Double-stranded RNA under force and torque: similarities to and striking differences from double-stranded DNA.

RNA plays myriad roles in the transmission and regulation of genetic information that are fundamentally constrained by its mechanical properties, including the elasticity and conformational transitions of the double-stranded (dsRNA) form. Although double-stranded DNA (dsDNA) mechanics have been dissected with exquisite precision, much less is known about dsRNA. Here we present a comprehensive characterization of dsRNA under external forces and torques using magnetic tweezers. We find that dsRNA has a force-torque phase diagram similar to that of dsDNA, including plectoneme formation, melting of the double helix induced by torque, a highly overwound state termed "P-RNA," and a highly underwound, left-handed state denoted "L-RNA." Beyond these similarities, our experiments reveal two unexpected behaviors of dsRNA: Unlike dsDNA, dsRNA shortens upon overwinding, and its characteristic transition rate at the plectonemic buckling transition is two orders of magnitude slower than for dsDNA. Our results challenge current models of nucleic acid mechanics, provide a baseline for modeling RNAs in biological contexts, and pave the way for new classes of magnetic tweezers experiments to dissect the role of twist and torque for RNA-protein interactions at the single-molecule level.

Unraveling single-stranded DNA in a solid-state nanopore.

Solid-state nanopores are an emerging class of single-molecule sensors. Whereas most studies so far focused on double-stranded DNA (dsDNA) molecules, exploration of single-stranded DNA (ssDNA) is of great interest as well, for example to employ such a nanopore device to read out the sequence. Here, we study the translocation of long random-sequence ssDNA through nanopores. Using atomic force microscopy, we observe the ssDNA to hybridize into a random coil, forming blobs of around 100 nm in diameter for 7 kb ssDNA. These large entangled structures have to unravel, when they arrive at the pore entrance. Indeed, we observe strong blockade events with a translocation time that is exponentially dependent on voltage, tau approximately e(-V/V(0)). Interestingly, this is very different than for dsDNA, for which tau approximately 1/V. We report translocations of ssDNA but also of ssDNA-dsDNA constructs where we compare the conductance-blockade levels for ssDNA versus dsDNA as a function of voltage.

Effective lead selection for improved protein production in Aspergillus niger based on integrated genomics.

The filamentous fungus Aspergillus niger is widely exploited for industrial production of enzymes and organic acids. An integrated genomics approach was developed to determine cellular responses of A. niger to protein production in well-controlled fermentations. Different protein extraction methods in combination with automated sample processing and protein identification allowed quantitative analysis of 898 proteins. Three different enzyme overproducing strains were compared to their isogenic fungal host strains. Clear differences in response to the amount and nature of the overproduced enzymes were observed. The corresponding genes of the differentially expressed proteins were studied using transcriptomics. Genes that were up-regulated both at the proteome and transcriptome level were selected as leads for generic strain improvement. Up-regulated proteins included proteins involved in carbon and nitrogen metabolism as well as (oxidative) stress response, and proteins involved in protein folding and endoplasmic reticulum-associated degradation (ERAD). Reduction of protein degradation through the removal of the ERAD factor doaA combined with overexpression of the oligosaccharyl transferase sttC in A. niger overproducing beta-glucuronidase (GUS) strains indeed resulted in a small increase in GUS expression.