Mass spectrometry of oligonucleotides

Expanding research in the field of modified oligonucleotides demands suitable analytical tools for size and purity verification of known compounds and accurate structure elucidation of unknowns. There is a need for characterization of the types and sites of modifications in oligonucleotides and to identify and sequence selected candidates originating from synthesis. The potential of electrospray tandem mass spectrometry (ESI-MS/MS) for structural characterization and sequencing of oligonucleotides is demonstrated. The fundamental behavior of DNA, RNA, and selected modified oligonucleotides in gas-phase is shown. Since gas-phase dissociation does not demand specific structural prerequisites, the method bears a great potential for rapid and most accurate characterization of modified oligonucleotides, e.g. from combinatorial libraries.     

RNAi activity of siRNAs modified with 2'-aminoalkyl-substituted fluorinated nucleobases

We recently reported that a 1'-deoxy-1'-(4,6-difluoro-1H-benzimidazol-1-yl)-2'-(beta-aminoethyl)-beta-d-ribofuranose nucleoside appears to be a universal nucleoside which does not differentiate between the four natural nucleosides A, C, G, and U in duplexes. Moreover, ribozymes modified with this nucleoside analog showed a better or at least equal catalytic activity relative to Watson-Crick mismatches.[1] Due to these data, we investigated the ability of this compound to tolerate Watson-Crick mismatches in order to avoid HIV escape mutations in RNA interference. The influence of this nucleoside analog on siRNA efficiency was analyzed with a proven siRNA targeting GFP.     

A new fast method for nanoLC-MALDI-TOF/TOF-MS analysis using monolithic columns for peptide preconcentration and separation in proteomic studies

A new fast method for identification and characterization of proteolytic digests of proteins by monolithic liquid chromatography coupled with mass spectrometry has been developed. The advantages of the monolithic columns are a high-pressure stability and low back pressure resulting in higher flow rates for capillary or nanosize columns simplifying the system handling. As was shown in several publications, such monolithic stationary phases are highly qualified for the analysis of peptides and proteins, but so far, only small volumes could be injected into the system, which might hamper the sample preparation leading to protein precipitation and partial loss of sample. To overcome the problem of small injection volumes, we established a system including a short monolithic trap column to allow preconcentration of the peptides. The injected sample is flushed at higher flow rates onto the trap column, bound to the stationary phase, and in this way concentrated in a few nanoliters before starting the separation. The expanded system was optimized and tested using different reference protein samples. Eluting peptides were detected by MALDI-TOF/TOF-MS and identified by database searching. The system is now a permanent part for proteome analysis in our lab, and as such, it was successfully applied for the detection of post-translational modifications and the analysis of membrane proteins. One example for these analyses is also included in this paper.     

Spatio-temporal variation in post-recovery dynamics in a large Peregrine Falcon (Falco peregrinus) population in the Jura mountains 2000–2020†

After spectacular population crashes in the 1960 and 1970s, Peregrine Falcon Falco peregrinus populations recovered worldwide, and in the 2000s many study populations were back to normal. However, post-recovery trends have been documented less well. We combined three long-term population studies covering the entire Jura mountain range (16 304 km²) to examine spatio-temporal variation in population dynamics during 2000–2020 in 420 known nesting sites in five regions: Ain, Jura and Doubs in France, and Jura Sud and Jura Nord in Switzerland. Every year about 60% of all sites were surveyed at least once, and this proportion increased over time. We used Bayesian occupancy modelling to investigate site persistence and colonization probabilities and correct status and trend estimates for bias due to non-random site coverage. Based on the average of two models correcting for preferential sampling, we estimated that population size peaked in 2002 (Jura), 2005 (Doubs), 2007 (Ain, Jura Nord) and in 2007 for the Jura as a whole. Thereafter, the population declined significantly in four regions and in the Jura as a whole, with the strongest decline found in the Jura Nord ( –38%, 95% credible interval –34%/–41%) during 2007–2020. Thus, we found considerable spatial heterogeneity in post-recovery trends and widespread post-recovery declines. Reasons for the latter may include Eagle Owl Bubo bubo predation, illegal persecution and human disturbance. Our results illustrate how quickly positive population trends may be reversed, such that continuous long-term monitoring even for species seemingly ‘out of danger’ remains invaluable. Our study also emphasizes the importance of correcting for both coverage bias in general and preferential sampling in particular when assessing population trends in studies where not every territory can be surveyed in all years.     

The regulatory role of CO2 on nutrient releases from ashed rice straw phytoliths

Biogeochemistry - Phytolith is widely known as a silica structure in numerous silicon (Si) accumulator plants, e.g., rice, and it contains various nutrients and other beneficial elements. When rice...     

Dwarf shrub hydraulics: two Vaccinium species (Vaccinium myrtillus,Vaccinium vitis‐idaea) of the European Alps compared

Vaccinium myrtillus and Vaccinium vitis‐idaea are two dwarf shrubs widespread in the European Alps. We studied the hydraulics of these species hypothesizing that (1) the hydraulic architecture of dwarf shrubs differs from trees, (2) hydraulic properties reflect the species' ecological amplitude and (3) hydraulic properties vary spatially and seasonally. Key hydraulic parameters (osmotic potential, turgor loss point, xylem hydraulic conductivity, vulnerability to drought‐induced embolism, stomata closure, drought‐induced cell damage and embolism repair) and related wood anatomical traits (conduit diameter and conduit wall reinforcement) were analyzed at four sites in Tyrol, Austria. Both species exhibited low hydraulic safety as well as low hydraulic efficiency. Fifty percentage embolism accumulated at ?2.08 (V. myrtillus) and ?1.97 MPa (V. vitis‐idaea), 88% stomata closure was at ?2.19 and ?2.35 MPa, respectively. After drought, both species showed embolism repair on re‐watering. Site‐specific variation within species was low, while seasonal changes in embolism resistance and turgor loss point were observed. Results indicate that studied Vaccinium species have a high risk for embolism formation. This is balanced by refilling capacities, which are probably based on the small growth height of dwarf shrubs. V. vitis‐idaea, which occurs on drier sites, showed more efficient repair and a lower turgor loss point than V. myrtillus.