Sequence- and Structure-Dependent Cytotoxicity of Phosphorothioate and 2'-O-Methyl Modified Single-Stranded Oligonucleotides.

Single-stranded oligonucleotides (SSOs) are a rapidly expanding class of therapeutics that comprises antisense oligonucleotides, microRNAs, and aptamers, with ten clinically approved molecules. Chemical modifications such as the phosphorothioate backbone and the 2'-O-methyl ribose can improve the stability and pharmacokinetic properties of therapeutic SSOs, but they can also lead to toxicity in vitro and in vivo through nonspecific interactions with cellular proteins, gene expression changes, disturbed RNA processing, and changes in nuclear structures and protein distribution. In this study, we screened a mini library of 277 phosphorothioate and 2'-O-methyl-modified SSOs, with or without mRNA complementarity, for cytotoxic properties in two cancer cell lines. Using circular dichroism, nucleic magnetic resonance, and molecular dynamics simulations, we show that phosphorothioate- and 2'-O-methyl-modified SSOs that form stable hairpin structures through Watson-Crick base pairing are more likely to be cytotoxic than those that exist in an extended conformation. In addition, moderate and highly cytotoxic SSOs in our dataset have a higher mean purine composition than pyrimidine. Overall, our study demonstrates a structure-cytotoxicity relationship and indicates that the formation of stable hairpins should be a consideration when designing SSOs toward optimal therapeutic profiles.

A distinct ssDNA/RNA binding interface in the Nsp9 protein from SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel, highly infectious RNA virus that belongs to the coronavirus family. Replication of the viral genome is a fundamental step in the virus life cycle and SARS-CoV-2 non-structural protein 9 (Nsp9) is shown to be essential for virus replication through its ability to bind RNA in the closely related SARS-CoV-1 strain. Two recent studies revealing the three-dimensional structure of Nsp9 from SARS-CoV-2 have demonstrated a high degree of similarity between Nsp9 proteins within the coronavirus family. However, the binding affinity to RNA is very low which, until now, has prevented the determination of the structural details of this interaction. In this study, we have utilized nuclear magnetic resonance spectroscopy (NMR) in combination with surface biolayer interferometry (BLI) to reveal a distinct binding interface for both ssDNA and RNA that is different to the one proposed in the recently solved SARS-CoV-2 replication and transcription complex (RTC) structure. Based on these data, we have proposed a structural model of a Nsp9-RNA complex, shedding light on the molecular details of these important interactions.

Mitochondrial capacity using NIRS and incomplete recovery curves: Proximal and Medial Vastus Lateralis muscle.

Near-Infrared Spectroscopy (NIRS) has been used to measure muscle mitochondrial capacity (mVO2max) as the recovery rate constant of muscle metabolism after exercise. The current method requires as many as 50 short ischemic occlusions to generate 2 recovery rate constants. PURPOSE: To determine the effectiveness of using a 6-occlusion protocol (Mito6) versus one with 22 occlusions (Mito22) to measure muscle mitochondrial capacity. METHOD: In two independent data sets (bicep n=7, forearm A n=23), recovery curves were analyzed independently using both the Mito6 and Mito22 analyses. A third data set (Forearm B, n=16) was generated on forearm muscles of healthy subjects using four Mito6 tests performed in succession. Recovery rate constants were generated using a MATLAB routine. RESULTS: When calculated from the same data set, the recovery rate constants were not significantly different between the Mito22 and Mito6 analyses for the bicep (1.43+0.33min-1, 1.43+0.35min-1, p=0.81) and the forearm A (1.97+0.40min-1, 1.97+0.43min-1, p=0.90). The correlation between Mito22 and Mito6 recovery rate constants was y=1.07x-0.09, R2=0.90 for the bicep data and 1.00x+0.01, R2=0.85 for the forearm A data. When performing the four Mito6 tests in the Forearm B study; recovery rate constants were not different between tests (1.50±0.51 min-1, 1.42±0.54 min-1, 1.26±0.41 min-1, 1.29±0.47 min-1, P>0.05). CONCLUSIONS: Muscle mitochondrial capacity was not different between the Mito6 analysis and the longer Mito22 analysis. The Mito6 protocol was considered more practical as it used fewer ischemic occlusion periods, and multiple tests could be performed in succession in less time. There were no order effects for the rate constants of four repeated Mito6 tests of mitochondrial capacity, supporting the use of multiple tests to improve accuracy.

Near Infrared Spectroscopy Measurements of Mitochondrial Capacity Using Partial Recovery Curves.

BACKGROUND: Near-infrared spectroscopy (NIRS) has been used to measure muscle mitochondrial capacity (mVO2max) as the recovery rate constant of muscle metabolism after exercise. The current method requires as many as 50 short ischemic occlusions to generate two recovery rate constants. PURPOSE: To determine the validity and repeatability of using a 6-occlusion protocol versus one with 22 occlusions to measure muscle mitochondrial capacity. The order effect of performing multiple Mito6 test was also evaluated. METHOD: In two independent data sets (bicep n = 7, forearm A n = 23), recovery curves were analyzed independently using both the 6 and 22 occlusion methods. A third data set (forearm B n = 16) was generated on the forearm muscles of healthy subjects using four 6-occlusion tests performed in succession. Recovery rate constants were generated using a MATLAB routine. RESULTS: When calculated from the same data set, the recovery rate constants were not significantly different between the 22 occlusion and 6 occlusion methods for the bicep (1.43 ± 0.33 min-1, 1.43 ± 0.35 min-1, p = 0.81) and the forearm A (1.97 ± 0.40 min-1, 1.97 ± 0.43 min-1, p = 0.90). Equivalence testing showed that the mean difference was not different than zero and the 90% confidence intervals were within 5% of the average rate constant. This was true for the Mito6 and the Mito5∗ approaches. Bland-Altman analysis showed a slope of 0.21 min-1 and an r of 0.045 for the bicep dataset and a slope of -0.01 min-1 and an r of 0.045 for the forearm A dataset. When performing the four 6-occlusion tests; recovery rate constants showed no order effects (1.50 ± 0.51 min-1, 1.42 ± 0.54 min-1, 1.26 ± 0.41 min-1, 1.29 ± 0.47 min-1, P > 0.05). CONCLUSION: The Mito6 analysis is a valid and repeatable approach to measure mitochondrial capacity. The Mito6 protocol used fewer ischemic occlusion periods and multiple tests could be performed in succession in less time, increasing the practicality of the NIRS mitochondrial capacity test. There were no order effects for the rate constants of four repeated 6-occlusion tests of mitochondrial capacity, supporting the use of multiple tests to improve accuracy.

Barrier-to-autointegration factor 1 (Banf1) regulates poly [ADP-ribose] polymerase 1 (PARP1) activity following oxidative DNA damage.

The DNA repair capacity of human cells declines with age, in a process that is not clearly understood. Mutation of the nuclear envelope protein barrier-to-autointegration factor 1 (Banf1) has previously been shown to cause a human progeroid disorder, Néstor-Guillermo progeria syndrome (NGPS). The underlying links between Banf1, DNA repair and the ageing process are unknown. Here, we report that Banf1 controls the DNA damage response to oxidative stress via regulation of poly [ADP-ribose] polymerase 1 (PARP1). Specifically, oxidative lesions promote direct binding of Banf1 to PARP1, a critical NAD+-dependent DNA repair protein, leading to inhibition of PARP1 auto-ADP-ribosylation and defective repair of oxidative lesions, in cells with increased Banf1. Consistent with this, cells from patients with NGPS have defective PARP1 activity and impaired repair of oxidative lesions. These data support a model whereby Banf1 is crucial to reset oxidative-stress-induced PARP1 activity. Together, these data offer insight into Banf1-regulated, PARP1-directed repair of oxidative lesions.

Ocean acidification at high latitudes: potential effects on functioning of the Antarctic bivalve Laternula elliptica.

Ocean acidification is a well recognised threat to marine ecosystems. High latitude regions are predicted to be particularly affected due to cold waters and naturally low carbonate saturation levels. This is of concern for organisms utilising calcium carbonate (CaCO(3)) to generate shells or skeletons. Studies of potential effects of future levels of pCO(2) on high latitude calcifiers are at present limited, and there is little understanding of their potential to acclimate to these changes. We describe a laboratory experiment to compare physiological and metabolic responses of a key benthic bivalve, Laternula elliptica, at pCO(2) levels of their natural environment (430 µatm, pH 7.99; based on field measurements) with those predicted for 2100 (735 µatm, pH 7.78) and glacial levels (187 µatm, pH 8.32). Adult L. elliptica basal metabolism (oxygen consumption rates) and heat shock protein HSP70 gene expression levels increased in response both to lowering and elevation of pH. Expression of chitin synthase (CHS), a key enzyme involved in synthesis of bivalve shells, was significantly up-regulated in individuals at pH 7.78, indicating L. elliptica were working harder to calcify in seawater undersaturated in aragonite (Ω(Ar) = 0.71), the CaCO(3) polymorph of which their shells are comprised. The different response variables were influenced by pH in differing ways, highlighting the importance of assessing a variety of factors to determine the likely impact of pH change. In combination, the results indicate a negative effect of ocean acidification on whole-organism functioning of L. elliptica over relatively short terms (weeks-months) that may be energetically difficult to maintain over longer time periods. Importantly, however, the observed changes in L. elliptica CHS gene expression provides evidence for biological control over the shell formation process, which may enable some degree of adaptation or acclimation to future ocean acidification scenarios.