Folding Double-Stranded DNA into Designed Shapes with Triplex-Forming Oligonucleotides.

The compaction and organization of genomic DNA is a central mechanism in eukaryotic cells, but engineered architectural control over double-stranded DNA (dsDNA) is notably challenging. Here, long dsDNA templates are folded into designed shapes via triplex-mediated self-assembly. Triplex-forming oligonucleotides (TFOs) bind purines in dsDNA via normal or reverse Hoogsteen interactions. In the triplex origami methodology, these non-canonical interactions are programmed to compact dsDNA (linear or plasmid) into well-defined objects, which demonstrate a variety of structural features: hollow and raster-filled, single- and multi-layered, with custom curvatures and geometries, and featuring lattice-free, square-, or honeycomb-pleated internal arrangements. Surprisingly, the length of integrated and free-standing dsDNA loops can be modulated with near-perfect efficiency; from hundreds down to only six bp (2 nm). The inherent rigidity of dsDNA promotes structural robustness and non-periodic structures of almost 25.000 nt are therefore formed with fewer unique starting materials, compared to other DNA-based self-assembly methods. Densely triplexed structures also resist degradation by DNase I. Triplex-mediated dsDNA folding is methodologically straightforward and orthogonal to Watson-Crick-based methods. Moreover, it enables unprecedented spatial control over dsDNA templates.

An albumin-angiotensin converting enzyme 2-based SARS-CoV-2 decoy with FcRn-driven half-life extension.

The emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutants and breakthrough infections despite available coronavirus disease 2019 (COVID-19) vaccines calls for antiviral therapeutics. The application of soluble angiotensin converting enzyme 2 (ACE2) as a SARS-CoV-2 decoy that reduces cell bound ACE2-mediated virus entry is limited by a short plasma half-life. This work presents a recombinant human albumin ACE2 genetic fusion (rHA-ACE2) to increase the plasma half-life by an FcRn-driven cellular recycling mechanism, investigated using a wild type (WT) albumin sequence and sequence engineered with null FcRn binding (NB). Binding of rHA-ACE2 fusions to SARS-CoV-2 spike protein subdomain 1 (S1) was demonstrated (WT-ACE2 KD = 32.8 nM and NB-ACE2 KD = 31.7 nM) using Bio-Layer Interferometry and dose-dependent in vitro inhibition of host cell infection of pseudotyped viruses displaying surface SARS-CoV-2 spike (S) protein. FcRn-mediated in vitro recycling was translated to a five times greater plasma half-life of WT-ACE2 (t½ ß = 13.5 h) than soluble ACE2 (t½ ß = 2.8 h) in humanised FcRn/albumin double transgenic mice. The rHA-ACE2-based SARS-CoV-2 decoy system exhibiting FcRn-driven circulatory half-life extension introduced in this work offers the potential to expand and improve the anti-COVID-19 anti-viral drug armoury. STATEMENT OF SIGNIFICANCE: The COVID-19 pandemic has highlighted the need for rapid development of efficient antiviral therapeutics to combat SARS-CoV-2 and new mutants to lower morbidity and mortality in severe cases, and for people that are unable to receive a vaccine. Here we report a therapeutic albumin ACE2 fusion protein (rHA-ACE2), that can bind SARS-CoV-2 S protein decorated virus-like particles to inhibit viral infection, and exhibits extended in vivo half-life compared to ACE2 alone. Employing ACE2 as a binding decoy for the virus is expected to efficiently inhibit all SARS-CoV-2 mutants as they all rely on binding with endogenous ACE2 for viral cell entry and, therefore, rHA-ACE2 constitutes a versatile addition to the therapeutic arsenal for combatting COVID-19.

Dynein links engulfment and execution of apoptosis via CED-4/Apaf1 in C. elegans.

Apoptosis ensures removal of damaged cells and helps shape organs during development by removing excessive cells. To prevent the intracellular content of the apoptotic cells causing damage to surrounding cells, apoptotic cells are quickly cleared by engulfment. Tight regulation of apoptosis and engulfment is needed to prevent several pathologies such as cancer, neurodegenerative and autoimmune diseases. There is increasing evidence that the engulfment machinery can regulate the execution of apoptosis. However, the underlying molecular mechanisms are poorly understood. We show that dynein mediates cell non-autonomous cross-talk between the engulfment and apoptotic programs in the Caenorhabditis elegans germline. Dynein is an ATP-powered microtubule-based molecular motor, built from several subunits. Dynein has many diverse functions including transport of cargo around the cell. We show that both dynein light chain 1 (DLC-1) and dynein heavy chain 1 (DHC-1) localize to the nuclear membrane inside apoptotic germ cells in C. elegans. Strikingly, lack of either DLC-1 or DHC-1 at the nuclear membrane inhibits physiological apoptosis specifically in mutants defective in engulfment. This suggests that a cell fate determining dialogue takes place between engulfing somatic sheath cells and apoptotic germ cells. The underlying mechanism involves the core apoptotic protein CED-4/Apaf1, as we find that DLC-1 and the engulfment protein CED-6/GULP are required for the localization of CED-4 to the nuclear membrane of germ cells. A better understanding of the communication between the engulfment machinery and the apoptotic program is essential for identifying novel therapeutic targets in diseases caused by inappropriate engulfment or apoptosis.

A novel excision selection method for isolation of antibodies binding antigens expressed specifically by rare cells in tissue sections.

There is a growing appreciation of single cell technologies to provide increased biological insight and allow development of improved therapeutics. The central dogma explains why single cell technologies is further advanced in studies targeting nucleic acids compared to proteins, as nucleic acid amplification makes experimental detection possible. Here we describe a novel method for single round phage display selection of antibody fragments from genetic libraries targeting antigens expressed by rare cells in tissue sections. We present and discuss the results of two selections of antibodies recognizing antigens expressed by perivascular cells surrounding capillaries located in a human brain section; with the aim of identifying biomarkers expressed by pericytes. The area targeted for selection was identified by a known biomarker and morphological appearance, however in situ hybridizations to nucleic acids can also be used for the identification of target cells. The antibody selections were performed directly on the tissue sections followed by excision of the target cells using a glass capillary attached to micromanipulation equipment. Antibodies bound to the target cells were characterized using ELISA, immunocytochemistry and immunohistochemistry. The described method will provide a valuable tool for the discovery of novel biomarkers on rare cells in all types of tissues.

Selection of cell-type specific antibodies on tissue-sections using phage display.

With the advent of modern technologies enabling single cell analysis, it has become clear that small sub-populations of cells or even single cells can drive the phenotypic appearance of tissue, both diseased and normal. Nucleic acid based technologies allowing single cell analysis has been faster to mature, while technologies aimed at analysing the proteome at a single cell level is still lacking behind, especially technologies which allow single cell analysis in tissue. Introducing methods, that allows such analysis, will pave the way for discovering new biomarkers with more clinical relevance, as these may be unique for microenvironments only present in tissue and will avoid artifacts introduced by in vitro studies. Here, we introduce a technology enabling biomarker identification on small sub-populations of cells within a tissue section. Phage antibody libraries are applied to the tissue sections, followed by washing to remove non-bound phage particles. To eliminate phage antibodies binding to antigens ubiquitously expressed and retrieve phage antibodies binding specifically to antigens expressed by the sub-population of cells, the area of interest is protected by a 'shadow stick'. The phage antibodies on the remaining areas on the slide are exposed to UV light, which introduces cross-links in the phage genome, thus rendering them non-replicable. In this work we applied the technology, guided by CD31 expressing endothelial cells, to isolate recombinant antibodies specifically binding biomarkers expressed either by the cell or in the microenvironment surrounding the endothelial cell.

Degradation of C-terminal tag sequences on domain antibodies purified from E. coli supernatant.

Expression of recombinant proteins often takes advantage of peptide tags expressed in fusion to allow easy detection and purification of the expressed proteins. However, as the fusion peptides most often are flexible appendages at the N- or C-terminal, proteolytic cleavage may result in removal of the tag sequence. Here, we evaluated the functionality and stability of 14 different combinations of commonly used tags for purification and detection of recombinant antibody fragments. The tag sequences were inserted in fusion with the c-terminal end of a domain antibody based on the HEL4 scaffold in a phagemid vector. This particular antibody fragment was able to refold on the membrane after blotting, allowing us to detect c-terminal tag breakdown by use of protein A in combination with detection of the tags in the specific constructs. The degradation of the c-terminal tags suggested specific sites to be particularly prone to proteolytic cleavage, leaving some of the tag combinations partially or completely degraded. This specific work illustrates the importance of tag design with regard to recombinant antibody expression in E. coli, but also aids the more general understanding of protein expression.

A novel heavy domain antibody library with functionally optimized complementarity determining regions.

Today a number of synthetic antibody libraries of different formats have been created and used for the selection of a large number of recombinant antibodies. One of the determining factors for successful isolation of recombinant antibodies from libraries lies in the quality of the libraries i.e. the number of correctly folded, functional antibodies contained in the library. Here, we describe the construction of a novel, high quality, synthetic single domain antibody library dubbed Predator. The library is based on the HEL4 domain antibody with the addition of recently reported mutations concerning the amino acid composition at positions critical for the folding characteristics and aggregation propensities of domain antibodies. As a unique feature, the CDR3 of the library was designed to mimic the natural human immune response by designating amino acids known to be prevalent in functional antibodies to the diversity in CDR3. CDR randomizations were performed using trinucleotide synthesis to avoid the presence of stop codons. Furthermore a novel cycle free elongation method was used for the conversion of the synthesized single stranded DNA containing the randomized CDRs into double stranded DNA of the library. In addition a modular approach has been adopted for the scaffold in which each CDR region is flanked by unique restrictions sites, allowing easy affinity maturation of selected clones by CDR shuffling. To validate the quality of the library, one round phage display selections were performed on purified antigens and highly complex antigen mixtures such as cultured eukaryotic cells resulting in several specific binders. The further characterization of some of the selected clones, however, indicates a reduction in thermodynamic stability caused by the inclusion the additional mutations to the HEL4 scaffold.

Raising antibodies against circulating foetal cells from maternal peripheral blood.

OBJECTIVE: Cells of foetal origin circulating in the maternal peripheral bloodstream present a unique source for non-invasive prenatal diagnostics. The aims of this study were to raise antibodies against identified circulating foetal cells from the maternal blood, test the properties of these antibodies and to determine the foetal cell type recognised by the antibodies. METHOD: Cells from a male foetus were identified in a maternal blood sample by FISH analysis of the X- and Y- chromosomes. The identified cells were subjected to phage display selection using a novel single cell selection strategy. Selected antibodies were tested by immunocytochemistry on foetal and adult tissue arrays, an endothelial cell line, and peripheral blood mononuclear cells. RESULTS: Three identified foetal cells subjected to antibody selection, yielded a total of 12 antibodies. Three antibodies gave distinct staining patterns on tissue arrays, and endothelial cells. One antibody, SF1.3, shows specific staining of a subpopulation of peripheral blood mononuclear cells, including a fraction of CD34 positive cells. CONCLUSION: These findings indicate that the identified foetal cells could have been progenitor cells of haematopoietic origin. The antibody SF1.3 could be a potential tool toward non-invasive prenatal diagnostics.