A crucial feature of differentiated cells is the rapid activation of enhancer-driven transcriptional programs in response to signals. The potential contributions of physicochemical properties of

We utilized a system for sequence-specific RNA base editing via Adenosine Deaminases acting on RNA (ADAR) enzymes with associated ADAR guide RNAs (adRNAs). We systematically engineered it to harness ADARs, and comprehensively evaluated its specificity and activity in vitro and in vivo via two mouse models of human disease. We anticipate this platform will enable tunable and reversible engineering of RNAs for diverse applications.

Polyketides are clinically important natural products that often require elaborate organic syntheses owing to their complex chemical structures. Here we report the multienzyme total synthesis of the Streptomyces maritimus enterocin and wailupemycin bacteriostatic agents in a single reaction vessel from simple benzoate and malonate substrates. To our knowledge, our results represent the first in vitro assembly of a complete type II polyketide synthase enzymatic pathway to natural products.

Mass spectrometry (MS) is an important tool for studying non-ribosomal peptide, polyketide, and fatty acid biosynthesis. Here we describe a new approach using multi-stage tandem MS on a common ion trap instrument to obtain high-resolution measurements of the masses of substrates and intermediates bound to phosphopantetheinylated (holo) carrier proteins. In particular, we report the chemical formulas of twelve diagnostic MS 3 fragments of the phosphopantetheine moiety ejected from holo carrier proteins during MS 2 . We demonstrate our method by observing the formation of holo-AcpC, a putative acyl carrier protein from Streptococcus agalactiae.The biosynthesis of polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) natural products, including the well-established therapeutic agents penicillin, vancomycin, and rapamycin, requires post-translational modification of carrier proteins by addition of a 4′-phosphopantetheine (PPant) arm. The sulfur at the free end of this moiety forms a thioester bond with substrates and intermediates at each step of the biosynthetic process. Because these substrates and intermediates increase the overall mass of the associated carrier proteins, the latter are ideal targets for "top-down" characterization of PKS and NRPS pathways by mass spectrometry (MS). 1 A top-down approach was recently shown to facilitate the study of phosphopantetheinyl-tethered substrates. 2 Thermal activation by infrared multiphoton dissociation (IRMPD) or collision-induced dissociation (CID) causes the holo form of acyl and peptidyl carrier proteins, which are found in PKS and NRPS pathways, respectively, to consistently "eject" their PPant arm, preserving the thioester linkage to the substrate ( Figure 1a). This "PPant ejection assay" allows the mass of substrates loaded onto carrier proteins to be readily deduced from the mass of the corresponding PPant fragments. 3 Specifically, when no substrate is linked to the PPant arm, the fragment ejected from the carrier protein has chemical formula C 11 H 21 N 2 O 3 S + , giving an MS 2 peak at m/z 261.1267. When an acyl or *Corresponding author. Tel.: +1 858 534 6607; email: pdorrestein@ucsd.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. peptidyl substrate is bound to the PPant arm, the PPant peak in the MS 2 spectrum is shifted by an amount equal to the mass of the substrate less a water molecule. 4 NIH Public AccessThe PPant ejection assay can greatly facilitate the characterization of NRPS and PKS systems by mass spectrometry, but demands instruments capable of both high sensitivity and high ...