12 research outputs found
DNA-Nanostructure-Guided Assembly of Proteins into Programmable Shapes
The development of
methods to synthesize artificial protein complexes
with precisely controlled configurations will enable diverse biological
and medical applications. Using DNA to link proteins provides programmability
that can be difficult to achieve with other methods. Here, we use
DNA origami as an âassemblerâ to guide the linking
of proteinâDNA conjugates using a series of oligonucleotide
hybridization and displacement operations. We constructed several
isomeric protein nanostructures, including a dimer, two types of trimer
structures, and three types of tetramer assemblies, on a DNA origami
platform by using a C3-symmetric building block composed of a protein
trimer modified with DNA handles. Our approach expands the scope for
the precise assembly of protein-based nanostructures and will enable
the formulation of functional protein complexes with stoichiometric
and geometric control
Atomistic Picture of OpeningâClosing Dynamics of DNA Holliday Junction Obtained by Molecular Simulations
Holliday junction (HJ) is a noncanonical four-way DNA
structure
with a prominent role in DNA repair, recombination, and DNA nanotechnology.
By rearranging its four arms, HJ can adopt either closed or open state.
With enzymes typically recognizing only a single state, acquiring
detailed knowledge of the rearrangement process is an important step
toward fully understanding the biological function of HJs. Here, we
carried out standard all-atom molecular dynamics (MD) simulations
of the spontaneous openingâclosing transitions, which revealed
complex conformational transitions of HJs with an involvement of previously
unconsidered âhalf-closedâ intermediates. Detailed free-energy
landscapes of the transitions were obtained by sophisticated enhanced
sampling simulations. Because the force field overstabilizes the closed
conformation of HJs, we developed a system-specific modification which
for the first time allows the observation of spontaneous openingâclosing
HJ transitions in unbiased MD simulations and opens the possibilities
for more accurate HJ computational studies of biological processes
and nanomaterials
Atomistic Picture of OpeningâClosing Dynamics of DNA Holliday Junction Obtained by Molecular Simulations
Holliday junction (HJ) is a noncanonical four-way DNA
structure
with a prominent role in DNA repair, recombination, and DNA nanotechnology.
By rearranging its four arms, HJ can adopt either closed or open state.
With enzymes typically recognizing only a single state, acquiring
detailed knowledge of the rearrangement process is an important step
toward fully understanding the biological function of HJs. Here, we
carried out standard all-atom molecular dynamics (MD) simulations
of the spontaneous openingâclosing transitions, which revealed
complex conformational transitions of HJs with an involvement of previously
unconsidered âhalf-closedâ intermediates. Detailed free-energy
landscapes of the transitions were obtained by sophisticated enhanced
sampling simulations. Because the force field overstabilizes the closed
conformation of HJs, we developed a system-specific modification which
for the first time allows the observation of spontaneous openingâclosing
HJ transitions in unbiased MD simulations and opens the possibilities
for more accurate HJ computational studies of biological processes
and nanomaterials
Tuning the Cavity Size and Chirality of Self-Assembling 3D DNA Crystals
The foundational goal of structural
DNA nanotechnologyî¸the
field that uses oligonucleotides as a molecular building block for
the programmable self-assembly of nanostructured systemsî¸was
to use DNA to construct three-dimensional (3D) lattices for solving
macromolecular structures. The programmable nature of DNA makes it
an ideal system for rationally constructing self-assembled crystals
and immobilizing guest molecules in a repeating 3D array through their
specific stereospatial interactions with the scaffold. In this work,
we have extended a previously described motif (4 Ă 5) by expanding
the structure to a system that links four double-helical layers; we
use a central weaving oligonucleotide containing a sequence of four
six-base repeats (4 Ă 6), forming a matrix of layers that are
organized and dictated by a series of Holliday junctions. In addition,
we have assembled mirror image crystals (l-DNA) with the
identical sequence that are completely resistant to nucleases. Bromine
and selenium derivatives were obtained for the l- and d-DNA forms, respectively, allowing phase determination for
both forms and solution of the resulting structures to 3.0 and 3.05
Ă
resolution. Both right- and left-handed forms crystallized
in the trigonal space groups with mirror image 3-fold helical screw
axes <i>P</i>3<sub>2</sub> and <i>P</i>3<sub>1</sub> for each motif, respectively. The structures reveal a highly organized
array of discrete and well-defined cavities that are suitable for
hosting guest molecules and allow us to dictate <i>a priori</i> the assembly of guestâDNA conjugates with a specified crystalline
hand
Creating a stem cell niche in the inner ear using self-assembling peptide amphiphiles
<div><p>The use of human embryonic stem cells (hESCs) for regeneration of the spiral ganglion will require techniques for promoting otic neuronal progenitor (ONP) differentiation, anchoring of cells to anatomically appropriate and specific niches, and long-term cell survival after transplantation. In this study, we used self-assembling peptide amphiphile (PA) molecules that display an IKVAV epitope (IKVAV-PA) to create a niche for hESC-derived ONPs that supported neuronal differentiation and survival both in vitro and in vivo after transplantation into rodent inner ears. A feature of the IKVAV-PA gel is its ability to form organized nanofibers that promote directed neurite growth. Culture of hESC-derived ONPs in IKVAV-PA gels did not alter cell proliferation or viability. However, the presence of IKVAV-PA gels increased the number of cells expressing the neuronal marker beta-III tubulin and improved neurite extension. The self-assembly properties of the IKVAV-PA gel allowed it to be injected as a liquid into the inner ear to create a biophysical niche for transplanted cells after gelation in vivo. Injection of ONPs combined with IKVAV-PA into the modiolus of X-SCID rats increased survival and localization of the cells around the injection site compared to controls. Human cadaveric temporal bone studies demonstrated the technical feasibility of a transmastoid surgical approach for clinical intracochlear injection of the IKVAV-PA/ONP combination. Combining stem cell transplantation with injection of self-assembling PA gels to create a supportive niche may improve clinical approaches to spiral ganglion regeneration.</p></div
Ex vivo cadaveric human temporal bone study.
<p><b>(A)</b>: Schematized midmodiolar cross-section of a mammalian cochlea, showing injection needle approaching the modiolus. <b>(B-C)</b>: Photomicrograph of the basal turn of the left cochlea in a cadaveric temporal bone <b>(B)</b> and in humans <b>(C)</b>. Black arrows indicate direction of injection with IKVAV-PA gels. Note the direction of the fine needle for injection of IKVAV gel containing hESCs. A blue-dotted line shows the cochleostomy site. A white dotted line: round window. <b>(D and E)</b>: Endoscopic IKVAV-PA gel injection into human modiolus. View of cochleostomy site using a 16-mm 0° rigid endoscope. A black dashed line marks cochleostomy boundary. A white dashed line indicates presumed plane of the scala vestibuli. <b>(F)</b>: Artistâs rendition of the superior view of the human skull base, with black arrow showing the direction of injection of IKVAV gels with hESCs and anatomical landmarks. Black square corresponds to sectioned area in subsequent figures. <b>(G-L)</b>: IKVAV-PA gels with hESCs in the IAC in two sets of human cadaveric temporal bones. <b>(G and J)</b>: Middle cranial fossa view of the human cadaveric temporal bone before the hESC injection. TAMRA-tagged IKVAV gels <b>(H)</b> and corresponding autofluorescence measurement observed in normal temporal bone tissue abutting the IAC <b>(I)</b>. TRA-1-81 tagged hESCs with magnified inset <b>(K)</b> and corresponding autofluorescence measurement <b>(L)</b>. Abbreviations: RW: round window; C: cochleostomy site; ST: scala tympani; SV: scala vestibule; M: modiolus; N: spinal needle; ACF: anterior cranial fossa; MCF: middle cranial fossa; PCF: posterior cranial fossa. Cranial nerves are denoted by Roman numerals.</p
Neuronal differentiation of hESC-derived late-stage ONPs in IKVAV and VVIAK 3D matrices.
<p><b>(Aa)</b>: An experimental paradigm on neuronal differentiation of hESC-derived mid-stage ONPs treated with S/R/E/F/I in IKVAV & VVIAK PA gels (3-D) and Matrigel⢠(2-D) for seven days. DIV: days in vitro. <b>(Ab)</b>: A photomicrograph of phase-contrasted image of a mid ONP treated with S/R/E/F/I in IKVAV-PA gels. A White arrow indicates a neurite. Scale = 20 Îźm. <b>(B)</b>: Immunocytochemistry of nestin and β-III tubulin for hESC-derived mid-ONPs treated with S/R/E/F/I in IKVAV-PA gels. DAPI stain is shown in blue. A white arrow indicates neurites. Scale bar: 20 Îźm. <b>(C)</b>: Quantification of nestin and β-III tubulin-immunopositive cells on derived mid-stage ONPs treated with S/R/E/F/I in IKVAV-PA gels, VVIAK-PA gels, and Matrigel⢠for 7 days. β-III: β-III tubulin. (<b>D</b>): Quantification of neurite-bearing cells on derived mid-stage ONPs treated with S/R/E/F/I. in IKVAV-PA gels, VVIAK-PA gels, and Matrigelâ˘. (<b>E</b>): Quantification of average neurite length on S/R/E/F/I-treated hESC-derived mid-stage ONPs. ** <i>p</i> < 0.01, * <i>p</i> < 0.05 by one-way ANOVA with Tukey-Kramerâs post-hoc test.</p
Schematic summary of the protocol and timeline for deriving the SGN lineage from undifferentiated hESCs.
<p>DIV: day in vitro; NNE: nonneuronal ectoderm; PPE: preplacodal ectoderm; ONP: otic neural progenitor; BMP4: bone morphogenetic protein 4; SHH: Sonic hedgehog; ATRA: all-trans retinoic acid; EGF: epidermal growth factor; BDNF: brain-derived neurotrophic factor; NT-3: neurotrophin 3; IGF-1: insulin-like growth factor 1; FGF2: fibroblast growth factor 2; E8: Essential 8<sup>â˘</sup> medium; N2B27-CDM: chemically defined medium containing N2 and B27 supplements; MACS: magnetic-activated cell sorting; FACS: fluorescence-activated cell sorting; p75: low-affinity neurotrophin receptor (p75<sup>NTR</sup>); IKVAV-PA: IKVAV-containing peptide amphiphile; VVIAK-PA: VVIAK-containing peptide amphiphile. Protocol adapted and modified from Matsuoka et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190150#pone.0190150.ref008" target="_blank">8</a>].</p
Immunocytochemical assessment of differentiation of mid-stage hESC-derived ONPs into late-stage ONPs.
<p><b>(A)</b>: An experimental paradigm. Immunocytochemistry of late-stage ONPs derived from H1, H7, and H9 undifferentiated ESCs for SOX2 <b>(B)</b>, PAX2 <b>(C)</b>, PAX8/NEUROD1 <b>(D)</b>, and GATA3/PAX8 <b>(E)</b>. Human hESC cell lines used were H7 (C), H9 (B), and H1 (D, E). Scale bars: 50 Îźm (B and D) and 20 Îźm (C and E). m-ONP: mid-stage ONPs; l-ONP: late-stage ONPs; SGN: spiral ganglion neurons; S/R/E/F/I: SHH/ATRA/EGF/FGF2/IGF-1.</p
Electron microscopy study of IKVAV-PA gels in vitro and ex vivo human cadaveric tissue.
<p>(<b>A</b>): SEM image of aligned IKVAV nanofibers in vitro (note predominant orientation in the direction of the shear force, indicated by red arrow). Scale bar = 1 Îźm. (<b>B</b>): Non-aligned IKVAV nanofibers in vitro showing a randomly oriented configuration due to fluid turbulence. Scale bar = 1 Îźm. (<b>C</b>): TEM image of self-assembled IKVAV-PA gel overlying the facial-vestibulocochlear nerve complex (indicated by a black circle and black arrows) following endoscopic transmastoid injection into the modiolus. Note the parallel alignment of self-assembled peptides indicated by the red arrow (11,000Ă magnification). Scale bar = 1 Îźm. (<b>D</b>): Control nerve specimen with no evidence of peptide scaffold for comparison (1,900Ă magnification). Scale bar = 1 Îźm. Note putative epineurium of the facial-vestibulocochlear nerve complex indicated by the white dotted line (C & D). Abbreviations: VIIVIII: facial-vestibulocochlear nerve complex.</p