The translation of DNA sequences into synthetic products is a key requirement of our approach to evolving synthetic molecules through iterated cycles of translation, selection, and amplification. Here we report general linker and purification strategies for sequence-specific DNA-templated synthesis that collectively enable the product of a DNA-templated reaction to be isolated and to undergo subsequent DNA-templated reactions. Using these strategies, we have achieved the first multistep nucleic acid-templated small-molecule syntheses to generate two different molecules. In addition to representing a method for translating DNA templates sequence-specifically into corresponding multistep synthetic products, our findings also provide experimental support for previously proposed models invoking multistep nucleic acid-templated synthesis as mediating the prebiotic translation of replicable information into the earliest functional molecules.
Abstract The all-protein cytosine base editor DdCBE uses TALE proteins and a double-stranded DNA-specific cytidine deaminase (DddA) to mediate targeted C•G-to-T•A editing. To improve editing efficiency and overcome the strict T C sequence-context constraint of DddA, we used phage-assisted non-continuous and continuous evolution to evolve DddA variants with improved activity and expanded targeting scope. Compared to canonical DdCBEs, base editors with evolved DddA6 improved mitochondrial DNA (mtDNA) editing efficiencies at T C by 3.3-fold on average. DdCBEs containing evolved DddA11 offered a broadened H C (H = A, C or T) sequence compatibility for both mitochondrial and nuclear base editing, increasing average editing efficiencies at A C and C C targets from less than 10% for canonical DdCBE to 15–30% and up to 50% in cell populations sorted to express both halves of DdCBE. We used these evolved DdCBEs to efficiently install disease-associated mtDNA mutations in human cells at non-T C target sites. DddA6 and DddA11 substantially increase the effectiveness and applicability of all-protein base editing.
Despite transformative advances in protein design with deep learning, the design of small-molecule-binding proteins and sensors for arbitrary ligands remains a grand challenge. Here we combine deep learning and physics-based methods to generate a family of proteins with diverse and designable pocket geometries, which we employ to computationally design binders for six chemically and structurally distinct small-molecule targets. Biophysical characterization of the designed binders revealed nanomolar to low micromolar binding affinities and atomic-level design accuracy. The bound ligands are exposed at one edge of the binding pocket, enabling the de novo design of chemically induced dimerization (CID) systems; we take advantage of this to create a biosensor with nanomolar sensitivity for cortisol. Our approach provides a general method to design proteins that bind and sense small molecules for a wide range of analytical, environmental, and biomedical applications.
Background: Children afflicted with Hutchinson-Gilford Progeria Syndrome (HGPS) die from progressive vascular disease at an average age of 14. Despite elucidation of the genetic cause—C1824T point ...
ABSTRACT Bacillus thuringiensis or Bt is a Gram-positive soil bacterium, widely and safely applied in the environment as an insecticide for combatting insect pests that damage crops and vector diseases. Dominant active ingredients made by Bt are insect-killing crystal (Cry) proteins released as crystalline inclusions upon bacterial sporulation. Some Bt Cry proteins, e . g ., Cry5B, target nematodes (roundworms) and show exceptional promise as anthelmintics (cures for parasitic nematode diseases). We have recently described IBaCC (for I nactivated Ba cteria with C ytosolic C rystal(s)) in which bioactive Bt Cry crystals (containing Cry5B) are fully contained within the cytosol of dead bacterial ghosts. Here we demonstrate that these IBaCC-trapped Cry5B crystals can be liberated and purified away from cellular constituents yielding P urified C ytosolic C rystals (PCC). Cry5B PCC contains ∼95% Cry5B protein out of the total protein content. Cry5B PCC is highly bioactive against parasitic nematode larvae and adults in vitro . Cry5B PCC is also highly active in vivo against experimental human hookworm and Ascaris infections in rodents. The process was scaled up to the 100 liter scale to produce PCC for a pilot study to treat two foals infected with the Ascarid, Parascaris spp. Single dose Cry5B PCC brought the fecal egg counts of both foals to zero. These studies describe the process for the scalable production of purified Bt crystals and define a new active pharmaceutical ingredient form of Bt Cry proteins. NON-TECHNICAL IMPORTANCE PARAGRAPH Bacillus thuringiensis crystal proteins are widely and safely used as insecticides. Recent studies show they also can cure gastrointestinal parasitic worm (nematode) infections when ingested. However, reproducible, scalable, and practical techniques for purifying these proteins have been lacking. Here, we address this severe limitation and present scalable and practical methods for large-scale purification of potently bioactive B. thuringiensis crystals and crystal proteins. The resultant product, called Purified Cytosolic Crystals (PCC), is highly compatible with ingestible drug delivery and formulation. Furthermore, there are growing applications in agriculture and insect control where access to large quantities of purified crystal proteins are desirable and where these methods will find great utility.
