Discovery, implications and initial use of semi-synthetic organisms with an expanded genetic alphabet/code.

Much recent interest has focused on developing proteins for human use, such as in medicine. However, natural proteins are made up of only a limited number of canonical amino acids with limited functionalities, and this makes the discovery of variants with some functions difficult. The ability to recombinantly express proteins containing non-canonical amino acids (ncAAs) with properties selected to impart the protein with desired properties is expected to dramatically improve the discovery of proteins with different functions.

Creation, Optimization, and Use of Semi-Synthetic Organisms that Store and Retrieve Increased Genetic Information.

With few exceptions, natural proteins are built from only 20 canonical (proteogenic) amino acids which limits the functionality and accordingly the properties they can possess. Genetic code expansion, i.e.

Transcriptional processing of an unnatural base pair by eukaryotic RNA polymerase II.

The development of unnatural base pairs (UBPs) has greatly increased the information storage capacity of DNA, allowing for transcription of unnatural RNA by the heterologously expressed T7 RNA polymerase (RNAP) in Escherichia coli. However, little is known about how UBPs are transcribed by cellular RNA polymerases. Here, we investigated how synthetic unnatural nucleotides, NaM and TPT3, are recognized by eukaryotic RNA polymerase II (Pol II) and found that Pol II is able to selectively recognize UBPs with high fidelity when dTPT3 is in the template strand and rNaMTP acts as the nucleotide substrate.

Efforts toward Further Integration of an Unnatural Base Pair into the Biology of a Semisynthetic Organism.

We have developed semisynthetic organisms (SSOs) that by virtue of a family of synthetic, unnatural base pairs (UBPs), store and retrieve increased information. To date, transcription in the SSOs has relied on heterologous expression of the RNA polymerase from T7 bacteriophage; here, we explore placing transcription under the control of the endogenous host multisubunit RNA polymerase. The results demonstrate that the RNA polymerase is able to transcribe DNA containing a UBP and that with the most optimal UBP identified to date it should be possible to select for increased uptake of unnatural triphosphates.

Genetic Code Expansion: Inception, Development, Commercialization.

Virtually all natural proteins are built from only 20 amino acids, and while this makes possible all the functions they perform, the ability to encode other amino acids selected for specific purposes promises to enable the discovery and production of proteins with novel functions, including therapeutic proteins with more optimal drug-like properties. The field of genetic code expansion (GCE) has for years enabled the production of such proteins for academic purposes and is now transitioning to commercialization for the production of more optimal protein therapeutics. Focusing on , we review the history and current state of the field.

Transcription and Reverse Transcription of an Expanded Genetic Alphabet In Vitro and in a Semisynthetic Organism.

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.

Initial Analysis of the Arylomycin D Antibiotics.

The arylomycins are a class of natural product antibiotics that inhibit bacterial type I signal peptidase and are under development as therapeutics. Four classes of arylomycins are known, arylomycins A-D. Previously, we reported the synthesis and analysis of representatives of the A, B, and C classes and showed that their spectrum of activity has the potential to be much broader than originally assumed.

New codons for efficient production of unnatural proteins in a semisynthetic organism.

Natural organisms use a four-letter genetic alphabet that makes available 64 triplet codons, of which 61 are sense codons used to encode proteins with the 20 canonical amino acids. We have shown that the unnatural nucleotides dNaM and dTPT3 can pair to form an unnatural base pair (UBP) and allow for the creation of semisynthetic organisms (SSOs) with additional sense codons. Here, we report a systematic analysis of the unnatural codons.

Nanopore Sequencing of an Expanded Genetic Alphabet Reveals High-Fidelity Replication of a Predominantly Hydrophobic Unnatural Base Pair.

Unnatural base pairs (UBPs) have been developed and used for a variety of applications as well as for the engineering of semisynthetic organisms (SSOs) that store and retrieve increased information. However, these applications are limited by the availability of methods to rapidly and accurately determine the sequence of unnatural DNA. Here we report the development and application of the MspA nanopore to sequence DNA containing the d-d UBP.

Selection of Aptamers with Large Hydrophobic 2'-Substituents.

Previously, we evolved a DNA polymerase, SFM4-3, for the recognition of substrates modified at their 2' positions with a fluoro, -methyl, or azido substituent. Here we use SFM4-3 to synthesize 2'-azido-modified DNA; we then use the azido group to attach different, large hydrophobic groups via click chemistry. We show that SFM4-3 recognizes the modified templates under standard conditions, producing natural DNA and thereby allowing amplification.

Progress toward Eukaryotic Semisynthetic Organisms: Translation of Unnatural Codons.

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.

Structure and Dynamics of Stacking Interactions in an Antibody Binding Site.

For years, antibodies (Abs) have been used as a paradigm for understanding how protein structure contributes to molecular recognition. However, with the ability to evolve Abs that recognize specific chromophores, they also have great potential as models for how protein dynamics contribute to molecular recognition. We previously raised murine Abs to different chromophores and, with the use of three-pulse photon echo peak shift spectroscopy, demonstrated that the immune system is capable of producing Abs with widely varying flexibility.

Optimization of Replication, Transcription, and Translation in a Semi-Synthetic Organism.

Previously, we reported the creation of a semi-synthetic organism (SSO) that stores and retrieves increased information by virtue of stably maintaining an unnatural base pair (UBP) in its DNA, transcribing the corresponding unnatural nucleotides into the codons and anticodons of mRNAs and tRNAs, and then using them to produce proteins containing noncanonical amino acids (ncAAs). Here we report a systematic extension of the effort to optimize the SSO by exploring a variety of deoxy- and ribonucleotide analogues. Importantly, this includes the first in vivo structure-activity relationship (SAR) analysis of unnatural ribonucleoside triphosphates.

Eight-Letter DNA.

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 (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.

Site-Specific Labeling of DNA via PCR with an Expanded Genetic Alphabet.

The polymerase chain reaction (PCR) is a universal and essential tool in molecular biology and biotechnology, but it is generally limited to the amplification of DNA with the four-letter genetic alphabet. Here, we describe PCR amplification with a six-letter alphabet that includes the two natural dA-dT and dG-dC base pairs and an unnatural base pair (UBP) formed between the synthetic nucleotides dNaM and d5SICS or dTPT3 or analogs of these synthetic nucleotides modified with linkers that allow for the site-specific labeling of the amplified DNA with different functional groups. Under standard conditions, the six-letter DNA may be amplified with high efficiency and with greater than 99.

Inhibition of Protein Secretion in and Sub-MIC Effects of Arylomycin Antibiotics.

At sufficient concentrations, antibiotics effectively eradicate many bacterial infections. However, during therapy, bacteria are unavoidably exposed to lower antibiotic concentrations, and sub-MIC exposure can result in a wide variety of other effects, including the induction of virulence, which can complicate therapy, or horizontal gene transfer (HGT), which can accelerate the spread of resistance genes. Bacterial type I signal peptidase (SPase) is an essential protein that acts at the final step of the general secretory pathway.

Progress Toward a Semi-Synthetic Organism with an Unrestricted Expanded Genetic Alphabet.

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 semisynthetic organisms (SSOs) that store increased genetic information and use it to produce proteins containing noncanonical amino acids. However, retention of the UBPs was poor in some sequence contexts.

Expansion of the genetic code via expansion of the genetic alphabet.

Current methods to expand the genetic code enable site-specific incorporation of non-canonical amino acids (ncAAs) into proteins in eukaryotic and prokaryotic cells. However, current methods are limited by the number of codons possible, their orthogonality, and possibly their effects on protein synthesis and folding. An alternative approach relies on unnatural base pairs to create a virtually unlimited number of genuinely new codons that are efficiently translated and highly orthogonal because they direct ncAA incorporation using forces other than the complementary hydrogen bonds employed by their natural counterparts.

Optimization of a β-Lactam Scaffold for Antibacterial Activity via the Inhibition of Bacterial Type I Signal Peptidase.

β-Lactam antibiotics, one of the most important class of human therapeutics, act via the inhibition of penicillin-binding proteins (PBPs). The unparalleled success in their development has inspired efforts to develop them as inhibitors of other targets. Bacterial type I signal peptidase is evolutionarily related to the PBPs, but the stereochemistry of its substrates and its catalytic mechanism suggest that β-lactams with the 5 stereochemistry, as opposed to the 5 stereochemistry of the traditional β-lactams, would be required for inhibition.

Evolved polymerases facilitate selection of fully 2'-OMe-modified aptamers.

RNA or DNA aptamers with 2'-OMe-modifications have been pursued to increase resistance to nucleases, but have been difficult to identify because the OMe groups ablate polymerase recognition. We recently reported evolution of the thermostable DNA polymerases SFM4-6 and SFM4-9, which enable the efficient "transcription" and "reverse transcription", respectively, of 2'-OMe oligonucleotides. With these polymerases, we now report the first selection of fully 2'-OMe modified aptamers, specifically aptamers that bind human neutrophil elastase (HNE).

Scalable Access to Arylomycins via C-H Functionalization Logic.

Arylomycins are a promising class of "latent" antibacterial natural products currently in preclinical development. Access to analogues within this family has previously required a lengthy route involving multiple functional group manipulations that is costly and time-intensive on scale. This study presents a simplified route predicated on simple C-H functionalization logic that is enabled by a Cu-mediated oxidative phenol coupling that mimics the putative biosynthesis.

A Tool for the Import of Natural and Unnatural Nucleoside Triphosphates into Bacteria.

Nucleoside triphosphates play a central role in biology, but efforts to study these roles have proven difficult because the levels of triphosphates are tightly regulated in a cell and because individual triphosphates can be difficult to label or modify. In addition, many synthetic biology efforts are focused on the development of unnatural nucleoside triphosphates that perform specific functions in the cellular environment. In general, both of these efforts would be facilitated by a general means to directly introduce desired triphosphates into cells.

Reprograming the Replisome of a Semisynthetic Organism for the Expansion of the Genetic Alphabet.

Semisynthetic organisms (SSOs) created from Escherichia coli can replicate a plasmid containing an unnatural base pair (UBP) formed between the synthetic nucleosides dNaM and dTPT3 (dNaM-dTPT3) when the corresponding unnatural triphosphates are imported via expression of a nucleoside triphosphate transporter. The UBP can also be transcribed and used to translate proteins containing unnatural amino acids. However, UBPs are not well retained in all sequences, limiting the information that can be encoded, and are invariably lost upon extended growth.