Biology subjects

Khakimzhan, A.

Publications and source records attributed to Khakimzhan, A..

4 recordsLinked to original sources

bioRxiv2024

Red Light Mediated Photo-Conversion of Silicon Rhodamines to Oxygen Rhodamines for Single-Molecule Microscopy

The rhodamine motif has been modified in myriad ways to produce probes with specific fluorescent and chemical properties optimal for a variety of microscopy experiments. Recently, far-red emitting silicon rhodamines have become popular in single-molecule localization microscopy (SMLM), since these dyes are membrane-permeable and can be used alongside red fluorophores for two-color imaging. While this has expanded multi-color SMLM imaging capabilities, we demonstrate that silicon rhodamines can create previously unreported photoproducts with significantly blueshifted emissions, which appear as bright single-molecule crosstalk in the red emission channel. We show that this fluorescence is caused by the replacement of the central silicon group with oxygen after 640 nm illumination, turning far-red silicon rhodamines (JFX650, JF669, etc.) into their red oxygen rhodamine counterparts (JFX554, JF571, etc.). While this blueshifted population can cause artifacts in two-color SMLM data, we demonstrate up to 16-fold reduction in crosstalk using oxygen-scavenging systems. We also leverage this far-red photoconversion to demonstrate UV-free photoactivated localization microscopy (PALM) without the need for additives, and with 5-fold higher efficiency than the Cy5 to Cy3 conversion. Finally, we demonstrate multiplexed pseudo two-color PALM in a single emission channel by separating localizations by their photo-activation wavelengths instead of their emission wavelengths. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/595223v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@141fe66org.highwire.dtl.DTLVardef@1a4f9f5org.highwire.dtl.DTLVardef@16605dorg.highwire.dtl.DTLVardef@1109255_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

Cell-free expression with a quartz crystal microbalance enables rapid, dynamic, and label-free characterization of membrane-interacting proteins

Integral and interacting membrane proteins (IIMPs) constitute a vast family of biomolecules that perform essential functions in all forms of life. However, characterizing their interactions with lipid bilayers remains limited due to challenges in purifying and reconstituting IIMPs in vitro or labeling IIMPs without disrupting their function in vivo. Here, we report TXTL-QCMD to dynamically characterize interactions between diverse IIMPs and membranes without protein purification or labeling. As part of TXTL-QCMD, IIMPs are synthesized using cell-free transcription-translation (TXTL), and their interactions with supported lipid bilayers are measured using a quartz crystal microbalance with dissipation (QCMD). TXTL-QCMD reconstitutes known IIMP-membrane dependencies, including specific association with prokaryotic or eukaryotic membranes or oscillating interactions by the E. coli Min system. Applying TXTL-QCMD to the recently discovered Zorya anti-phage system unamenable to labeling, we discovered that ZorA and ZorB integrate together within the lipids found at the poles of bacteria while ZorE diffuses freely on the non-pole membrane. These efforts establish the potential of TXTL-QCMD to broadly characterize the large diversity of IIMPs.

biophysics↗

bioRxiv2024

On-chip large-scale-integration and 2D collective modes of genetically programmed artificial cells

The on-chip large-scale-integration of genetically programmed artificial cells capable of exhibiting collective modes is an important goal for fundamental research and technology. Here, we report assembly of a 2D layout of 1024 monolithic DNA compartments as artificial cells on a 5-millimeter square silicon chip. Homeostatic cell-free protein synthesis reactions driven by genetic circuits occur inside the compartments. We created a reaction-diffusion system with a 30x30 square lattice of artificial cells interconnected by thin capillaries for diffusion of products. Driving the system by a genetic oscillator revealed emergent collective modes of synchrony and propagating phase waves in 2D, with dynamics controlled by geometry. This demonstrates a class of nonequilibrium autonomous systems, where chemical energy consumed to make proteins induces 2D collective patterns of gene expression on multicellular scales, with applications in biological computing, sensing, and materials synthesis.

biophysics↗

bioRxiv2021

Critical role of DNA unwinding and Cas9 conformational changes in modelling off-target effects

CRISPR-Cas9 off-target effects interfere with the ability to accurately perform genetic edits. To predict off-target effects CRISPR-Cas9 researchers perform high throughput guide RNA mismatch and bulge experiments and then use the data to fit thermodynamic binding models. While impactful from an engineering perspective such models are not based on the experimentally observed target interrogation process and thus incorrectly measure the energetic effects mismatches have on the system. In this work we convert an experimentally deduced qualitive model of target interrogation to a linear ODE model and demonstrate that the mismatch tolerance patterns observed in experiments do not need to be caused by differences in energetic penalties of mismatches but rather are emergent effects of the timing and coordination of target DNA unwinding and Cas9 conformational changes.

biophysics↗