Efficient selection of nucleic acid aptamers with high affinity and specificity for a broad range of targets remains challenging. Historically, aptamer selections have been protracted and tedious processes, often requiring double-digit rounds of selection to converge nucleic acid pools into a small number of prospective high-affinity aptamers. More recently, the use of microfluidic devices and specialized equipment has helped streamline the aptamer selection process, but these platforms are not necessarily accessible to the broad research community. Here, we demonstrate that aptamers with high affinity and moderate specificity can be obtained with a conventional selection workflow that is modified to include facile methods for increasing partitioning and enhancing selection stringency. This process exposes an immobilized protein target to a single-stranded DNA library, followed by washing with buffer that contains the undesired off-target(s), with both steps occurring under constant perfusion using a standard peristaltic pump. Prospective aptamers are then eluted, amplified by an emulsion polymerase chain reaction, regenerated to single strands by enzymatic digestion, and resubjected to the selection procedure. We validated this selection scheme using the platelet-derived growth factor (PDGF) family, whereby we successfully isolated nanomolar affinity aptamers against PDGF-BB with specificity comparable to an aptamer selected using a microfluidics-based approach.
Yeast has been a versatile model
host for complex and valuable
natural product biosynthesis via the reconstruction of heterologous
biosynthetic pathways. Recent advances in natural product pathway
elucidation have uncovered many large and complicated plant pathways
that contain 10–30 genes for the biosynthesis of structurally
complex, valuable natural products. However, the ability to reconstruct
ultralong pathways efficiently in yeast does not match the increasing
demand for valuable plant natural product biomanufacturing. Here,
we developed a one-pot, multigene pathway integration method in yeast,
named MULTI-SCULPT for multiplex integration via selective, CRISPR-mediated,
ultralong pathway transformation. Leveraging multilocus genomic disruption
via CRISPR/Cas9, newly developed native and synthetic genetic parts,
and fine-tuned gene integration and characterization methods, we managed
to integrate 21 DNA inserts that contain a 12-gene plant isoflavone
biosynthetic pathway into yeast with a 90–100% success rate
in 12 days. This method enables fast and efficient ultralong biosynthetic
pathway integration and can allow for the fast iterative integration
of even longer pathways in the future. Ultimately, this method will
accelerate combinatorial optimization of elucidated plant natural
product pathways and accelerate putative pathway characterization
heterologously.
Discovering natural product biosynthetic pathways from medicinal plants is challenging and laborious, largely due to the complexity of the transcriptomics-driven pathway prediction process. Here we developed a novel approach that captures the protein-level connections between enzymes for pathway discovery with improved accuracy. We proved that heterologous protein-protein interaction screening in yeast enabled the efficient discovery of both dynamic plant enzyme complexes and the pathways they organize. This approach discovered complexes and pathways in the monoterpene indole alkaloid metabolism of a medicinal plant, kratom with high success rate. Screening using a strictosidine β-D-glucosidase (MsSGD1) against 19 medium-chain dehydrogenase/reductases (MsMDRs) identified five MsSGD1-MsMDR complexes. Three out of the five interacting MsMDRs were then proven functional, while the remaining 14 non-interacting candidates did not show obvious activities. The work discovered three branched pathways by combining transcriptomics, metabolomics, and heterologous PPI screening and demonstrated a new plant pathway discovery strategy.
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