All natural organisms store genetic information in a four-letter, twobase-pair genetic alphabet. The expansion of the genetic alphabet with two synthetic unnatural nucleotides that selectively pair to form an unnatural base pair (UBP) would increase the information storage potential of DNA, and semisynthetic organisms (SSOs) that stably harbor this expanded alphabet would thereby have the potential to store and retrieve increased information. Toward this goal, we previously reported that Escherichia coli grown in the presence of the unnatural nucleoside triphosphates dNaMTP and d5SICSTP, and provided with the means to import them via expression of a plasmid-borne nucleoside triphosphate transporter, replicates DNA containing a single dNaM-d5SICS UBP. Although this represented an important proof-of-concept, the nascent SSO grew poorly and, more problematically, required growth under controlled conditions and even then was unable to indefinitely store the unnatural information, which is clearly a prerequisite for true semisynthetic life. Here, to fortify and vivify the nascent SSO, we engineered the transporter, used a more chemically optimized UBP, and harnessed the power of the bacterial immune response by using Cas9 to eliminate DNA that had lost the UBP. The optimized SSO grows robustly, constitutively imports the unnatural triphosphates, and is able to indefinitely retain multiple UBPs in virtually any sequence context. This SSO is thus a form of life that can stably store genetic information using a six-letter, three-base-pair alphabet.T he natural genetic alphabet is composed of four letters whose selective pairing to form two base pairs underlies the storage and retrieval of virtually all biological information. This alphabet is essentially conserved throughout nature, and has been since the last common ancestor of all life on Earth. Significant effort has been directed toward the development of an unnatural base pair (UBP), formed between two synthetic nucleotides, that functions alongside its natural counterparts (1-3), which would represent a remarkable integration of a man-made, synthetic component into one of life's most central processes. Moreover, semisynthetic organisms (SSOs) that stably harbor such a UBP in their DNA could store and potentially retrieve the increased information, and thereby lay the foundation for achieving the central goal of synthetic biology: the creation of new life forms and functions (4).For over 15 years, we have sought to develop such a UBP (1), and these efforts eventually yielded a family of predominantly hydrophobic UBPs, with that formed between dNaM and d5SICS (dNaM-d5SICS; Fig. 1A) being a particularly promising example (5-7). Despite lacking complementary hydrogen bonding, we demonstrated that the dNaM-d5SICS UBP is well replicated by a variety of DNA polymerases in vitro (7-10), and that this efficient replication is mediated by a unique mechanism that draws upon interbase hydrophobic and packing interactions (11,12). These efforts then culminated in the first pro...

We have created a bacterial semisynthetic organism (SSO) that retains an unnatural base pair (UBP) in its DNA, transcribes it into mRNA and tRNA with cognate unnatural codons and anticodons, and after the tRNA is charged with a noncanonical amino acid synthesizes proteins containing the noncanonical amino acid. Here, we report the first progress toward the creation of eukaryotic SSOs. After demonstrating proof-of-concept with human HEK293 cells, we show that a variety of different unnatural codon–anticodon pairs can efficiently mediate the synthesis of unnatural proteins in CHO cells. Interestingly, we find that there are both similarities and significant differences between how the prokaryotic and eukaryotic ribosomes recognize the UBP, with the eukaryotic ribosome appearing more tolerant. The results represent the first progress toward eukaryotic SSOs and, in fact, suggest that such SSOs might be able to retain more unnatural information than their bacterial counterparts.

Through the development of unnatural base pairs that are compatible with native DNA and RNA polymerases and the ribosome, we have expanded the genetic alphabet and enabled in vitro and in vivo production of proteins containing noncanonical amino acids. However, the absence of assays to characterize transcription has prevented the deconvolution of the contributions of transcription and translation to the reduced performance of some unnatural codons. Here we show that RNA containing the unnatural nucleotides is efficiently reverse transcribed into cDNA, and we develop an assay to measure the combined fidelity of transcription and reverse transcription. With this assay, we examine the performance of a wide variety of unnatural codons, both in vitro and in the in vivo environment of a semisynthetic organism. We find that transcription is generally efficient, decoding at the ribosome is generally more challenging, and, correspondingly, sequence-dependent translation efficiency is the origin of variable codon performance.

Acute myeloid leukemia (AML) is a hematologic malignancy that is characterized by clonal proliferation of myeloid blasts. Despite the progress that has been made in the treatment of various malignant hematopoietic diseases, the effective treatment of AML remains very challenging. Differentiation therapy has emerged as a promising approach for leukemia treatment, and new and effective chemical agents to trigger the differentiation of AML cells, especially drug-resistant cells, are urgently required. Herein, the natural product jungermannenone C, a tetracyclic diterpenoid isolated from liverworts, is reported to induce cell differentiation in AML cells. Interestingly, the unnatural enantiomer of jungermannenone C (1) was found to be more potent than jungermannenone C in inducing cell differentiation. Furthermore, compound 1 targets peroxiredoxins I and II by selectively binding to the conserved cysteine residues and leads to cellular reactive oxygen species accumulation. Accordingly, ent-jungermannenone C (1) shows potential for further investigation as an effective differentiation therapy against AML.

Mass photometry (MP) was used to investigate the assembly of myristoylated full-length HIV-1 Gag (myr-Gag) and vRNA 5′ UTR fragment in a supported lipid bilayer (SLB) model system. The MP trajectories demonstrated that Gag trimerization on the membrane is a key step of early Gag assembly in the presence of vRNA. Growth of myr-Gag oligomers requires vRNA, occuring by addition of 1 or 2 monomers at a time from solution. These data support a model where formation of the Gag hexamers characteristic of the immature capsid lattice occurs by a gradual edge expansion, following a trimeric nucleation event. These dynamic single molecule data involving protein, RNA, and lipid components together, provide novel and fundamental insights into the initiation of virus capsid assembly.