Towards an Understanding of the Molecular Mechanisms of Variable Unnatural Base-Pair Behavior: A Biophysical Analysis of dNaM-dTPT3.

The series of unnatural base pairs (UBPs) developed by the Romesberg lab, which pair via hydrophobic and packing interactions have been replicated, transcribed, and translated inside of a living organism. However, as to why these UBPs exhibit variable fidelity and efficiency when used in different contexts is not clear. In an effort to gain some insights, we investigated the thermal stability and pairing selectivity of the (d)NaM-(d)TPT3 UBP in 11nt duplexes via UV spectroscopy and the effects on helical structure via CD spectroscopy. We observed that while the duplexes containing a UBP are less stable than fully natural duplexes, they are generally more stable than duplexes containing natural mispairs. This work provides the first insights connecting the thermal stability of the (d)NaM-(d)TPT3 UBP to the molecular mechanisms for varying replication fidelity in different sequence contexts in DNA, asymmetrical transcription fidelity, and codon:anticodon interactions and can assist in future UBP development.

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. This review outlines progress and achievements made towards developing a functional unnatural base pair and its use to generate semi-synthetic organisms with an expanded genetic alphabet that serves as the basis of an expanded genetic code.

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

ß-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 ß-lactams with the 5S stereochemistry, as opposed to the 5R stereochemistry of the traditional ß-lactams, would be required for inhibition. We report the synthesis and evaluation of a variety of 5S penem derivatives and identify several with promising activity against both a Gram-positive and a Gram-negative bacterial pathogen. To our knowledge these are the first 5S ß-lactams to possess significant antibacterial activity and the first ß-lactams imparted with antibacterial activity via optimization of the inhibition of a target other than a PBP. Along with the privileged status of their scaffold and the promise of bacterial signal peptidase I (SPase) as a target, this activity makes these compounds promising leads for development as novel therapeutics.

Synthetic Biology Parts for the Storage of Increased Genetic Information in Cells.

To bestow cells with novel forms and functions, the goal of synthetic biology, we have developed the unnatural nucleoside triphosphates dNaMTP and dTPT3TP, which form an unnatural base pair (UBP) and expand the genetic alphabet. While the UBP may be retained in the DNA of a living cell, its retention is sequence-dependent. We now report a steady-state kinetic characterization of the rate with which the Klenow fragment of E. coli DNA polymerase I synthesizes the UBP and its mispairs in a variety of sequence contexts. Correct UBP synthesis is as efficient as for a natural base pair, except in one sequence context, and in vitro performance is correlated with in vivo performance. The data elucidate the determinants of efficient UBP synthesis, show that the dNaM-dTPT3 UBP is the first generally recognized natural-like base pair, and importantly, demonstrate that dNaMTP and dTPT3TP are well optimized and standardized parts for the expansion of the genetic alphabet.

Single-cell mass spectrometry with multi-solvent extraction identifies metabolic differences between left and right blastomeres in the 8-cell frog (Xenopus) embryo.

Single-cell metabolic mass spectrometry enables the discovery (untargeted) analysis of small molecules in individual cells. Using single-cell capillary electrophoresis high-resolution mass spectrometry (CE-HRMS), we recently uncovered small-molecule differences between embryonic cells located along the animal-vegetal and dorsal-ventral axes of the 16-cell frog (Xenopus laevis) embryo, raising the question whether metabolic cell heterogeneity also exists along the left-right body axis. To address this question, we here advance single-cell CE-HRMS for identifying and quantifying metabolites in higher analytical sensitivity, and then use the methodology to compare metabolite production between left and right cells. Our strategy utilizes multiple solvents with complementary physicochemical properties to extract small molecules from single cells and improve electrophoretic separation, increasing metabolite ion signals for quantification and tandem HRMS. As a result, we were able to identify 55 different small molecules in D1 cells that were isolated from 8-cell embryos. To quantify metabolite production between left and right cells, we analyzed n = 24 different D1 cells in technical duplicate-triplicate measurements. Statistical and multivariate analysis based on 80 of the most repeatedly quantified compounds revealed 10 distinct metabolites that were significantly differentially accumulated in the left or right cells (p < 0.05 and fold change ≥1.5). These metabolites were enriched in the arginine-proline metabolic pathway in the right, but not the left D1 cells. Besides providing analytical benefits for single-cell HRMS, this work provides new metabolic data on the establishment of normal body asymmetry in the early developing embryo.