Biology subjects

Krasnogor, N.

Publications and source records attributed to Krasnogor, N..

10 recordsLinked to original sources

bioRxiv2025

Optimizing DNA origami assembly through selection of scaffold sequences that minimise off-target interactions

DNA origami is a mainstay of DNA nanotechnology and several efforts have been devoted to understanding how various factors of the self-assembly reaction affect the final yield of the target origami structure. This study analyses how base sequence affects origami yield through the generation of off-target side reactions during selfassembly. Off-target bindings are an under-explored phenomenon and can potentially introduce unwanted assembly barriers and kinetic traps in the origami folding pathway. We developed a multi-objective computational approach that takes a given origami design and scores different scaffold sequences (and their complementary staples) for the prevalence of four different types of off-target binding events. Using our method on DNA origami, we can select bad regions of biological sequences (like lambda DNA phage) that, when used as origami scaffold sequences, have an excessive number of off-target side reactions for each shape. We show, using high-resolution atomic force microscopy (AFM), that these scaffold sequences largely fail to fold into the target triangle or rectangle structure in vitro, despite the scaffold sequence having a fully complementary staple set present. Conversely, using our method we can also select good regions of biological sequences. These sequences are deficient in off-target reactions and when used as origami scaffolds, fold more successfully into their target structures as characterised by AFM. These results have been validated in "blind" folding experiments at two different laboratories in which the experimenters did not know which scaffolds were good or bad folders. To further investigate assembly behaviour, optical tweezers experiments revealed distinct mechanical response profiles, correlating with scaffold-specific off-target interactions. While variants with higher GC content show a high mean unfolding force, variants with lower off-target binding demonstrated more uniform force-extension curves. Our analysis confirmed that high off-target binding leads to increased structural heterogeneity, as seen in the clustering behaviour of unfolding traces of OT experiments. Over-all, our work demonstrates how the off-target reactions implicit in base sequences can derail the origami self-assembly process if sufficiently prevalent, and we provide a software tool to select scaffold sequences that minimise off-target reactions for any DNA origami design.

bioengineering↗

bioRxiv2023

Reverse Engineering DNA Origami Nanostructure Designs from Raw Scaffold and Staple Sequence Lists

Designs for scaffolded DNA origami nanostructures are commonly and minimally published as the list of DNA staple and scaffold sequences required. In nearly all cases, high-level editable design files (e.g. caDNAno) which generated the low-level sequences are not made available. This de facto raw sequence exchange format allows published origami designs to be re-attempted in the laboratory by other groups, but effectively stops designs from being significantly modified or re-purposed for new future applications. To make the raw sequence exchange format more accessible to further design and engineering, in this work we propose the first algorithmic solution to the inverse problem of converting staple/scaffold sequences back to a guide schematic resembling the original origami schematic. The guide schematic can be used to aid the manual re-input of an origami into a CAD tool like caDNAno, hence recovering a high-level editable design file. Creation of a guide schematic can also be used to double check that a list of staple strand sequences does not have errors and indeed does assemble into a desired origami nanostructure prior to costly laboratory experimentation. We tested our reverse algorithm on 36 diverse origami designs from the literature and found that 29 origamis (81%) had a good quality guide schematic recovered from raw sequences. Our software is made available at https://revnano.readthedocs.io.

bioinformatics↗

bioRxiv2022

Light-up split Broccoli aptamer as a versatile tool for RNA assembly monitoring in cell-free TX-TL system, hybrid RNA/DNA origami tagging and DNA biosensing

Binary light-up aptamers are intriguing and emerging tools with potential in different fields. Herein, we demonstrate the versatility of a split Broccoli aptamer system able to turn on the fluorescence signal only in the presence of a complementary sequence. First, an RNA three-way junction harbouring the split system was assembled in an E. coli based cell-free TX-TL system where the folding of the functional aptamer is demonstrated. Then, the same strategy is introduced into a bio-orthogonal hybrid RNA/DNA rectangle origami characterized by atomic force microscopy: the activation of the split system through the origami self-assembly is demonstrated. Finally, our system is successfully used to detect femtomoles of a Campylobacter spp. DNA target sequence. Potential applications of our system include real-time monitoring of the self-assembly of nucleic acid-based devices in vivo and of intracellular delivery of therapeutic nanostructures, as well as in vitro and in vivo detection of different DNA/RNA target.

synthetic biology↗

bioRxiv2021

Targetron-assisted delivery of exogenous DNA sequences into Pseudomonas putida through CRISPR-aided counterselection

Genome editing methods based on Group II introns (known as Targetron technology) have been long used as a gene knock-out strategy in a wide range of organisms in a fashion independent of homologous recombination. Yet, their utility as delivery systems has been typically suboptimal because of their reduced efficiency of insertion when they carry exogenous sequences. We show that this limitation can be tackled and Targetron adapted as a general tool in Gram-negative bacteria. To this end, a set of broad host range standardized vectors were designed for conditional expression of the Ll.LtrB intron. After testing the correct functionality of these plasmids in Escherichia coli and Pseudomonas putida, we created a library of Ll.LtrB variants carrying cargo DNA sequences of different lengths to benchmark the capacity of intron-mediated delivery in these bacteria. Next, we combined CRISPR/Cas9-facilitated counterselection to increase the chances of finding genomic sites inserted with the thereby engineered introns. By following this pipeline, we were able to insert exogenous sequences of up to 600 bp at specific genomic locations in wild-type P. putida KT2440 and its {Delta}recA derivative. Finally, we were able to apply this technology to successfully tag this strain with an orthogonal short sequence (barcode) that acts as a unique identifier for tracking this microorganism in biotechnological settings. The results with P. putida exemplified the value of the Targetron approach for unrestricted delivery of small DNA fragments to the genomes of Gram-negative bacteria for a suite of genome editing endeavours.

synthetic biology↗

bioRxiv2021

Versioning Biological Cells for Trustworthy Cell Engineering

"Full-stack" biotechnology platforms for cell line (re)programming are on the horizon, due mostly to (a) advances in gene synthesis and editing techniques as well as (b) the growing integration with informatics, the internet of things and automation. These emerging platforms will accelerate the production and consumption of biological products. Hence, transparency, traceability and -ultimately-trustworthiness is required -from cradle to grave- for engineered cell lines and their engineering processes. We report here the first version control system for cell engineering that integrates a new cloud-based version control software for cell lines digital footprint with molecular barcoding of living samples. We argue that version control for cell engineering marks a significant step towards more open, reproducible, easier to trace and share, and more trustworthy engineering biology. One Sentence SummaryWe demonstrate a transparent and open way of engineering and sharing cell lines.

synthetic biology↗

bioRxiv2019

Co-transcriptional folding of a bio-orthogonal fluorescent scaffolded RNA origami

The scaffolded origami technique has provided an attractive tool for engineering nucleic acid nanostructures. This paper demonstrates scaffolded RNA origami folding in vitro in which all components are transcribed simultaneously in a single-pot reaction. Double-stranded DNA sequences are transcribed by T7 RNA polymerase into scaffold and staple strands able to correctly fold in high yield into the nanoribbon. Synthesis is successfully confirmed by atomic force microscopy and the unpurified transcription reaction mixture is analyzed by an in gel-imaging assay where the transcribed RNA nanoribbons are able to capture the specific dye through the reconstituted split Broccoli aptamer showing a clear green fluorescent band. Finally, we simulate the RNA origami in silico using the nucleotide-level coarse-grained model oxRNA to investigate the thermodynamic stability of the assembled nanostructure in isothermal conditions over a period of time. Our work suggests that the scaffolded origami technique is a valid, and potentially more powerful, assembly alternative to the single-stranded origami technique for future in vivo applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/864678v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@ff817aorg.highwire.dtl.DTLVardef@8da463org.highwire.dtl.DTLVardef@12381b7org.highwire.dtl.DTLVardef@194733_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2019

Linking Engineered Cells to Their Digital Twins: a Version Control System for Strain Engineering

1.As DNA sequencing and synthesis become cheaper and more easily accessible, the scale and complexity of biological engineering projects is set to grow. Yet, although there is an accelerating convergence between biotechnology and computing science, a deficit in software and laboratory techniques diminishes the ability to make biotechnology more agile, reproducible and transparent while, at the same time, limiting the security and safety of synthetic biology constructs. To partially address some of these problems, this paper presents an approach for physically linking engineered cells to their digital footprint - we called it digital twinning. This enables the tracking of the entire engineering history of a cell line in a specialised version control system for collaborative strain engineering.

synthetic biology↗

bioRxiv2019

NIHBA: A Network Interdiction Approach with Hybrid Benders Algorithm for Strain Design

Flux balance analysis (FBA) based bilevel optimisation has been a great success in redesigning metabolic networks for biochemical overproduction. To date, many computational approaches have been developed to solve the resulting bilevel optimisation problems. However, most of them are of limited use due to biased optimality principle, poor scalability with the size of metabolic networks, potential numeric issues, or low quantity of design solutions in a single run. In this work, we have employed a network interdiction model free of growth optimality assumptions, a special case of bilevel optimisation, for computational strain design and have developed a hybrid Benders algorithm (HBA) that deals with complicating binary variables in the model, thereby achieving high efficiency without numeric issues in search of best design strategies. More importantly, HBA can list solutions that meet users production requirements during the search, making it possible to obtain numerous design strategies at a small runtime overhead (typically[~] 1 hour).

bioengineering↗

bioRxiv2018

Easybiotics: a GUI for 3D physical modelling of multi-species bacterial populations

Motivation3D physical modelling is a powerful computational technique which allows for the simulation of complex systems such as consortia of mixed bacterial species. The complexities in physical modelling reside in the knowledge intensive model building process and the computational expense in calculating their numerical solutions. These models can offer insights into microbiology, both in understanding natural systems and as design tools for developing novel synthetic bacteria. Developing a robust synthetic system typically requires multiple iterations around the specify->design->build->test cycle to meet specifications. This process is laborious and expensive for both the computational and laboratory aspects, hence any improvement in any of the workflow steps would be welcomed. We have previously introduced Simbiotics (Naylor, 2017) a powerful and flexible platform for designing and analysing three-dimensional simulations of mixed species bacterial populations. Simbiotics requires programming experience to use which creates barriers to entry for use of the tool.\n\nResultsIn the spirit of enabling biologists who may not have programming skills to install and utilise Simbiotics, we present in this application note Easybiotics, a user-friendly graphical user interface for Simbiotics. Users may design, simulate and analyse models from within the GUI, with features such as live graph plotting and parameter sweeps. Easybiotics provides full access to all of Simbiotics simulation features, such as cell growth, motility and gene regulation.\n\nAvailabilityEasybiotics and Simbiotics are free to use under the GPL3.0 license, and can be found at: https://bitbucket.org/simbiotics/simbiotics/wiki/Home\n\nContactnatalio.krasnogor@newcastle.ac.uk

bioinformatics↗

bioRxiv2017

Human iPSC-derived RPE and retinal organoids reveal impaired alternative splicing of genes involved in pre-mRNA splicing in PRPF31 autosomal dominant retinitis pigmentosa

Mutations in pre-mRNA processing factors (PRPFs) cause 40% of autosomal dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed PRPFs cause retinal disease. To understand the molecular basis of this phenotype, we have generated RP type 11 (PRPF31-mutated) patient-specific retinal organoids and retinal pigment epithelium (RPE) from induced pluripotent stem cells (iPSC). Impaired alternative splicing of genes encoding pre-mRNA splicing proteins occurred in patient-specific retinal cells and Prpf31+/- mouse retinae, but not fibroblasts and iPSCs, providing mechanistic insights into retinal-specific phenotypes of PRPFs. RPE was the most affected, characterised by loss of apical-basal polarity, reduced trans-epithelial resistance, phagocytic capacity, microvilli, and cilia length and incidence. Disrupted cilia morphology was observed in patient-derived-photoreceptors that displayed progressive features associated with degeneration and cell stress. In situ gene-editing of a pathogenic mutation rescued key structural and functional phenotypes in RPE and photoreceptors, providing proof-of-concept for future therapeutic strategies.\n\neTOCPRPF31 is a ubiquitously expressed pre-mRNA processing factor that when mutated causes autosomal dominant RP. Using a patient-specific iPSC approach, Buskin and Zhu et al. show that retinal-specific defects result from altered splicing of genes involved in the splicing process itself, leading to impaired splicing, loss of RPE polarity and diminished phagocytic ability as well as reduced cilia incidence and length in both photoreceptors and RPE.\n\nHighlightsO_LISuccessful generation of iPSC-derived RPE and photoreceptors from four RP type 11 patients\nC_LIO_LIRPE cells express the mutant PRPF31 protein and show the lowest expression of wildtype protein\nC_LIO_LIPRPF31 mutations result in altered splicing of genes involved in pre-mRNA splicing in RPE and retinal organoids\nC_LIO_LIPrpf31 haploinsufficiency results in altered splicing of genes involved in pre-mRNA splicing in mouse retina\nC_LIO_LIRPE cells display loss of polarity, reduced barrier function and phagocytosis\nC_LIO_LIPhotoreceptors display shorter and fewer cilia and degenerative features\nC_LIO_LIRPE cells display most abnormalities suggesting they might be the primary site of pathogenesis\nC_LIO_LIIn situ gene editing corrects the mutation and rescues key phenotypes\nC_LI

genetics↗