Shorter Is Better: The α-(l)-Threofuranosyl Nucleic Acid Modification Improves Stability, Potency, Safety, and Ago2 Binding and Mitigates Off-Target Effects of Small Interfering RNAs.

Chemical modifications are necessary to ensure the metabolic stability and efficacy of oligonucleotide-based therapeutics. Here, we describe analyses of the α-(l)-threofuranosyl nucleic acid (TNA) modification, which has a shorter 3'-2' internucleotide linkage than the natural DNA and RNA, in the context of small interfering RNAs (siRNAs). The TNA modification enhanced nuclease resistance more than 2'-O-methyl or 2'-fluoro ribose modifications. TNA-containing siRNAs were prepared as triantennary N-acetylgalactosamine conjugates and were tested in cultured cells and mice. With the exceptions of position 2 of the antisense strand and position 11 of the sense strand, the TNA modification did not inhibit the activity of the RNA interference machinery. In a rat toxicology study, TNA placed at position 7 of the antisense strand of the siRNA mitigated off-target effects, likely due to the decrease in the thermodynamic binding affinity relative to the 2'-O-methyl residue. Analysis of the crystal structure of an RNA octamer with a single TNA on each strand showed that the tetrose sugar adopts a C4'-exo pucker. Computational models of siRNA antisense strands containing TNA bound to Argonaute 2 suggest that TNA is well accommodated in the region kinked by the enzyme. The combined data indicate that the TNA nucleotides are promising modifications expected to increase the potency, duration of action, and safety of siRNAs.

Nutrition-Related Information Shared by Latine Influencers: A YouTube Content Analysis.

Latines are the fastest growing populace in the United States. Latine is a new, inclusive term for Hispanic and Latino populations regardless of gender identity. When compared with non-Latine counterparts, Latines have higher prevalence rates of obesity, diabetes, chronic liver disease, and kidney disease, which are associated with poor dietary behaviors. More research is warranted into the factors behind Latines' understanding of nutrition and potential sources of health information that influence dietary behaviors. This study describes the nutrition-related health information shared through YouTube by English-speaking Latine individuals between the ages of 18 and 49. For this content analysis, a cross-sectional study design was used. A four-step search strategy identified eligible YouTube channels and their corresponding video blogs (vlogs): discovery, screening, eligibility, and included. NVivo 1.0 was used to qualitatively code the nutrition-related information. A total of 68 vlogs were identified and reviewed. Five main themes emerged from the data (what vloggers discussed): Nutrition Philosophies, Inaccurate Information, Product Promotion, Recommendations based on the 2020-2025 Dietary Guidelines for Americans (DGA), and Recommendations not based on the 2020-2025 DGA. Although some of the nutrition-related information shared followed the 2020-2025 DGA, not all information were in line with these guidelines. Misinformation can undermine the scientific work done by health professionals and can threaten the health and lives of the citizenry by creating barriers for accessing, understanding, and utilizing evidenced-informed guidance in making health decisions. This study revealed that more research is warranted into specific aspects of social media and how they influence health behavior.

Expanding RNAi therapeutics to extrahepatic tissues with lipophilic conjugates.

Therapeutics based on short interfering RNAs (siRNAs) delivered to hepatocytes have been approved, but new delivery solutions are needed to target additional organs. Here we show that conjugation of 2'-O-hexadecyl (C16) to siRNAs enables safe, potent and durable silencing in the central nervous system (CNS), eye and lung in rodents and non-human primates with broad cell type specificity. We show that intrathecally or intracerebroventricularly delivered C16-siRNAs were active across CNS regions and cell types, with sustained RNA interference (RNAi) activity for at least 3 months. Similarly, intravitreal administration to the eye or intranasal administration to the lung resulted in a potent and durable knockdown. The preclinical efficacy of an siRNA targeting the amyloid precursor protein was evaluated through intracerebroventricular dosing in a mouse model of Alzheimer's disease, resulting in amelioration of physiological and behavioral deficits. Altogether, C16 conjugation of siRNAs has the potential for safe therapeutic silencing of target genes outside the liver with infrequent dosing.

From bench to bedside: Improving the clinical safety of GalNAc-siRNA conjugates using seed-pairing destabilization.

Preclinical mechanistic studies have pointed towards RNA interference-mediated off-target effects as a major driver of hepatotoxicity for GalNAc-siRNA conjugates. Here, we demonstrate that a single glycol nucleic acid or 2'-5'-RNA modification can substantially reduce small interfering RNA (siRNA) seed-mediated binding to off-target transcripts while maintaining on-target activity. In siRNAs with established hepatotoxicity driven by off-target effects, these novel designs with seed-pairing destabilization, termed enhanced stabilization chemistry plus (ESC+), demonstrated a substantially improved therapeutic window in rats. In contrast, siRNAs thermally destabilized to a similar extent by the incorporation of multiple DNA nucleotides in the seed region showed little to no improvement in rat safety suggesting that factors in addition to global thermodynamics play a role in off-target mitigation. We utilized the ESC+ strategy to improve the safety of ALN-HBV, which exhibited dose-dependent, transient and asymptomatic alanine aminotransferase elevations in healthy volunteers. The redesigned ALN-HBV02 (VIR-2218) showed improved specificity with comparable on-target activity and the program was reintroduced into clinical development.

siRNAs containing 2'-fluorinated Northern-methanocarbacyclic (2'-F-NMC) nucleotides: in vitro and in vivo RNAi activity and inability of mitochondrial polymerases to incorporate 2'-F-NMC NTPs.

We recently reported the synthesis of 2'-fluorinated Northern-methanocarbacyclic (2'-F-NMC) nucleotides, which are based on a bicyclo[3.1.0]hexane scaffold. Here, we analyzed RNAi-mediated gene silencing activity in cell culture and demonstrated that a single incorporation of 2'-F-NMC within the guide or passenger strand of the tri-N-acetylgalactosamine-conjugated siRNA targeting mouse Ttr was generally well tolerated. Exceptions were incorporation of 2'-F-NMC into the guide strand at positions 1 and 2, which resulted in a loss of the in vitro activity. Activity at position 1 was recovered when the guide strand was modified with a 5' phosphate, suggesting that the 2'-F-NMC is a poor substrate for 5' kinases. In mice, the 2'-F-NMC-modified siRNAs had comparable RNAi potencies to the parent siRNA. 2'-F-NMC residues in the guide seed region position 7 and at positions 10, 11 and 12 were well tolerated. Surprisingly, when the 5'-phosphate mimic 5'-(E)-vinylphosphonate was attached to the 2'-F-NMC at the position 1 of the guide strand, activity was considerably reduced. The steric constraints of the bicyclic 2'-F-NMC may impair formation of hydrogen-bonding interactions between the vinylphosphonate and the MID domain of Ago2. Molecular modeling studies explain the position- and conformation-dependent RNAi-mediated gene silencing activity of 2'-F-NMC. Finally, the 5'-triphosphate of 2'-F-NMC is not a substrate for mitochondrial RNA and DNA polymerases, indicating that metabolites should not be toxic.

Synthesis, chirality-dependent conformational and biological properties of siRNAs containing 5'-(R)- and 5'-(S)-C-methyl-guanosine.

Various chemical modifications have been identified that enhance potency of small interfering RNAs (siRNAs) and that reduce off-target effects, immune stimulation, and toxicities of metabolites of these therapeutic agents. We previously described 5'-C-methyl pyrimidine nucleotides also modified at the 2' position of the sugar. Here, we describe the synthesis of 2'-position unmodified 5'-(R)- and 5'-(S)-C-methyl guanosine and evaluation of these nucleotides in the context of siRNA. The (R) isomer provided protection from 5' exonuclease and the (S) isomer provided protection from 3' exonuclease in the context of a terminally modified oligonucleotide. siRNA potency was maintained when these modifications were incorporated at the tested positions of sense and antisense strands. Moreover, the corresponding 5' triphosphates were not substrates for mitochondrial DNA polymerase. Models generated based on crystal structures of 5' and 3' exonuclease oligonucleotide complexes with 5'-(R)- and 5'-(S)-C-methyl substituents attached to the 5'- and 3'-terminal nucleotides, respectively, provided insight into the origins of the observed protections. Structural properties of 5'-(R)-C-methyl guanosine incorporated into an RNA octamer were analysed by X-ray crystallography, and the structure explains the loss in duplex thermal stability for the (R) isomer compared with the (S) isomer. Finally, the effect of 5'-C-methylation on endoribonuclease activity has been explained.

Platform for quantitative multiscale imaging of tissue composition.

Changes in the multi-level physical structure of biological features going from cellular to tissue level composition is a key factor in many major diseases. However, we are only beginning to understand the role of these structural changes because there are few dedicated multiscale imaging platforms with sensitivity at both the cellular and macrostructural spatial scale. A single platform reduces bias and complications from multiple sample preparation methods and can ease image registration. In order to address these needs, we have developed a multiscale imaging system using a range of imaging modalities sensitive to tissue composition: Ultrasound, Second Harmonic Generation Microscopy, Multiphoton Microscopy, Optical Coherence Tomography, and Enhanced Backscattering. This paper details the system design, the calibration for each modality, and a demonstration experiment imaging a rabbit eye.

Does regional diversity recover after disturbance? A field experiment in constructed ponds.

The effects of disturbance on local species diversity have been well documented, but less recognized is the possibility that disturbances can alter diversity at regional spatial scales. Since regional diversity can dictate which species are available for recolonization of degraded sites, the loss of diversity at regional scales may impede the recovery of biodiversity following a disturbance. To examine this we used a chemical disturbance of rotenone, a piscicide commonly used for fish removal in aquatic habitats, on small fishless freshwater ponds. We focused on the non-target effects of rotenone on aquatic invertebrates with the goal of assessing biodiversity loss and recovery at both local (within-pond) and regional (across ponds) spatial scales. We found that rotenone caused significant, large, but short-term losses of species at both local and regional spatial scales. Using a null model of random extinction, we determined that species were selectively removed from communities relative to what would be expected if species loss occurred randomly. Despite this selective loss of biodiversity, species diversity at both local and regional spatial scales recovered to reference levels one year after the addition of rotenone. The rapid recovery of local and regional diversity in this study was surprising considering the large loss of regional species diversity, however many aquatic invertebrates disperse readily or have resting stages that may persist through disturbances. We emphasize the importance of considering spatial scale when quantifying the impacts of a disturbance on an ecosystem, as well as considering how regional species loss can influence recovery from disturbance.

Polymer chain organization in tensile-stretched poly(ethylene oxide)-based polymer electrolytes.

Polymer chain orientation in tensile-stretched poly(ethylene oxide)-lithium trifluoromethanesulfonate polymer electrolytes are investigated with polarized infrared spectroscopy as a function of the degree of strain and salt composition (ether oxygen atom to lithium ion ratios of 20:1, 15:1, and 10:1). The 1359 and 1352 cm(-1) bands are used to probe the crystalline PEO and P(EO)(3)LiCF(3)SO(3) domains, respectively, allowing a direct comparison of chain orientation for the two phases. Two-dimensional correlation FT-IR spectroscopy indicates that the two crystalline domains align at the same rate as the polymer electrolytes are stretched. Quantitative measurements of polymer chain orientation obtained through dichroic infrared spectroscopy show that chain orientation predominantly occurs between strain values of 150% and 250%, regardless of salt composition investigated. There are few changes in chain orientation for either phase when the films are further elongated to a strain of 300%; however, the PEO domains are slightly more oriented at the high strain values. The spectroscopic data are consistent with stretching-induced melt-recrystallization of the unoriented crystalline domains in the solution-cast polymer films. Stretching the films pulls polymer chains from the crystalline domains, which subsequently recrystallize with the polymer helices parallel to the stretch direction. If lithium ion conduction in crystalline polymer electrolytes is viewed as consisting of two major components (facile intra-chain lithium ion conduction and slow helix-to-helix inter-grain hopping), then alignment of the polymer helices will affect the ion conduction pathways for these materials by reducing the number of inter-grain hops required to migrate through the polymer electrolyte.

Identification and characterization of native proteins of Escherichia coli BL-21 that display affinity towards Immobilized Metal Affinity Chromatography and Hydrophobic Interaction Chromatography Matrices.

The purpose of this study was to identify and characterize Escherichia coli proteins which display affinity towards both Immobilized Metal Affinity Chromatography (IMAC) and Hydrophobic Interaction Chromatography (HIC). Co(II) IMAC was chosen as the primary capture step, followed by HIC employing different concentrations of salt to promote adsorption. Results provided insight on this rather small pool of E. coli proteins. Nine out of the ten have isoelectric values less than six, and half are considered nonessential. These data indicate that the combination of IMAC and HIC could be developed as a potent method for the purification of recombinant proteins by judicious choice of the salt concentration used to promote HIC, the development of E. coli strain(s) deficient in certain genomic proteins, and the design of an IMAC-HIC affinity tail for recombinant protein isolation based on the very proteins deleted from the genome.