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The DNA polymerase I from Geobacillus stearothermophilus (also known as Bst DNAP) is widely used in isothermal amplification reactions, where its strand displacement ability is prized. More robust versions of this enzyme should be enabled for diagnostic applications, especially for carrying out higher temperature reactions that might proceed more quickly. To this end, we appended a short fusion domain from the actin-binding protein villin that improved both stability and purification of the enzyme. In parallel, we have developed a machine learning algorithm that assesses the relative fit of individual amino acids to their chemical microenvironments at any position in a protein and applied this algorithm to predict sequence substitutions in Bst DNAP. The top predicted variants had greatly improved thermotolerance (heating prior to assay), and upon combination, the mutations showed additive thermostability, with denaturation temperatures up to 2.5 °C higher than the parental enzyme. The increased thermostability of the enzyme allowed faster loop-mediated isothermal amplification assays to be carried out at 73 °C, where both Bst DNAP and its improved commercial counterpart Bst 2.0 are inactivated. Overall, this is one of the first examples of the application of machine learning approaches to the thermostabilization of an enzyme.

The DNA polymerase I from Geobacillus stearothermophilus (also known as Bst DNAP) is widely used in isothermal amplification reactions, where its strand displacement ability is prized. More robust versions of this enzyme should be enabled for diagnostic applications, especially for carrying out higher temperature reactions that might proceed more quickly. To this end, we appended a short fusion domain from the actin-binding protein villin that improved both stability and purification of the enzyme. In parallel, we have developed a machine learning algorithm that assesses the relative fit of individual amino acids to their chemical microenvironments at any position in a protein and applied this algorithm to predict sequence substitutions in Bst DNAP. The top predicted variants had greatly improved thermotolerance (heating prior to assay), and upon combination, the mutations showed additive thermostability, with denaturation temperatures up to 2.5 °C higher than the parental enzyme. The increased thermostability of the enzyme allowed faster loop-mediated isothermal amplification assays to be carried out at 73 °C, where both Bst DNAP and its improved commercial counterpart Bst 2.0 are inactivated. Overall, this is one of the first examples of the application of machine learning approaches to the thermostabilization of an enzyme.

Taq DNA polymerase, one of the first thermostable DNA polymerases to be discovered, has been typecast as a DNA-dependent DNA polymerase commonly employed for PCR. However, Taq polymerase belongs to the same DNA polymerase superfamily as the Molony murine leukemia virus reverse transcriptase and has in the past been shown to possess reverse transcriptase activity. We report optimized buffer and salt compositions that promote the reverse transcriptase activity of Taq DNA polymerase, and thereby allow it to be used as the sole enzyme in TaqMan RT-qPCR reactions. We demonstrate the utility of Taq-alone RT-qPCR reactions by executing CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays that could detect as few as 2 copies/μL of input viral genomic RNA.

One of the most remarkable, but typically unremarked, aspects of the translation apparatus is the pleiotropic pliability of tRNA. This humble cloverleaf/L-shaped molecule must implement the first genetic code, via base pairing and wobble interactions, but is also largely responsible for the specificity of the second genetic code, the pairings between amino acids, tRNA synthetases, and tRNAs. Despite the overarching similarities between tRNAs, they must nonetheless be specifically recognized by cognate tRNA synthetases and largely rejected by noncognate synthetases. Conversely, despite the differences between tRNAs that allow such discrimination, they must be uniformly accepted by the ribosome, in part via the machinations of the translation elongation factors, which work with a diverse coterie of tRNA-amino acid conjugates to balance binding and loading. While it is easy to ascribe both discrimination and acceptance to the individual proteins (synthetases and EF-Tu/eEF-1) that recognize tRNAs, there is a large body of evidence that suggests that the sequences, structures, and dynamics of tRNAs are instrumental in the choices these proteins make.

Strand-displacing polymerases are a crucial component of isothermal amplification (IA) reactions, where the lack of thermal cycling reduces equipment needs and improves the time to answer, especially for point-of-care applications. In order to improve the function of strand-displacing polymerases, we have developed an emulsion-based directed evolution scheme, high-temperature isothermal compartmentalized self-replication (HTI-CSR) that does not rely on thermal cycling. Starting from an algorithm-optimized shuffled library of exonuclease-deficient Family A polymerases from Geobacillus stearothermophilus (Bst LF) and Thermus aquaticus (Klentaq), we have applied HTI-CSR to generate a more thermostable strand-displacing polymerase variant that performs well in loop-mediated isothermal amplification and rolling circle amplification, even after thermal challenges of up to 95 °C that lead to better primer annealing. The new enzyme (v5.9) is also capable of a variety of new reactions, including isothermal hyperbranched rolling circle amplification. The HTI-CSR method should now prove useful for evolving additional beneficial phenotypes in strand-displacing polymerases.

A bridge containing a rigid trans-stilbene group, −P(O)(O-)O(CH_2)_3NHC(O) C_6H_4CH CHC_6H_4C(O)NH(CH_2)_3OP(O)(O-)−, has been incorporated into several oligonucleotide sequences based on the minimal Rev Binding Element (RBE) of HIV-1. This bridge was found to be as effective as a UUCG tetraloop in stabilizing short RNA duplex structures containing mismatched bases and bulged out nucleotide residues and to be more effective than either a TTTT loop or a triethyleneglycol linker in stabilizing similar DNA structures. Evaluation of stilbene-containing RNA RBE sequences of varying length for their ability to bind the Rev protein of HIV-1 showed that a 22-nucleotide stilbenedicarboxamide conjugate bound Rev almost as well as a 94-base fragment of the Rev Responsive Element (RRE). A DNA hairpin mimetic with the same sequence was incapable of Rev binding. Taken together, these experiments serve as an example for how in vitroselection and chemical modification can be combined to generate high-affinity mimetics of nucleic acid sequence and structure.

The interactions between the mouse major urinary protein isoform MUP-I and the pheromone 2-sec-butyl-4,5-dihydrothiazole have been characterized in solution. (15)N-labeled and (15)N, (13)C-doubly-labeled recombinant MUP-I were produced in a bacterial expression system and purified to homogeneity. Racemic 2-sec-butyl-4, 5-dihydrothiazole was produced synthetically. An equilibrium diffusion assay and NMR titration revealed that both enantiomers of the pheromone bind to the recombinant protein with a stoichiometry of 1 equiv of protein to 1 equiv of racemic pheromone. A micromolar dissociation constant and slow-exchange regime dissociation kinetics were determined for the pheromone-protein complex. (1)H, (15)N, and (13)C chemical shifts of MUP-I were assigned using triple resonance and (15)N-correlated 3D NMR experiments. Changes in protein (1)H(N) and (15)N(H) chemical shifts upon addition of pheromone were used to identify the ligand binding site. Several amide signals, corresponding to residues on one side of the binding site, were split into two peaks in the saturated protein-ligand complex. Similarly, two overlapping ligand spin systems were present in isotope-filtered NMR spectra of labeled protein bound to unlabeled pheromone. The two sets of peaks were attributed to the two possible chiralities of the pheromone. Intermolecular NOEs indicated that the orientation of the pheromone in the MUP-I binding cavity is opposite to that modeled in a previous X-ray structure.