of single-walled carbon nanotube (SWNT) allow economical utilization of nanotube properties in electronics applications. Recent advances have enabled production of micrometer scale SWNT transistors and sensors but scaling these devices down to the nanoscale, and improving the coupling of SWNTs to other nanoscale components, may require techniques that can generate a greater degree of nanoscale geometric order than has thus far been achieved. Here, we introduce linker-induced surface assembly, a new technique that uses small structured DNA linkers to assemble solution dispersed nanotubes into parallel arrays on charged surfaces. Parts of our linkers act as spacers to precisely control the internanotube separation distance down to <3 nm and can serve as scaffolds to position components such as proteins between adjacent parallel nanotubes. The resulting arrays can then be stamped onto other substrates. Our results demonstrate a new paradigm for the self-assembly of anisotropic colloidal nanomaterials into ordered structures and provide a potentially simple, low cost, and scalable route for preparation of exquisitely structured parallel SWNT films with applications in high-performance nanoscale switches, sensors, and meta-materials.

Thermal decomposition of condensed-phase nitromethane from molecular dynamics from ReaxFF reactive dynamics.

We studied the thermal decomposition and subsequent reaction of the energetic material nitromethane (CH(3)NO(2)) using molecular dynamics with ReaxFF, a first principles-based reactive force field. We characterize the chemistry of liquid and solid nitromethane at high temperatures (2000-3000 K) and density 1.97 g/cm(3) for times up to 200 ps. At T = 3000 K the first reaction in the decomposition of nitromethane is an intermolecular proton transfer leading to CH(3)NOOH and CH(2)NO(2). For lower temperatures (T = 2500 and 2000 K) the first reaction during decomposition is often an isomerization reaction involving the scission of the C-N bond the formation of a C-O bond to form methyl nitrate (CH(3)ONO). Also at very early times we observe intramolecular proton transfer events. The main product of these reactions is H(2)O which starts forming following those initiation steps. The appearance of H(2)O marks the beginning of the exothermic chemistry. Recent quantum-mechanics-based molecular dynamics simulations on the chemical reactions and time scales for decomposition of a crystalline sample heated to T = 3000 K for a few picoseconds are in excellent agreement with our results, providing an important, direct validation of ReaxFF.

A novel selenium and copper-containing peptide with both superoxide dismutase and glutathione peroxidase activities.

Superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT) play crucial roles in balancing the production and decomposition of reactive oxygen species (ROS) in living organisms. These enzymes act cooperatively and synergistically to scavenge ROS. In order to imitate the synergism of these enzymes, we designed and synthesized a novel 32-mer peptide (32P) on the basis of the previous 15-mer peptide with GPX activity and a 17-mer peptide with SOD activity. Upon the selenation and chelation of copper, the 32-mer peptide is converted to a new Se- and Cu-containing 32-mer peptide (Se-Cu-32P) and displays both SOD and GPX activities and its kinetics was studied. Moreover, the novel peptide was demonstrated to be able to better protect vero cells from the injury induced by xanthine oxidase (XOD)/xanthine/Fe2+ damage system than its parents. Thus, this bifunctional enzyme imitated the synergism of SOD and GPX and could be a better candidate of therapeutic medicine.

Self-assembly of carbon nanotubes into two-dimensional geometries using DNA origami templates.

A central challenge in nanotechnology is the parallel fabrication of complex geometries for nanodevices. Here we report a general method for arranging single-walled carbon nanotubes in two dimensions using DNA origami-a technique in which a long single strand of DNA is folded into a predetermined shape. We synthesize rectangular origami templates ( approximately 75 nm x 95 nm) that display two lines of single-stranded DNA 'hooks' in a cross pattern with approximately 6 nm resolution. The perpendicular lines of hooks serve as sequence-specific binding sites for two types of nanotubes, each functionalized non-covalently with a distinct DNA linker molecule. The hook-binding domain of each linker is protected to ensure efficient hybridization. When origami templates and DNA-functionalized nanotubes are mixed, strand displacement-mediated deprotection and binding aligns the nanotubes into cross-junctions. Of several cross-junctions synthesized by this method, one demonstrated stable field-effect transistor-like behaviour. In such organizations of electronic components, DNA origami serves as a programmable nanobreadboard; thus, DNA origami may allow the rapid prototyping of complex nanotube-based structures.

Coupling of Raman radial breathing modes in double-wall carbon nanotubes and bundles of nanotubes.

Measurements of the radial breathing modes from Raman Spectroscopy have been most useful in characterizing the diameters of single-wall carbon nanotubes (SWNT), where there is a simple monotonic relationship between frequency and diameter. Similar correlations have also been used to predict sizes for double and multiple wall nanotubes and for bundles of SWNT. However this can lead to significant errors because the relationship between frequencies and diameter is much more complicated for DWNT. This is because of couplings between the vibrations of various walls. To provide guidance in such assignments we used the GraFF atomistic force field to predict the in-phase and counter-phase radial breathing modes (RBMs) of double wall carbon nanotubes (DWNTs) over a broad range of inner and outer diameters and chiralities. We then developed an analytical model to describe the RBMs of dispersed DWNTs. This enables the inner and outer shell diameters to be extracted from pairs of RBM peaks. We find that nanotubes bundles show significant dependent peak broadening and shifting compared to dispersed nanotubes. For bundles of SWNT and DWNT, the relationships are much more complicated.