918 research outputs found
Locked nucleic acid oligomers as handles for single molecule manipulation.
Single-molecule manipulation (SMM) techniques use applied force, and measured elastic response, to reveal microscopic physical parameters of individual biomolecules and details of biomolecular interactions. A major hurdle in the application of these techniques is the labeling method needed to immobilize biomolecules on solid supports. A simple, minimally-perturbative labeling strategy would significantly broaden the possible applications of SMM experiments, perhaps even allowing the study of native biomolecular structures. To accomplish this, we investigate the use of functionalized locked nucleic acid (LNA) oligomers as biomolecular handles that permit sequence-specific binding and immobilization of DNA. We find these probes form bonds with DNA with high specificity but with varied stability in response to the direction of applied mechanical force: when loaded in a shear orientation, the bound LNA oligomers were measured to be two orders of magnitude more stable than when loaded in a peeling, or unzipping, orientation. Our results show that LNA provides a simple, stable means to functionalize dsDNA for manipulation. We provide design rules that will facilitate their use in future experiments
Single-molecule stretching shows glycosylation sets tension in the hyaluronan-aggrecan bottlebrush
Large bottlebrush complexes formed from the polysaccharide hyaluronan (HA)
and the proteoglycan aggrecan contribute to cartilage compression resistance
and are necessary for healthy joint function. A variety of mechanical forces
act on these complexes in the cartilage extracellular matrix, motivating the
need for a quantitative description which links their structure and mechanical
response. Studies using electron microscopy have imaged the HA-aggrecan brush
but require adsorption to a surface, dramatically altering the complex from its
native conformation. We use magnetic tweezers force spectroscopy to measure
changes in extension and mechanical response of an HA chain as aggrecan
monomers bind and form a bottlebrush. This technique directly measures changes
undergone by a single complex with time and under varying solution conditions.
Upon addition of aggrecan, we find a large swelling effect manifests when the
HA chain is under very low external tension (i.e. stretching forces less than
~1 pN). We use models of force-extension behavior to show that repulsion
between the aggrecans induces an internal tension in the HA chain. Through
reference to theories of bottlebrush polymer behavior, we demonstrate that the
experimental values of internal tension are consistent with a polydisperse
aggrecan population, likely caused by varying degrees of glycosylation. By
enzymatically deglycosylating aggrecan, we show that aggrecan glycosylation is
the structural feature which causes HA stiffening. We then construct a simple
stochastic binding model to show that variable glycosylation leads to a wide
distribution of internal tensions in HA, causing variations in the mechanics at
much longer length-scales. Our results provide a mechanistic picture of how
flexibility and size of HA and aggrecan lead to the brush architecture and
mechanical properties of this important component of cartilage
Ciz1 cooperates with cyclin-A-CDK2 to activate mammalian DNA replication in vitro
Initiation of mammalian DNA replication can be reconstituted from isolated G1-phase nuclei and cell extracts, supplemented with cyclin-dependent protein kinases (CDKs). Under these conditions, cyclin E supports pre-replication complex assembly, whereas cyclin-A-associated kinase acts later to terminate assembly and activate DNA replication. The mechanism by which these events are coordinated is unknown. Here, we show that the replication factor Ciz1 interacts with cyclins E and A sequentially through distinct cyclin-binding motifs. Cyclin A displaces cyclin E from Ciz1 in a manner that is dependent on functional domains that are essential for its role in DNA replication. Furthermore, in cell-free assays, recombinant cyclin-A-CDK2 complexes and recombinant Ciz1 cooperate to promote initiation of DNA replication in late G1-phase nuclei. In addition, Ciz1 supports immobilization of cyclin A in isolated nuclei and depletion of Ciz1 by RNAi impairs immobilization, suggesting that Ciz1 promotes initiation by helping to target the kinase to a specific subnuclear compartment. We propose that Ciz1 acts to coordinate the functions of cyclins E and A in the nucleus, by delivering cyclin-A-associated kinase to sites that are specified by cyclin E, helping to ensure that they execute their functions in the same place and in the correct order
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