Evolution of Functionally Enhanced α-l-Threofuranosyl Nucleic Acid Aptamers.

Synthetic genetic polymers (xeno-nucleic acids, XNAs) have the potential to transition aptamers from laboratory tools to therapeutic agents, but additional functionality is needed to compete with antibodies. Here, we describe the evolution of a biologically stable artificial genetic system composed of α-l-threofuranosyl nucleic acid (TNA) that facilitates the production of backbone- and base-modified aptamers termed "threomers" that function as high quality protein capture reagents. Threomers were discovered against two prototypical protein targets implicated in human diseases through a combination of in vitro selection and next-generation sequencing using uracil nucleotides that are uniformly equipped with aromatic side chains commonly found in the paratope of antibody-antigen crystal structures. Kinetic measurements reveal that the side chain modifications are critical for generating threomers with slow off-rate binding kinetics. These findings expand the chemical space of evolvable non-natural genetic systems to include functional groups that enhance protein target binding by mimicking the structural properties of traditional antibodies.

Functional Comparison of Laboratory-Evolved XNA Polymerases for Synthetic Biology.

Artificial genetic polymers (XNAs) have enormous potential as new materials for synthetic biology, biotechnology, and molecular medicine; yet, very little is known about the biochemical properties of XNA polymerases that have been developed to synthesize and reverse-transcribe XNA polymers. Here, we compare the substrate specificity, thermal stability, reverse transcriptase activity, and fidelity of laboratory-evolved polymerases that were established to synthesize RNA, 2'-fluoroarabino nucleic acid (FANA), arabino nucleic acid (ANA), hexitol nucleic acid (HNA), threose nucleic acid (TNA), and phosphonomethylthreosyl nucleic acid (PMT). We find that the mutations acquired to facilitate XNA synthesis increase the tolerance of the enzymes for sugar-modified substrates with some sacrifice to protein-folding stability. Bst DNA polymerase was found to have weak reverse transcriptase activity on ANA and uncontrolled reverse transcriptase activity on HNA, differing from its known recognition of FANA and TNA templates. These data benchmark the activity of current XNA polymerases and provide opportunities for generating new polymerase variants that function with greater activity and substrate specificity.

Optimising complementary soft tissue synchrotron X-ray microtomography for reversibly-stained central nervous system samples.

Synchrotron radiation microtomography (SRµCT) is a nominally non-destructive 3D imaging technique which can visualise the internal structures of whole soft tissues. As a multi-stage technique, the cumulative benefits of optimising sample preparation, scanning parameters and signal processing can improve SRµCT imaging efficiency, image quality, accuracy and ultimately, data utility. By evaluating different sample preparations (embedding media, tissue stains), imaging (projection number, propagation distance) and reconstruction (artefact correction, phase retrieval) parameters, a novel methodology (combining reversible iodine stain, wax embedding and inline phase contrast) was optimised for fast (~12 minutes), high-resolution (3.2-4.8 µm diameter capillaries resolved) imaging of the full diameter of a 3.5 mm length of rat spinal cord. White-grey matter macro-features and micro-features such as motoneurons and capillary-level vasculature could then be completely segmented from the imaged volume for analysis through the shallow machine learning SuRVoS Workbench. Imaged spinal cord tissue was preserved for subsequent histology, establishing a complementary SRµCT methodology that can be applied to study spinal cord pathologies or other nervous system tissues such as ganglia, nerves and brain. Further, our 'single-scan iterative downsampling' approach and side-by-side comparisons of mounting options, sample stains and phase contrast parameters should inform efficient, effective future soft tissue SRµCT experiment design.