| 1. |
- Höök, Fredrik, 1966, et al.
(författare)
-
Supported lipid bilayers, tethered lipid vesicles, and vesicle fusion investigated using gravimetric, plasmonic, and microscopy techniques
- 2007
-
Ingår i: Biointerphases. - : American Vacuum Society. - 1559-4106 .- 1934-8630. ; 3:2
-
Tidskriftsartikel (refereegranskat)abstract
- This article summarizes our most recent contributions to the rapidly growing field of supported lipid assemblies with emphasis on current studies addressing both fundamental and applied aspects of supported lipid bilayer (SLB) and tethered lipid vesicles (TLVs) to be utilized in sensing applications. The new insights obtained from combining the quartz crystal microbalance with dissipation monitoring technique with surface plasmon resonance are described, and we also present recent studies in which nanoplasmonic sensing has been used in studies of SLBs and TLVs. To gain full control over the spatial arrangement of TLVs in both two and three dimensions, we have developed a method for site-selective and sequence-specific sorting of DNA-tagged vesicles to surfaces modified with complementary DNA. The combination of this method with nanoplasmonic sensing formats is covered as well as the possibility of using DNA-modified vesicles for the detection of unlabeled DNA targets on the single-molecule level. Finally, a new method for membrane fusion induced by hybridization of vesicle-anchored DNA is demonstrated, including new results on content mixing obtained with vesicle populations encapsulating short, complementary DNA strands.
|
|
| 2. |
|
|
| 3. |
- Klenkar, Goran, et al.
(författare)
-
Addressable adsorption of lipid vesicles and subsequent protein interaction studies
- 2008
-
Ingår i: Biointerphases. - : American Vacuum Society. - 1559-4106 .- 1934-8630. ; 3:2, s. 29-37
-
Tidskriftsartikel (refereegranskat)abstract
- We demonstrate a convenient chip platform for the addressable immobilization of protein-loaded vesicles on a microarray for parallelized, high-throughput analysis of lipid-protein systems. Self-sorting of the vesicles on the microarray was achieved through DNA bar coding of the vesicles and their hybridization to complementary strands, which are preimmobilized in defined array positions on the chip. Imaging surface plasmon resonance in ellipsometric mode was used to monitor vesicle immobilization, protein tethering, protein-protein interactions, and chip regeneration. The immobilization strategy proved highly specific and stable and presents a mild method for the anchoring of vesicles to predefined areas of a surface, while unspecific adsorption to both noncomplementary regions and background areas is nonexistent or, alternatively, undetectable. Furthermore, histidine-tagged receptors have been stably and functionally immobilized via bis-nitrilotriacetic acid chelators already present in the vesicle membranes. It was discovered though that online loading of proteins to immobilized vesicles leads to cross contamination of previously loaded vesicles and that it was necessary to load the vesicles offline in order to obtain pure protein populations on the vesicles. We have used this cross-binding effect to our benefit by coimmobilizing two receptor subunits in different ratios on the vesicle surface and successfully demonstrated ternary complex formation with their ligand. This approach is suitable for mechanistic studies of complex multicomponent analyses involving membrane-bound systems.
|
|
| 4. |
- Simonsson, Lisa, 1982, et al.
(författare)
-
Site-Specific DNA-Controlled Fusion of Single Lipid Vesicles to Supported Lipid Bilayers
- 2010
-
Ingår i: ChemPhysChem. - : Wiley. - 1439-7641 .- 1439-4235. ; 11:5, s. 1011-1017
-
Tidskriftsartikel (refereegranskat)abstract
- We investigate the Ca2+-triggered fusion of lipid vesicles site-selectively tethered to a DNA-modified supported lipid bilayer array, with the DNA strands designed such that hybridization occurs in a zipperlike fashion. Prior to the addition of Ca2+, which is observed to induce docking and subsequent fusion (within 200 ms), the vesicles display lateral mobility determined by the number of tethers. Fusion is observed to require around ten DNA strands per vesicle, but does not occur at higher DNA coverage. However, despite the fact that fusion was restricted to occurring for vesicles tethered with around ten DNA strands, there is no correlation between single-vesicle diffusivity and fusogenicity. A possible scenario for the DNA-induced fusion machinery, consistent with these observations, is that prior to Ca2+-induced docking, the vesicles diffuse with a small number (2-4) of DNA tethers. Upon addition of Ca2+, the vesicles dock, presumably due to bridging of lipid head groups. Fusion then occurs under conditions where 10-16 DNA tethers form and rearrange at the rim of the contact region between a docked vesicle and the SLB. The time required for this rearrangement, which may include both DNA hybridization and dehybridization during zipping, is expected to represent the observed docking and fusion time of less than 200 ms.
|
|
| 5. |
- Stengel, Gudrun, et al.
(författare)
-
Ambivalent Incorporation of the Fluorescent Cytosine Analogues tC and tCo by Human DNA Polymerase alpha and Klenow Fragment
- 2009
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 1520-4995 .- 0006-2960. ; 48:31, s. 7547-7555
-
Tidskriftsartikel (refereegranskat)abstract
- Westudied the incorporation of the fluorescent cytidine analogues 1,3-diaza-2-oxophenothiazine (tC) and 1,3-diaza-2-oxophenoxazine (tCo) by human DNA polymerase R and Klenow fragment of DNA polymerase I (Escherichia coli). These tricyclic nucleobases possess the regular hydrogen bonding interface of cytosine but are significantly expanded in size toward the major groove. Despite the size alteration, both DNA polymerases insert dtCTP and dtCoTP with remarkable catalytic efficiency. Polymerization opposite guanine is comparable to the insertion of dCTP, while the insertion opposite adenine is only ∼4-11 times less efficient than the formation of a T-A base pair. Both enzymes readily extend the formed tC(o)-G and tC(o)-A base pairs and can incorporate at least four consecutive nucleotide analogues. Consistent with these results, both DNA polymerases efficiently polymerize dGTP and dATP when tC and tCo are in the template strand. Klenow fragment inserts dGTP with a 4-9-fold higher probability than dATP, while polymerase R favors dGTP over dATP by a factor of 30-65. Overall, the properties of tC(o) as a templating base and as an incoming nucleotide are surprisingly symmetrical and may be universal for A and B family DNA polymerases. This finding suggests that the aptitude for ambivalent base pairing is a consequence of the electronic properties of tC(o). © 2009 American Chemical Society.
|
|
| 6. |
|
|
| 7. |
- Stengel, Gudrun, et al.
(författare)
-
Conformational dynamics of DNA polymerase probed with a novel fluorescent DNA base analogue
- 2007
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 1520-4995 .- 0006-2960. ; 46:43, s. 12289-12297
-
Tidskriftsartikel (refereegranskat)abstract
- DNA polymerases discriminate between correct and incorrect nucleotide substrates during a "nonchemical" step that precedes phosphodiester bond formation in the enzymatic cycle of nucleotide incorporation. Despite the importance of this process in polymerase fidelity, the precise nature of the molecular events involved remains unknown. Here we report a fluorescence resonance energy transfer (FRET) system that monitors conformational changes of a polymerase-DNA complex during selection and binding of nucleotide substrates. This system utilizes the fluorescent base analogue 1,3-diaza-2oxophenothiazine (tC) as the FRET donor and Alexa-555 (A555) as the acceptor. The tC donor was incorporated within a model DNA primer/template in place of a normal base, adjacent to the primer 3' terminus, while the A555 acceptor was attached to an engineered cysteine residue (C751) located in the fingers subdomain of the Klenow fragment (KF) polymerase. The FRET efficiency increased significantly following binding of a correct nucleotide substrate to the KF-DNA complex, showing that the fingers had closed over the active site. Fluorescence anisotropy titrations utilizing tC as a reporter indicated that the DNA was more tightly bound by the polymerase under these conditions, consistent with the formation of a closed ternary complex. The rate of the nucleotide-induced conformational transition, measured in stopped-flow FRET experiments, closely matched the rate of correct nucleotide incorporation, measured in rapid quench-flow experiments, indicating that the conformational change was the rate-limiting step in the overall cycle of nucleotide incorporation for the labeled KF-DNA system. Taken together, these results indicate that the FRET system can be used to probe enzyme conformational changes that are linked to the biochemical function of DNA polymerase.
|
|
| 8. |
- Stengel, Gudrun, et al.
(författare)
-
Determinants for Membrane Fusion Induced by Cholesterol-Modified DNA Zippers.
- 2008
-
Ingår i: The Journal of Physical Chemistry Part B. - : American Chemical Society (ACS). - 1520-5207 .- 1520-6106. ; 112:28, s. 8264-8274
-
Tidskriftsartikel (refereegranskat)abstract
- Intracellular membrane fusion is coordinated by membrane-anchored fusion proteins. The cytosolic domains of these proteins form a specific complex that pulls the membranes into close proximity. Although some results indicate that membrane merger can be accomplished solely on the basis of proximity, others emphasize the importance of bilayer stress exerted by transmembrane peptides. In a reductionist approach, we recently introduced a fusion machinery built from cholesterol-modified DNA zippers to mimic fusion protein function. Aiming to further optimize DNA-mediated fusion, we varied in this work length and number of DNA strands and used either one or two cholesterol groups for membrane anchoring of DNA. The results reveal that the use of two cholesterol anchors is essential to prevent cDNA strands from shuttling to the same membrane, which leads to vesicle release instead of membrane merger. A surface coverage of 6-13 DNA strands was a precondition for efficient fusion, whereas fusion was insensitive to DNA length within the tested range. Besides lipid mixing, we also demonstrate DNA-induced content mixing of large unilamellar vesicles composed of the most abundant cellular lipids phosphatidylcholine, phosphatidylethanolamine, cholesterol, and sphingomyelin. Taken together, DNA-mediated fusion emerges as a promising tool for the functionalization of artificial and biological membranes and may help to dissect the functional role of fusion proteins.
|
|
| 9. |
- Stengel, Gudrun, et al.
(författare)
-
DNA-induced programmable fusion of phospholipid vesicles
- 2007
-
Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 129:31, s. 9584-9584
-
Tidskriftsartikel (refereegranskat)abstract
- In nature, the fusion of phospholipid vesicles is regulated and catalyzed by highly specialized SNARE proteins. A key step in this process is to bring about close apposition of the lipid bilayers that are destined to fuse. Inspired by nature's use of molecular recognition between receptor proteins, we developed a method to force bilayers into proximity by the selective hybridization of membrane-anchored DNA strands. We demonstrate that this forced bilayer contact triggers vesicle fusion.
|
|
| 10. |
- Stengel, Gudrun, et al.
(författare)
-
DNA-mediated fusion of lipid vesicles
- 2006
-
Ingår i: Book of abstracts: 2nd Intl Symp on Semicond Nanowires, Lund, Sweden (2006).
-
Konferensbidrag (refereegranskat)
|
|
