We have developed a family of unnatural base pairs (UBPs), exemplified by the pair formed between dNaM and dTPT3, for which pairing is mediated not by complementary hydrogen bonding, but by hydrophobic and packing forces. These UBPs enabled the creation of the first semi-synthetic organisms (SSOs) that store increased genetic information and use it to produce proteins containing non-canonical amino acids. However, retention of the UBPs was poor in some sequence contexts. Here, to optimize the SSO we synthesize two novel benzothiophene-based dNaM analogs, dPTMO and dMTMO, and characterize the corresponding UBPs, dPTMO-dTPT3 and dMTMO-dTPT3. We demonstrate that these UBPs perform similarly to, or slightly worse than dNaM-dTPT3 in vitro. However, in the in vivo environment of an SSO, retention of dMTMO-dTPT3, and especially dPTMO-dTPT3, is significantly higher than that of dNaM-dTPT3. This more optimal in vivo retention results from better replication, as opposed to more efficient import of the requisite unnatural nucleoside triphosphates. Modeling studies suggest that the more optimal replication results from specific internucleobase interactions mediated by the thiophene sulfur atoms. Finally, we show that dMTMO and dPTMO efficiently template the transcription of RNA containing TPT3 and that their improved retention in DNA results in more efficient production of proteins with non-canonical amino acids. This is the first instance of using performance within the SSO as part of the UBP evaluation and optimization process. From a general perspective, the results demonstrate the importance of evaluating synthetic biology “parts” in their in vivo context and further demonstrate the ability of hydrophobic and packing interactions to replace the complementary hydrogen-bonding that underlies the replication of natural base pairs. From a more practical perspective, the identification of dMTMO-dTPT3 and especially dPTMO-dTPT3 represents significant progress towards the development of SSOs with an unrestricted ability to store and retrieve increased information.

AlphaFold2 is a promising new tool for researchers to predict protein structures and generate high-quality models, with low backbone and global rootmean-square deviation (RMSD) when compared with experimental structures. However, it is unclear if the structures predicted by AlphaFold2 will be valuable targets of docking. To address this question, we redocked ligands in the PDBbind datasets against the experimental co-crystallized receptor structures and against the AlphaFold2 structures using AutoDock-GPU. We find that the quality measure provided during structure prediction is not a good predictor of docking performance, despite accurately reflecting the quality of the alpha carbon alignment with experimental structures. Removing low-confidence regions of the predicted structure and making side chains flexible improves the docking outcomes. Overall, despite high-quality prediction of backbone conformation, fine structural details limit the naive application of AlphaFold2 models as docking targets.

Small molecules containing cyclopropane-heteroatom linkages are commonly needed in medicinal chemistry campaigns yet are problematic to prepare using existing methods. To address this issue, a scalable Chan-Lam cyclopropylation reaction using potassium cyclopropyl trifluoroborate has been developed. With phenol nucleophiles, the reaction effects O-cyclopropylation, whereas with 2-pyridones, 2-hydroxybenzimidazoles, and 2-aminopyridines the reaction brings about N-cyclopropylation. The transformation is catalyzed by Cu(OAc) and 1,10-phenanthroline and employs 1 atm of O as the terminal oxidant. This method is operationally convenient to perform and provides a simple, strategic disconnection toward the synthesis of cyclopropyl aryl ethers and cyclopropyl amine derivatives bearing an array of functional groups.

Hydrogen-bonds (H-bonds) between backbone N-H donors and CO acceptors are central to our understanding of protein structure and stability. However, while interactions between backbone N atoms and the N-H of the following residue are also common, they have been ignored as potential H-bonds due to their bent geometry and the assumption that the amide N is a poor H-bond acceptor. Recently, we reported indirect experimental evidence that these interactions constitute functional H-bonds. We now report a combined atoms in molecules and noncovalent interaction theoretical analysis of electron density that unambiguously supports the characterization of these interactions as H-bonds. The calculations further suggest that the N-H···N H-bonds are largely electrostatic in nature and, importantly, that they make a significant contribution to stability. Thus, given their apparently frequent occurrence, N-H···N H-bonds likely make critical, but previously unrecognized, contributions to protein structure and function.

Steve Benner and collaborators have recently reported an analysis of DNA containing eight nucleotide letters-the four natural letters dG, dC, dA, and dT, and four additional letters, called dP, dZ, dS, and dB. Their analysis demonstrates that the additional letters do not perturb the structure or stability of the base pairs formed between the natural letters, and remarkably, that the new base pairs, dP-dZ and dS-dB, behave virtually identically to the natural base pairs. This unprecedented result convincingly demonstrates that the thermodynamic and structural behavior previously thought to be the purview of only natural DNA is in fact not unique and can be imparted to suitably designed synthetic components. In addition, the first evidence that the eight letter DNA can be transcribed into RNA by a mutant RNA polymerase is presented, paving the way for the transfer of increased information from one biopolymer to another. Along with others working to develop unnatural DNA base pairs for both in vitro and in vivo applications, this work reporesents an important step toward the expansion of the genetic alphabet, a central goal of synthetic biology, and has profound implications for our understanding of the molecules and forces that can make life possible.

Partitioning of bioactive molecules, including drugs, into cell membranes may produce indiscriminate changes in membrane protein function. As a guide to safe drug development, it therefore becomes important to be able to predict the bilayer-perturbing potency of hydrophobic/amphiphilic drugs candidates. Toward this end, we exploited gramicidin channels as molecular force probes and developed in silico and in vitro assays to measure drugs’ bilayer-modifying potency. We examined eight drug-like molecules that were found to enhance or suppress gramicidin channel function in a thick 1,2-dierucoyl- sn -glycero-3-phosphocholine (DC 22:1 PC) but not in thin 1,2-dioleoyl- sn -glycero-3-phosphocholine (DC 18:1 PC) lipid bilayer. The mechanism underlying this difference was attributable to the changes in gramicidin dimerization free energy by drug-induced perturbations of lipid bilayer physical properties and bilayer–gramicidin interactions. The combined in silico and in vitro approaches, which allow for predicting the perturbing effects of drug candidates on membrane protein function, have implications for preclinical drug safety assessment.

Background and Purpose Rabbit aneurysm models are used for the testing of embolization devices and elucidating the mechanisms of human intracranial aneurysm growth and healing. We used RNA-sequencing technology to identify genes relevant to induced rabbit aneurysm biology and to identify genes and pathways of potential clinical interest. This process included sequencing microRNAs, which are important regulatory noncoding RNAs. Materials and Methods Elastase-induced saccular aneurysms were created at the origin of the right common carotid artery in 6 rabbits. Messenger RNA and microRNA were isolated from the aneurysm and from the control left common carotid artery at 12 weeks and processed by using RNA-sequencing technology. The results from RNA sequencing were analyzed by using the Ingenuity Pathway Analysis tool. Results A total of 9396 genes were analyzed by using RNA sequencing, 648 (6.9%) of which were found to be significantly differentially expressed between the aneurysms and control tissues (P<.05; false-discovery rate<0.01; fold change, >2 or <.5). Of these genes, 614 were mapped successfully, 143 were down-regulated, and 471 were up-regulated in the aneurysms as compared with controls. Using the same criteria for significance, 3 microRNAs were identified as down-regulated and 5 were identified as up-regulated. Pathway analysis associated these genes with inflammatory response, cellular migration, and coagulation, among other functions and pathologies. Conclusion RNA-sequencing analysis of rabbit aneurysms revealed differential regulation of some key pathways, including inflammation and antigen presentation. ANKRD1 and TACR1 were identified as genes of interest in the regulation of matrix metalloproteinases.

Glycine is a major neurotransmitter involved in several fundamental neuronal processes. The identity of the metabotropic receptor mediating slow neuromodulatory effects of glycine is unknown. We identified an orphan G protein–coupled receptor, GPR158, as a metabotropic glycine receptor (mGlyR). Glycine and a related modulator, taurine, directly bind to a Cache domain of GPR158, and this event inhibits the activity of the intracellular signaling complex regulator of G protein signaling 7–G protein β5 (RGS7-Gβ5), which is associated with the receptor. Glycine signals through mGlyR to inhibit production of the second messenger adenosine 3′,5′-monophosphate. We further show that glycine, but not taurine, acts through mGlyR to regulate neuronal excitability in cortical neurons. These results identify a major neuromodulatory system involved in mediating metabotropic effects of glycine, with implications for understanding cognition and affective states.