Biology subjects

Krieg, E.

Publications and source records attributed to Krieg, E..

9 recordsLinked to original sources

bioRxiv2026

Mapping 3D cellular mechanical activity with matrix-embedded DNA force-history probes

Cells continuously integrate mechanical cues from the surrounding extracellular matrix to control fundamental biological processes such as proliferation and migration. Yet, studying the mechanical activity of cells in 3D over time is challenging, low-throughput, and requires specialized equipment. Here, we introduce DNA-based force-history probes, which convert transient pico-Newton forces into cumulative fluorescent signals within a mechanically adjustable DNA-crosslinked cell culture matrix. The probes provide control over signal lifetimes, allowing stress patterns to be recorded over minutes to days with tunable temporal memory. We use this system to visualize the mechanical activity of breast cancer spheroids and map the trajectories of migrating cancer cells. By combining different fluorophores and DNA-encoded signal lifetimes, we produce dual-color probes that associate temporal information to mechanical events. Overall, force-history probes provide an endpoint-readable record of mechanical cell-matrix activity, offering new opportunities for studying cell function in physiology and disease.

bioengineering↗

bioRxiv2026

Programmable DNA Origami Caps for Site-Selective Functionalization of Microtubule Tips and Lattice Defects

Microtubules are central components of cytoskeletal transport systems and have been widely repurposed as active elements in motor-driven nanodevices. However, site-specific functionalization of stabilized microtubules remains a fundamental challenge, as the tubulin lattice presents chemically indistinguishable binding sites along its length. Here we report a strategy for selective end-functionalization of stabilized microtubules using DNA origami nanostructures. By coupling DNA origami to Fab fragments targeting acetylated -tubulin Lys40 within the microtubule lumen, and exploiting steric exclusion of the origami from the lattice interior, binding is confined to accessible sites at microtubule ends and lattice defects. Using a six-helix bundle origami as a minimal construct, we demonstrate selective tip labelling of gliding microtubules without perturbing kinesin-driven motility. The same structures additionally mark lattice defects, enabling dynamic visualization of defect sites during transport. Furthermore, we show that tip-bound origami can hybridize with complementary DNA strands to capture cargo from surfaces in motion, establishing programmable, end-specific loading. This approach introduces a generalizable route to spatially controlled functionalization of cytoskeletal filaments, enabling new capabilities in molecular transport, nanoscale assembly, and the study of microtubule integrity and repair.

bioengineering↗

bioRxiv2025

LASSO: versatile and selective biomolecule pulldown with combinatorial DNA-crosslinked polymers

Current methods for sequence-selective biomolecule isolation suffer from high cost, off-target effects, and limited flexibility. Here, we introduce LASSO (crossLink-Assisted Sequence-Selective isOlation), a versatile platform using programmable polymer phase separation to capture biomolecules under native conditions. LASSO relies on combinatorial crosslinker libraries--diverse mixtures of DNA strands that collectively trigger the formation of highly swollen polymer agglomerates with near-zero background binding. We demonstrate >80% pulldown efficiency for diverse targets, including DNA, SARS-CoV-2 RNA, and human thrombin. LASSO provides 8-20x higher binding capacity (4 nmol/mg polymer) than commercial microbeads. In RNA-seq workflows, LASSO depleted ribosomal RNA with 86% efficiency while yielding up to 7x fewer off-target outliers (p<0.001) versus state-of-the-art magnetic beads (riboPOOLs) and RNase H (NEBNext) kits. Thrombin was captured via switchable aptamers with 92% efficiency, and a gentle release mechanism allowed the subsequent isolation of 72% enzymatically active proteins from the polymer. LASSOs cost-effectiveness ($0.96/sample vs. $46-$51 for commercial kits), long-term stability (7+ years), simple usage, and modularity position it to transform diagnostics, transcriptomics, and bionanotechnology workflows.

bioengineering↗

bioRxiv2025

Programmable viscoelastic hydrogels uncover mechano-sensing timescales and direct cell polarity

Mechanical interactions between cells and their three-dimensional environment govern fundamental processes in tissue development and disease. Yet, how cells interrogate and respond to mechanical cues remains incompletely understood. Reproducing the mechanical properties of natural tissues in vitro is a key challenge, as these tissues exhibit complex viscoelastic behaviors and undergo continuous remodeling throughout an organisms development. Here, we leverage principles of dynamic DNA nanotechnology to introduce programmable mechanical cues into a synthetic hydrogel matrix that guides and interrogates the development of embedded cells. By systematically modulating matrix stress relaxation and stiffness, we uncover two distinct timescales of mechano-sensitive processes controlling apical-basal polarity in Madin-Darby Canine Kidney (MDCK) cells: the fast timescale (1-3 min) is linked to rapid integrin-mediated signaling, while the slower process (3-9 h) is associated with cytoskeletal reinforcements. Our analysis highlights that the commonly reported matrix "stiffness" value, often measured as the storage modulus at [~]1 Hz, has limited physiological relevance. These findings prompted us to develop a novel switchable DNA crosslinker that enables dynamic changes in viscoelasticity during ongoing cell culture. This controlled matrix reconfiguration induces reversible cell polarity inversions and guides the morphogenesis of complex multicellular structures. The material platform therefore offers unprecedented control over the mechanical microenvironment, opening new avenues for advanced applications in biophysics, tissue engineering, and disease modeling.

bioengineering↗

bioRxiv2024

Assembling a true "Olympic Gel" from >16,000 combinatorial DNA rings

Olympic gels are an elusive form of soft matter, comprising a three-dimensional network of mechanically interlocked cyclic molecules. In the absence of defined network junctions, the high conformational freedom of the molecules was previously theorized to confer unique mechanical properties to Olympic gels, such as non-linear elasticity and unconventional swelling characteristics. However, the synthesis of an Olympic gel exhibiting these intriguing features is challenging, since unintended crosslinking and polymerization processes are often favored over cyclization. Here, we report the successful assembly of a true Olympic gel from a library of DNA rings comprising more than 16,000 distinct molecules. Each of these rings contains a unique sequence domain that can be enzymatically activated to produce reactive termini that favor intramolecular cyclization. We characterized the genetic, mechanical, and structural characteristics of the material by next-generation sequencing, oscillatory rheology, large-scale computational simulations, atomic force microscopy, and cryogenic electron microscopy. Our results confirm the formation of a stable Olympic gel, which exhibits unique swelling behavior and an elastic response that is exclusively determined by entanglements, yet persists on long time scales. By combining concepts from polymer physics, synthetic biology, and DNA nanotechnology, this new material class provides a flexible experimental platform for future studies into the effects of network topology on macroscopic material properties and its function as a carrier of genetic information in biological and biomimetic systems. This work moreover demonstrates that exotic material properties can emerge in systems with a high compositional complexity that is more reminiscent of biological than synthetic matter.

bioengineering↗

bioRxiv2024

A spring-loaded and leakage-tolerant synthetic gene switch for in-vitro detection of DNA and RNA

Nucleic acid tests (NATs) are essential for biomedical diagnostics. Traditional NATs, often complex and expensive, have prompted the exploration of Toehold-Mediated Strand Displacement (TMSD) circuits as an economical alternative. However, the wide application of TMSD-based reactions is limited by leakage--the spurious activation of the reaction leading to high background signals and false positives. Here we introduce a new TMSD cascade that recognizes a custom nucleic acid input and generates an amplified output. The system is based on a pair of thermodynamically spring-loaded DNA modules. The binding of a predefined nucleic acid target triggers an intermolecular reaction that activates a T7 promoter, leading to the perpetual transcription of a fluorescent aptamer that can be detected by a smartphone camera. The system is designed to permit the selective depletion of leakage byproducts to achieve high sensitivity and zero-background signal in the absence of the correct trigger. Using Zika virus (ZIKV)- and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-derived nucleic acid sequences, we show that the assay generates a reliable target-specific readout. Native RNA can be directly detected under isothermal conditions, without requiring reverse transcription, with a sensitivity as low as 200 attomole. The modularity of the assay allows easy re-programming for the detection of other targets by exchanging a single sequence domain. This work provides a low-complexity and high-fidelity synthetic biology tool for point-of-care diagnostics and for the construction of more complex biomolecular computations.

synthetic biology↗

bioRxiv2022

Dynamic matrices with DNA-encoded viscoelasticity for advanced cell and organoid culture

3D cell and organoid cultures, which allow in vitro studies of organogenesis and carcinogenesis, rely on the mechanical support of viscoelastic matrices. However, commonly used matrix materials lack rational design and control over key cell-instructive properties. Herein, we report a class of fully synthetic hydrogels based on novel DNA libraries that self-assemble with ultra-high molecular weight polymers, forming a dynamic DNA-crosslinked matrix (DyNAtrix). DyNAtrix enables, for the first time, computationally predictable, systematic, and independent control over critical viscoelasticity parameters by merely changing DNA sequence information without affecting the compositional features of the system. This approach enables: (1) thermodynamic and kinetic control over network formation; (2) adjustable heat-activation for the homogeneous embedding of mammalian cells; and (3) dynamic tuning of stress relaxation times over several orders of magnitude, recapitulating the mechanical characteristics of living tissues. DyNAtrix is self-healing, printable, exhibits high stability, cyto-and hemocompatibility, and controllable degradation. DyNAtrix-based 3D cultures of human mesenchymal stromal cells, pluripotent stem cells, canine kidney cysts, and human placental organoids exhibit high viability (on par or superior to reference matrices), proliferation, and morphogenesis over several days to weeks. DyNAtrix thus represents a programmable and versatile precision matrix, paving the way for advanced approaches to biomechanics, biophysics, and tissue engineering. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/510936v2_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1a9c486org.highwire.dtl.DTLVardef@a578d7org.highwire.dtl.DTLVardef@1a08f7dorg.highwire.dtl.DTLVardef@2c64af_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2019

A smart polymer for sequence-selective binding, pulldown and release of DNA targets

Selective isolation of DNA is crucial for applications in biology, bionanotechnology, clinical diagnostics and forensics. We herein report a smart methanol-responsive polymer (MeRPy) that can be programmed to bind and separate single- as well as double-stranded DNA targets. Captured targets are quickly isolated and released back into solution by denaturation (sequence-agnostic) or toehold-mediated strand displacement (sequence-selective). The latter mode allows 99.8% efficient removal of unwanted sequences and 79% recovery of highly pure target sequences. We applied MeRPy for the depletion of insulin, glucagon, and transthyretin cDNA from clinical next-generation sequencing (NGS) libraries. This step improved data quality for low-abundance transcripts in expression profiles of pancreatic tissues. Its low cost, scalability, high stability and ease of use make MeRPy suitable for diverse applications in research and clinical laboratories, including enhancement of NGS libraries, extraction of DNA from biological samples, preparative-scale DNA isolations, and sorting of DNA-labeled non-nucleic acid targets.

molecular biology↗

bioRxiv2019

Rapid and scalable in vitro production of single-stranded DNA

We present a rapid, scalable, user-friendly method for in vitro production of high-purity single-stranded DNA (ssDNA) ranging from 89-3315 nucleotides in length. PCR with a forward primer bearing a methanol-responsive polymer generates a tagged amplicon that enables selective precipitation of the modified strand under denaturing conditions. We demonstrate that the recovered ssDNA can be used for CRISPR/Cas9 homology-directed repair in human cells, DNA-origami folding, and fluorescent in situ hybridization.

molecular biology↗