DNA Display III. Solid-Phase Organic Synthesis on Unprotected DNA

DNA-directed synthesis represents a powerful new tool for molecular discovery. Its ultimate utility, however, hinges upon the diversity of chemical reactions that can be executed in the presence of unprotected DNA. We present a solid-phase reaction format that makes possible the use of standard organic reaction conditions and common reagents to facilitate chemical transformations on unprotected DNA supports. We demonstrate the feasibility of this strategy by comprehensively adapting solid-phase 9-fluorenylmethyoxycarbonyl–based peptide synthesis to be DNA-compatible, and we describe a set of tools for the adaptation of other chemistries. Efficient peptide coupling to DNA was observed for all 33 amino acids tested, and polypeptides as long as 12 amino acids were synthesized on DNA supports. Beyond the direct implications for synthesis of peptide–DNA conjugates, the methods described offer a general strategy for organic synthesis on unprotected DNA. Their employment can facilitate the generation of chemically diverse DNA-encoded molecular populations amenable to in vitro evolution and genetic manipulation

Progenitor identification and SARS-CoV-2 infection in human distal lung organoids

The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange. Three-dimensional in vitro human distal lung culture systems would strongly facilitate investigation of pathologies including interstitial lung disease, cancer, and SARS-CoV-2-associated COVID-19 pneumonia. We generated long-term feeder-free, chemically defined culture of distal lung progenitors as organoids derived from single adult human alveolar epithelial type II (AT2) or KRT5+ basal cells. AT2 organoids exhibited AT1 transdifferentiation potential while basal cell organoids developed lumens lined by differentiated club and ciliated cells. Single cell analysis of basal organoid KRT5+ cells revealed a distinct ITGA6+ITGB4+ mitotic population whose proliferation further segregated to a TNFRSF12Ahi subfraction comprising ~10% of KRT5+ basal cells, residing in clusters within terminal bronchioles and exhibiting enriched clonogenic organoid growth activity. Distal lung organoids were created with apical-out polarity to display ACE2 on the exposed external surface, facilitating SARS-CoV-2 infection of AT2 and basal cultures and identifying club cells as a novel target population. This long-term, feeder-free organoid culture of human distal lung, coupled with single cell analysis, identifies unsuspected basal cell functional heterogeneity and establishes a facile in vitro organoid model for human distal lung infections including COVID-19-associated pneumonia

The human body at cellular resolution: the NIH Human Biomolecular Atlas Program

Abstract: Transformative technologies are enabling the construction of three-dimensional maps of tissues with unprecedented spatial and molecular resolution. Over the next seven years, the NIH Common Fund Human Biomolecular Atlas Program (HuBMAP) intends to develop a widely accessible framework for comprehensively mapping the human body at single-cell resolution by supporting technology development, data acquisition, and detailed spatial mapping. HuBMAP will integrate its efforts with other funding agencies, programs, consortia, and the biomedical research community at large towards the shared vision of a comprehensive, accessible three-dimensional molecular and cellular atlas of the human body, in health and under various disease conditions

Peptide–DNA Conjugate As Template for DNA Synthesis

<p>5′ PEG amino-modified 340-base ssDNA was loaded onto two DEAE Sepharose columns. The pentapeptide [Leu]enkephalin was synthesized on one column using EDC/HOAt and Fmoc–amino acids. The DNA was eluted, desalted, and used as template for radiolabeled primer extension reactions. Denaturing polyacrylamide gel electrophoresis analysis of reaction products shows no difference between ssDNA (control) and [Leu]enkephalin–ssDNA ([Leu]enk) templates.</p

Gold nanocrystal labels provide a sequence–to–3D structure map in SAXS reconstructions

Small-angle x-ray scattering (SAXS) is a powerful technique to probe the structure of biological macromolecules and their complexes under virtually arbitrary solution conditions, without the need for crystallization. While it is possible to reconstruct molecular shapes from SAXS data ab initio, the resulting electron density maps have a resolution of ~1 nm and are often insufficient to reliably assign secondary structure elements or domains. We show that SAXS data of gold-labeled samples significantly enhance the information content of SAXS measurements, allowing the unambiguous assignment of macromolecular sequence motifs to specific locations within a SAXS structure. We first demonstrate our approach for site-specifically internally and end-labeled DNA and an RNA motif. In addition, we present a protocol for highly uniform and site-specific labeling of proteins with small (~1.4 nm diameter) gold particles and apply our method to the signaling protein calmodulin. In all cases, the position of the small gold probes can be reliably identified in low-resolution electron density maps. Enhancing low-resolution measurements by site-selective gold labeling provides an attractive approach to aid modeling of a large range of macromolecular systems

DNA Support Structure and Modified Amino Acids

<div><p>(A) Peptide synthesis is carried out on DNA modified with a 5′ C12 amino (NC20) or a 5′ PEG amino (NP20) linker.</p> <p>(B) Fluorescent lysine derivative (compound 1, Fmoc-Lys[coumarin]-OH) and BME/DBU labile protecting groups for lysine (compound 2, Fmoc-Lys[Ns]-OH), arginine (compound 3, Fmoc-Arg[Ns]-OH), and histidine (compound 4, Fmoc-His[CNP]-OH).</p></div

Peptide Coupling to DNA Supports

<div><p>(A) Fmoc-based peptide coupling reaction to an aminated 20-base oligonucleotide (NC20) where X represents a succinimidyl or EDC/HOAt-activated ester.</p> <p>(B) HPLC chromatograms of a nonaminated 10-base (10mer) and an aminated 20-base (NC20) oligonucleotide. HPLC traces show DEAE column load (solid black) and elute (broken black). DEAE column elutes after succinimidyl ester–mediated (solid red) or EDC/HOAt-mediated (broken red) Fmoc-Leu coupling and Fmoc deprotection are shown. The resulting amino acid–DNA conjugate is denoted (Leu-NC20).</p> <p>(C) Chemical transformations are carried out using small DEAE Sepharose columns and syringes (left). Washes are facilitated by a vacuum manifold with chemically resistant stopcocks (right).</p></div

Absolute Intramolecular Distance Measurements with Angstrom-Resolution Using Anomalous Small-Angle X‑ray Scattering

Accurate determination of molecular distances is fundamental to understanding the structure, dynamics, and conformational ensembles of biological macromolecules. Here we present a method to determine the full distance distribution between small (∼7 Å radius) gold labels attached to macromolecules with very high-precision (≤1 Å) and on an absolute distance scale. Our method uses anomalous small-angle X-ray scattering close to a gold absorption edge to separate the gold–gold interference pattern from other scattering contributions. Results for 10–30 bp DNA constructs achieve excellent signal-to-noise and are in good agreement with previous results obtained by single-energy SAXS measurements without requiring the preparation and measurement of single labeled and unlabeled samples. The use of small gold labels in combination with ASAXS read out provides an attractive approach to determining molecular distance distributions that will be applicable to a broad range of macromolecular systems

The human body at cellular resolution: the NIH Human Biomolecular Atlas Program

Transformative technologies are enabling the construction of three dimensional (3D) maps of tissues with unprecedented spatial and molecular resolution. Over the next seven years, the NIH Common Fund Human Biomolecular Atlas Program (HuBMAP) intends to develop a widely accessible framework for comprehensively mapping the human body at single-cell resolution by supporting technology development, data acquisition, and detailed spatial mapping. HuBMAP will integrate its efforts with other funding agencies, programs, consortia, and the biomedical research community at large towards the shared vision of a comprehensive, accessible 3D molecular and cellular atlas of the human body, in health and various disease settings