The monoclonal anti-dsRNA antibody J2 binds double-stranded RNAs (dsRNA) in an apparently sequence-nonspecific way. The mAb only recognizes antigens with double-stranded regions of at least 40 bp and its affinity to poly(A) poly(U) and to dsRNAs with mixed base pair composition is about tenfold higher than to poly(I) poly(C). Because no specific binding site could be determined, the number, the exact dimensions, and other distinct features of the binding sites on a given antigen are difficult to evaluate by biochemical methods. We therefore employed scanning force microscopy (SFM) as a method to analyze antibody-dsRNA interaction and protein-RNA binding in general. Several in vitro-synthesized dsRNA substrates, generated from the Dictyostelium PSV-A gene, were used. In addition to the expected sequence-nonspecific binding, imaging of the complexes indicated preferential binding of antibodies to the ends of dsRNA molecules as well as to certain internal sites. Analysis of 2,000 bound antibodies suggested that the consensus sequence of a preferential internal binding site is A 2 N 9 A 3 N 9 A 2 , thus presenting A residues on one face of the helix. The site was verified by site-directed mutagenesis, which abolished preferential binding to this region. The data demonstrate that SFM can be efficiently used to identify and characterize binding sites for proteins with no or incomplete sequence specificity. This is especially the case for many proteins involved in RNA metabolism.

Cost parameters for VSC HVDC transmission infrastructure have been gathered from an extensive collection of techno-economic sources. These cost parameter sets have been converted to a common format, based on a linear investment cost model depending on the branch length and the power rating of cable systems and converter stations. In addition, an average parameter set was determined as the arithmetic mean of the collected parameter sets, and included in the study. The uniform format allowed for a comparison of the parameter sets with each other, which revealed large differences between the cost parameter sets. The identified disparity between the parameter sets reflects a high level of uncertainty which can only in part be explained by a varying focus and modelling approach of their sources. This implies limitations regarding the validity of the parameters sets as well as of the results from grid expansion studies carried out on the basis of these parameter sets. Comprehensive cost reference data has been collected from realised and contracted VSC HVDC projects (back-to-back, interconnector, and offshore wind connection). The cost parameter sets have been evaluated against the reference project cost data. This evaluation has again shown large variations between the parameter sets. On average, the cost for back-to-back systems are slightly underestimated, interconnectors are overestimated, and offshore wind connections are heavily underestimated. To clearly state the validity and limitations of this comparison and evaluation, the applied methodology with its compromises and drawbacks is discussed in detail. Considering the interest in and momentum of offshore grid development, as well as the number of offshore grid investment and evaluation studies being conducted, both the availability of reliable cost reference data and the validity of investment model cost parameters need continuing attention

Scanning force microscopy (SFM) can be used to image biomolecules at high resolution. Here we demonstrate that singlemolecule analysis by SFM complements biochemical data on RNA protein binding and can provide information that cannot be obtained by the usual biochemical methods. We have used this method to study the interaction between the RNA editing enzyme ADAR2 and RNA transcripts containing selective and nonselective editing sites. The natural selectively edited R/G site from glutamate receptor subunit B (GluR-B) was inserted into an RNA backbone molecule consisting of a completely doublestranded (ds) central part and incompletely paired ends derived from potato spindle tuber viroid (PSTVd). This molecule was efficiently edited at the R/G site, but promiscuous editing occurred at nonselective sites in the completely double-stranded region. The construct was also used to analyze binding of ADAR2 to wild-type and modified R/G editing sites in relation to binding at other nonselectively edited sites. Editing analysis together with SFM allow us to differentiate between binding and enzymatic activity. ADAR2 has been reported to have a general affinity to dsRNA. However, we show that there is a prominent bias for stable binding at sites selectively edited over other edited sites. On the other hand, promiscuous editing at nonselective sites apparently results from transient binding of the enzyme to the substrate. Furthermore, we find distinct sites with nonproductive binding of the enzyme.

Scanning force spectroscopy was used to measure the mechanical properties of double stranded RNA molecules in comparison with DNA. We find that, similar to the B-S transition in DNA, RNA molecules are stretched from the assumed A' conformation to a stretched conformation by applying a defined force (plateau force). The force depends on the G + C content of the RNA and is distinct from that required for the B-S transition of a homologous DNA molecule. After the conformational change, DNA can be further extended by a factor of 0.7 +/- 0.2 (S-factor) before melting occurs and the binding of the molecule to the cantilever is finally disrupted. For RNA, the S-factor was higher (1.0 +/- 0.2) and more variable. Experiments to measure secondary structures in single stranded RNA yielded a large number of different force-distance curves, suggesting disruption and stretching of various secondary structures. Oriented attachment of the molecules to the substrate, a defined pick-up point and an increased resolution of the instrument could provide the means to analyse RNA secondary structures by scanning force spectroscopy.

We have used an RNA consisting of the potato spindle tuber viroid (PSTVd) and 240 bp of double-stranded RNA derived from the GUS gene as a backbone for scanning force microscope (SFM) studies on RNA binding proteins. The in vitro transcribed RNA forms a rod-like structure of apparent 130 nm in length with a completely base paired central part flanked by the incompletely paired viroid helix with bulges on both sides. The termini of the molecule consist of loops such that no blunt or staggered RNA ends are exposed. Suitable, asymmetrical restriction sites in the construct allow for the insertion of sequences of interest, e. g. protein binding sites. We have inserted the IRE (iron responsive element) sequence into the construct and have used in vitro transcripts to study binding of IRE-BP. Relative binding frequencies show that 70% of the protein binds to the expected site in the molecule while only a slightly enhanced binding is observed at the termini. In the GUS-PSTVd-IRE backbone, the orientation of the molecule is easily determined by IRE-BP binding. It thus provides a versatile tool to study specific as well as preferential interaction of other proteins with sequences or structures inserted into a different part of the molecule.