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- Leino, Mattias, 1989-
(författare)
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Advancing DNA-based proximity methods
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cellular functions are governed by intricate chains of interactions between proteins. In order to properly understand cellular biology one must look into, not only protein function, but also its interacting network. Furthermore, due to the large heterogeneity between cells within cultures and tissue samples it is important to retain spatial information to enable investigations on a single cell level. In order to achieve this, in situ methods play a large importance in further elucidation of these interacting networks.In order to investigate interactions between macromolecules such as proteins and nucleic acids, many outstanding methods have been developed. Some focusing on larger scale analysis, some on live cell imaging and some on detecting novel interactions. Our own group has focused on in situ methods utilizing DNA conjugated antibodies. DNA itself is a great macromolecule to work with, it can be produced synthetically, DNA hybridization is highly predictable and there is a large repertoire of DNA-modifying enzymes. This has been used in the development of methods such as proximity ligation assay (PLA) and Proximity-dependent initiation of hybridization chain reaction (ProxHCR).Both methods utilize antibodies conjugated with DNA in order to detect proximity events between two proteins. PLA utilizes ligation to confirm proximity, while ProxHCR utilizes a chain of strand displacements to do the same. Both methods work well, but no method is beyond further optimization.For PLA, a general concern lies in the formation of incorrectly interacting probes, resulting in incorrect ligations that yield linear fragments, incapable of producing visible signal. As a result PLA can produce a substantial amount of false negatives. To address this, we produced a similar method, Unfold, to streamline the probe interactions and ligations to improve efficiency.For ProxHCR the original method required overly stringent reactions conditions to allow for efficient strand displacements and thus strong signal. Furthermore, signal strength was further compromised by oligonucleotide quality. To improve these issues, the ProxHCR method was completely redesigned and oligonucleotide quality along with signal strength was improved by further purification.Both optimizations resulted in more efficient and versatile methods suitable for routine lab work and potential diagnostic use.
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| 2. |
- Leino, Mattias, 1989-, et al.
(författare)
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Purification of detection hairpins improves the performance of proximity-dependent initiation of hybridization chain reaction
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- To accurately diagnose cancer and other aberrant cellular conditions, investigations of several parameters are required. Not only morphology, protein and gene expression but also more complex investigations, such as protein modifications, translocations and interactions can provide essential data. In order to investigate such cellular events, several molecular tools have been developed. Among them is proximity-dependent initiation of hybridization chain reaction (ProxHCR), an inexpensive tool developed to provide accurate analysis of proximity events within cells. While previous versions of ProxHCR has been capable of detecting protein-proximity, lacking signal strength has resulted in poor ability to properly quantify imaged results. Here we show how further purification of the HCR detection hairpins considerably increase signal strength. The increased signal strength allow for accurate quantification of proximity events in line with the established PLA method. Furthermore we show how ProxHCR can be used to track PDGFR-β phosphorylation and successive recruitment of GRB2 and PI3K over time. These results show how ProxHCR can be a valuable, enzyme free alternative to other proximity analysis tools.
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| 3. |
- Leino, Mattias, 1989-, et al.
(författare)
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Purification of DNA oligonucleotides to improve hybridization chain reaction performance
- 2023
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Ingår i: New Biotechnology. - : Elsevier. - 1871-6784 .- 1876-4347. ; 76, s. 33-40
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Tidskriftsartikel (refereegranskat)abstract
- Hybridization chain-reaction (HCR) is technique to generate a linear polymerization of oligonucleotide hairpins, used in multiple molecular biology methods. The HCR reaction is dependent on that every hairpin is metastable in the absence of a triggering oligonucleotide and that every hairpin can continue the polymerization, which places a strong demand on oligonucleotide quality. In this paper we show how further purification can greatly increase polymerization potential. We found that a single extra PAGE-purification could greatly enhance hairpin polymerization both in solution and in situ. Purification using a ligation-based method further improved polymerization, yielding in situ immunoHCR stains at least 3.4-times stronger than non-purified control. This demonstrates the importance of not only good sequence design of the oligonucleotide hairpins, but also the demand for high quality oligonucleotides to accomplish a potent and specific HCR.
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| 4. |
- Leino, Mattias, 1989-, et al.
(författare)
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The effects of artificial E-cadherin matrix-induced embryonic stem cell scattering on paxillin and RhoA activation via a-catenin
- 2014
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Ingår i: Biomaterials. - : Elsevier. - 0142-9612 .- 1878-5905. ; 35:6, s. 1797-1806
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Tidskriftsartikel (refereegranskat)abstract
- Mechanical forces have been shown to affect stem cell behavior in a large array of ways. However, our understanding of how these mechanical cues may regulate the behavior of embryonic stem cells (ESCs) remains in its infancy. Here, we aim to clarify the effect of cell scattering on the regulation of Rho family GTPases Rac1 and RhoA as well as paxillin. Allowing ESCs to spread and scatter on a synthetically designed E-cadherin substratum causes phosphorylation of paxillin on consensus phosphorylation sites leading to activation of Rac1 and inactivation of RhoA. By culturing cells in presence of RhoA activator or growing cells to a highly confluent state reverses the effect of cell scattering phenotype. Knockdown of Ecadherin-adapter protein a-catenin revealed that it negatively affects paxillin phosphorylation and upregulates RhoA activity in compact cellular aggregates. Collectively these results indicate that cell scattering might cause a conformational change of a-catenin limiting its capacity to inhibit paxillin phosphorylation that causes an increase in Rac1 activation and RhoA deactivation. Understanding how synthetically designed extracellular matrix affect ESC signaling through mechanical cues brings a new aspect for stem cell engineers to develop technologies for controlling cell function.
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| 5. |
- Wenson, Leonie, et al.
(författare)
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The method developer's guide to oligonucleotide design
- 2024
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Ingår i: Expert Review of Proteomics. - : Taylor & Francis. - 1478-9450 .- 1744-8387. ; 21:1-3, s. 65-80
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Forskningsöversikt (refereegranskat)abstract
- IntroductionDevelopment of new methods is essential to make great leaps in science, opening up new avenues for research, but the process behind method development is seldom described.Areas coveredOver the last twenty years we have been developing several new methods, such as in situ PLA, proxHCR, and MolBoolean, using oligonucleotide-conjugated antibodies to visualize protein-protein interactions. Herein, we describe the rationale behind the oligonucleotide systems of these methods. The main objective of this paper is to provide researchers with a description on how we thought when we designed those methods. We also describe in detail how the methods work and how one should interpret results.Expert opinionUnderstanding how the methods work is important in selecting an appropriate method for your experiments. We also hope that this paper may be an inspiration for young researchers to enter the field of method development. Seeing a problem is a motivation to develop a solution.
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