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1. Sequencing by avidity enables high accuracy with low reagent consumption

Author

Arslan, Sinan, Garcia, Francisco J., Guo, Minghao, Kellinger, Matthew W., Kruglyak, Semyon, LeVieux, Jake A., Mah, Adeline H., Wang, Haosen, Zhao, Junhua, Zhou, Chunhong, Altomare, Andrew, Bailey, John, Byrne, Matthew B., Chang, Chiting, Chen, Steve X., Cho, Byungrae, Dennler, Claudia N., Dien, Vivian T., Fuller, Derek, Kelley, Ryan, Khandan, Omid, Klein, Michael G., Kim, Michael, Lajoie, Bryan R., Lin, Bill, Liu, Yu, Lopez, Tyler, Mains, Peter T., Price, Andrew D., Robertson, Samantha R., Taylor-Weiner, Hermes, Tippana, Ramreddy, Tomaney, Austin B., Zhang, Su, Abtahi, Minna, Ambroso, Mark R., Bajari, Rosita, Bellizzi, Ava M., Benitez, Chris B., Berard, Daniel R., Berti, Lorenzo, Blease, Kelly N., Blum, Angela P., Boddicker, Andrew M., Bondar, Leo, Brown, Chris, Bui, Chris A., Calleja-Aguirre, Juan, Cappa, Kevin, Chan, Joshua, Chang, Victor W., Charov, Katherine, Chen, Xiyi, Constandse, Rodger M., Damron, Weston, Dawood, Mariam, DeBuono, Nicole, Dimalanta, John D., Edoli, Laure, Elango, Keerthana, Faustino, Nikka, Feng, Chao, Ferrari, Matthew, Frankie, Keith, Fries, Adam, Galloway, Anne, Gavrila, Vlad, Gemmen, Gregory J., Ghadiali, James, Ghorbani, Arash, Goddard, Logan A., Guetter, Adriana Roginski, Hendricks, Garren L., Hentschel, Jendrik, Honigfort, Daniel J., Hsieh, Yun-Ting, Hwang Fu, Yu-Hsien, Im, Scott K., Jin, Chaoyi, Kabu, Shradha, Kincade, Daniel E., Levy, Shawn, Li, Yu, Liang, Vincent K., Light, William H., Lipsher, Jonathan B., Liu, Tsung-li, Long, Grace, Ma, Rui, Mailloux, John M., Mandla, Kyle A., Martinez, Anyssa R., Mass, Max, McKean, Daniel T., Meron, Michael, Miller, Edmund A., Moh, Celyne S., Moore, Rachel K., Moreno, Juan, Neysmith, Jordan M., Niman, Cassandra S., Nunez, Jesus M., Ojeda, Micah T., Ortiz, Sara Espinosa, Owens, Jenna, Piland, Geoffrey, Proctor, Daniel J., Purba, Josua B., Ray, Michael, Rong, Daisong, Saade, Virginia M., Saha, Sanchari, Tomas, Gustav Santo, Scheidler, Nicholas, Sirajudeen, Luqmanal H., Snow, Samantha, Stengel, Gudrun, Stinson, Ryan, Stone, Michael J., Sundseth, Keoni J., Thai, Eileen, Thompson, Connor J., Tjioe, Marco, Trejo, Christy L., Trieger, Greg, Truong, Diane Ni, Tse, Ben, Voiles, Benjamin, Vuong, Henry, Wong, Jennifer C., Wu, Chiung-Ting, Yu, Hua, Yu, Yingxian, Yu, Ming, Zhang, Xi, Zhao, Da, Zheng, Genhua, He, Molly, and Previte, Michael

Published
2024
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2. Sequencing by avidity enables high accuracy with low reagent consumption

Author

Arslan, Sinan, primary, Garcia, Francisco J., additional, Guo, Minghao, additional, Kellinger, Matthew W., additional, Kruglyak, Semyon, additional, LeVieux, Jake A., additional, Mah, Adeline H., additional, Wang, Haosen, additional, Zhao, Junhua, additional, Zhou, Chunhong, additional, Altomare, Andrew, additional, Bailey, John, additional, Byrne, Matthew B., additional, Chang, Chiting, additional, Chen, Steve X., additional, Cho, Byungrae, additional, Dennler, Claudia N., additional, Dien, Vivian T., additional, Fuller, Derek, additional, Kelley, Ryan, additional, Khandan, Omid, additional, Klein, Michael G., additional, Kim, Michael, additional, Lajoie, Bryan R., additional, Lin, Bill, additional, Liu, Yu, additional, Lopez, Tyler, additional, Mains, Peter T., additional, Price, Andrew D., additional, Robertson, Samantha R., additional, Taylor-Weiner, Hermes, additional, Tippana, Ramreddy, additional, Tomaney, Austin B., additional, Zhang, Su, additional, Abtahi, Minna, additional, Ambroso, Mark R., additional, Bajari, Rosita, additional, Bellizzi, Ava M., additional, Benitez, Chris B., additional, Berard, Daniel R., additional, Berti, Lorenzo, additional, Blease, Kelly N., additional, Blum, Angela P., additional, Boddicker, Andrew M., additional, Bondar, Leo, additional, Brown, Chris, additional, Bui, Chris A., additional, Calleja-Aguirre, Juan, additional, Cappa, Kevin, additional, Chan, Joshua, additional, Chang, Victor W., additional, Charov, Katherine, additional, Chen, Xiyi, additional, Constandse, Rodger M., additional, Damron, Weston, additional, Dawood, Mariam, additional, DeBuono, Nicole, additional, Dimalanta, John D., additional, Edoli, Laure, additional, Elango, Keerthana, additional, Faustino, Nikka, additional, Feng, Chao, additional, Ferrari, Matthew, additional, Frankie, Keith, additional, Fries, Adam, additional, Galloway, Anne, additional, Gavrila, Vlad, additional, Gemmen, Gregory J., additional, Ghadiali, James, additional, Ghorbani, Arash, additional, Goddard, Logan A., additional, Guetter, Adriana Roginski, additional, Hendricks, Garren L., additional, Hentschel, Jendrik, additional, Honigfort, Daniel J., additional, Hsieh, Yun-Ting, additional, Hwang Fu, Yu-Hsien, additional, Im, Scott K., additional, Jin, Chaoyi, additional, Kabu, Shradha, additional, Kincade, Daniel E., additional, Levy, Shawn, additional, Li, Yu, additional, Liang, Vincent K., additional, Light, William H., additional, Lipsher, Jonathan B., additional, Liu, Tsung-li, additional, Long, Grace, additional, Ma, Rui, additional, Mailloux, John M., additional, Mandla, Kyle A., additional, Martinez, Anyssa R., additional, Mass, Max, additional, McKean, Daniel T., additional, Meron, Michael, additional, Miller, Edmund A., additional, Moh, Celyne S., additional, Moore, Rachel K., additional, Moreno, Juan, additional, Neysmith, Jordan M., additional, Niman, Cassandra S., additional, Nunez, Jesus M., additional, Ojeda, Micah T., additional, Ortiz, Sara Espinosa, additional, Owens, Jenna, additional, Piland, Geoffrey, additional, Proctor, Daniel J., additional, Purba, Josua B., additional, Ray, Michael, additional, Rong, Daisong, additional, Saade, Virginia M., additional, Saha, Sanchari, additional, Tomas, Gustav Santo, additional, Scheidler, Nicholas, additional, Sirajudeen, Luqmanal H., additional, Snow, Samantha, additional, Stengel, Gudrun, additional, Stinson, Ryan, additional, Stone, Michael J., additional, Sundseth, Keoni J., additional, Thai, Eileen, additional, Thompson, Connor J., additional, Tjioe, Marco, additional, Trejo, Christy L., additional, Trieger, Greg, additional, Truong, Diane Ni, additional, Tse, Ben, additional, Voiles, Benjamin, additional, Vuong, Henry, additional, Wong, Jennifer C., additional, Wu, Chiung-Ting, additional, Yu, Hua, additional, Yu, Yingxian, additional, Yu, Ming, additional, Zhang, Xi, additional, Zhao, Da, additional, Zheng, Genhua, additional, He, Molly, additional, and Previte, Michael, additional

Published
2023
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5. Lipid membranes carrying lipophilic cholesterol-based oligonucleotides-characterization and application on layer-by-layer coated particles

Author

Bunge, Andreas, Loew, Martin, Pescador, Paula, Arbuzova, Anna, Brodersen, Nicolai, Jing Kang, Dahne, Lars, Liebscher, Jrgen, Herrmann, Andreas, Stengel, Gudrun, and Huster, Daniel

Subjects
Cholesterol -- Chemical properties, Lipid membranes -- Composition, Oligonucleotides -- Chemical properties, Chemicals, plastics and rubber industries
Published
2009

6. Effect of transition metal ions on the fluorescence and Taqcatalyzed PCR of tricyclic cytidine analogues

Author

Stengel, Gudrun, Purse, Byron, and Kuchta, Robert D.

Subjects
Ions, Phenothiazines, Oxazines, Biocatalysis, Transition Elements, Humans, Taq Polymerase, DNA, Polymerase Chain Reaction, Sensitivity and Specificity, Article, Fluorescence
Abstract

The cytosine analogs 1,3-diaza-2-oxophenothiazine (tC) and 1,3-diaza-2-oxophenoxazine (tCo) stand out among fluorescent bases due to their unquenched fluorescence emission in double-stranded DNA. Recently, we reported a method for the generation of densely tCo-labeled DNA by polymerase chain reaction (PCR) that relied on the use of the extremely thermostable Deep Vent polymerase. We have now developed a protocol that employs the more commonly used Taq polymerase. Supplementing the PCR with Mn(2+) or Co(2+) ions dramatically increased the amount of tCo triphosphate (dtCoTP) incorporated and, thus, enhanced the brightness of the PCR products. The resulting PCR products could be easily detected in gels based on their intrinsic fluorescence. The Mn(2+) ions modulate the PCR by improving the bypass of template tCo and the overall catalytic efficiency. In contrast to the lower fidelity during tCo bypass, Mn(2+) improved the ability of Taq polymerase to distinguish between dtCoTP and dTTP when copying a template dA. Interestingly, Mn(2+) ions hardly affect the fluorescence emission of tC(o), whereas the coordination of Co(2+) ions with the phosphate groups of DNA and nucleotides statically quenches tC(o) fluorescence with small reciprocal Stern-Vollmer constants of 10-300μM.

Published
2011

7. High Density Labeling of PCR Products with the Fluorescent Analogue tCo

Author

Stengel, Gudrun, Urban, Milan, Purse, Byron W., and Kuchta, Robert D.

Subjects
Oxazines, DNA, DNA-Directed DNA Polymerase, Base Pairing, Polymerase Chain Reaction, Article, Fluorescent Dyes
Abstract

Fluorescent DNA of high molecular weight is an important tool for studying the physical properties of DNA and DNA-protein interactions, and it plays a key role in modern biotechnology for DNA sequencing and detection. While several DNA polymerases can incorporate large numbers of dye-linked nucleotides into primed DNA templates, the amplification of the resulting densely labeled DNA strands by polymerase chain reaction (PCR) is problematic. Here, we report a method for high density labeling of DNA in PCR reactions employing the 5'-triphosphate of 1,3-diaza-2-oxo-phenoxazine (tCo) and Deep Vent DNA polymerase. tCo is a fluorescent cytosine analogue that absorbs and emits light at 365 and 460 nm, respectively. We obtained PCR products that were fluorescent enough to directly visualize them in a gel by excitation with long UV light, thus eliminating the need for staining with ethidium bromide. Reactions with Taq polymerase failed to produce PCR products in the presence of only small amounts of dtCoTP. A comparative kinetic study of Taq and Deep Vent polymerase revealed that Taq polymerase, although it inserts dtCoTP with high efficiency opposite G, is prone to forming mutagenic tCo-A base pairs and does not efficiently extend base pairs containing tCo. These kinetics features explain the poor outcome of the PCR reactions with Taq polymerase. Since tCo substitutes structurally for cytosine, the presented labeling method is believed to be less invasive than labeling with dye-linked nucleotides and, therefore, produces DNA that is ideally suited for biophysical studies.

Published
2009

8. Plastic Trash goes Biohybrid'-Rapid and Selective Functionalization of Inert Plastic Surfaces with Biomolecules

Author

Schiller, Stefan M, Kambhampati, Dev, Stengel, Gudrun, Dolderer, Jürgen, Nielsen, Peter E., Förch, Renate, Knoll, W, Schiller, Stefan M, Kambhampati, Dev, Stengel, Gudrun, Dolderer, Jürgen, Nielsen, Peter E., Förch, Renate, and Knoll, W

Abstract

Udgivelsesdato: jan 2010, The covalent functionalization of "inert" polymers such as polypropylene with biomolecules for biocompatible or biosensor surfaces is challenging. Here we present a powerful approach to covalently modify "inert" macromolecular surfaces with biomacromolecules reusing old plastic material. A special emphasis was placed on easily accessible materials and a process which is easy, fast, efficient, cheap, and reliable. "Plastic trash" (lids from Eppendorf® pipet tip containers) was used as a polymer substrate to demonstrate the use/reuse of commercial packing material to covalently modify this material with a thin reactive plasma polymerized maleic anhydride nanolayer network, which can be subsequently modified with biomolecules for various applications, e.g., in tissue engineering and as biochips

Published
2010

9. Addressable adsorption of lipid vesicles and subsequent protein interaction studies

Author

Klenkar, Goran, Brian, Björn, Ederth, Thomas, Stengel, Gudrun, Hook, Fredrik, Piehler, Jacob, Liedberg, Bo, Klenkar, Goran, Brian, Björn, Ederth, Thomas, Stengel, Gudrun, Hook, Fredrik, Piehler, Jacob, and Liedberg, Bo

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.

Published
2008
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10. Supported lipid bilayers, tethered lipid vesicles, and vesicle fusion investigated using gravimetric, plasmonic, and microscopy techniques

Author

Hook, Fredrik, Stengel, Gudrun, Dahlin, Andreas B., Gunnarsson, Anders, Jonsson, Magnus P., Jonsson, Peter, Reimhult, Erik, Simonsson, Lisa, Svedhem, Sofia, Hook, Fredrik, Stengel, Gudrun, Dahlin, Andreas B., Gunnarsson, Anders, Jonsson, Magnus P., Jonsson, Peter, Reimhult, Erik, Simonsson, Lisa, and Svedhem, Sofia

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.

Published
2008
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18. Mapping multiple RNA modifications simultaneously by proximity barcode sequencing.

Author

Sendinc E, Yu H, Hwang Fu YH, Santos J, Johnson Z, Kirstein JR, Niu J, Chabot MB, Cantu VA, Džakula Ž, Lam Q, Anmangandla A, Burcham TS, Davis EM, Miles ZD, Price AD, Purse BW, Gregory RI, and Stengel G

Abstract

RNA is subject to a multitude of different chemical modifications that collectively represent the epitranscriptome. Individual RNA modifications including N6-methyladenosine (m 6 A) on mRNA play essential roles in the posttranscriptional control of gene expression. Recent technological advances have enabled the transcriptome-wide mapping of certain RNA modifications, to reveal their broad relevance and characteristic distribution patterns. However, convenient methods that enable the simultaneous mapping of multiple different RNA marks within the same sample are generally lacking. Here we present EpiPlex RNA modification profiling, a bead-based proximity barcoding assay with sequencing readout that expands the scope of molecular recognition-based RNA modification detection to multiple targets, while providing relative quantification and enabling low RNA input. Measuring signal intensity against spike-in controls provides relative quantification, indicative of the RNA mod abundance at each locus. We report on changes in the modification status of HEK293T cells upon treatment with pharmacological inhibitors separately targeting METTL3, the dominant m 6 A writer enzyme, and the EIF4A3 component of the exon junction complex (EJC). The treatments resulted in decreased or increased m 6 A levels, respectively, without effect on inosine levels. Inhibiting the helicase activity of EIF4A3 and EIF4A3 knockdown both cause a significant increase of m 6 A sites near exon junctions, consistent with the previously reported role of EIF4A3 in shaping the m 6 A landscape. Thus, EpiPlex offers a reliable and convenient method for simultaneous mapping of multiple RNA modifications to facilitate epitranscriptome studies.

Published
2024
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