The detection and quantification of short nucleic acid sequences has many potential applications in studying biological processes, monitoring disease initiation and progression, and evaluating environmental systems, but is challenging by nature. We present here an assay based on the solid-state nanopore platform for the identification of specific sequences in solution. We demonstrate that hybridization of a target nucleic acid with a synthetic probe molecule enables discrimination between duplex and single-stranded molecules with high efficacy. Our approach requires limited preparation of samples and yields an unambiguous translocation event rate enhancement that can be used to determine the presence and abundance of a single sequence within a background of non-target oligonucleotides.

Many regulated epigenetic elements and base lesions found in genomic DNA can both directly impact gene expression and play a role in disease processes. However, due to their non-canonical nature, they are challenging to assess with conventional technologies. Here, we present a new approach for the targeted detection of diverse modified bases in DNA. We first use enzymatic components of the DNA base excision repair pathway to install an individual affinity label at each location of a selected modified base with high yield. We then probe the resulting material with a solid-state nanopore assay capable of discriminating labeled DNA from unlabeled. The technique features exceptional modularity via selection of targeting enzymes, which we establish through the detection of four DNA base elements: uracil, 8-oxoguanine, T:G mismatch, and the methyladenine analog 1,N6-ethenoadenine. Our results demonstrate the potential for quantitative nanopore assessment of a broad range of base modifications.

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The biotin-streptavidin bond is the strongest noncovalent bond in nature and is thus used extensively in biotechnology applications. However, the difficulty of releasing the bond without high temperatures or corrosive solutions can be a barrier to applications involving nucleic acids and other delicate substrates. Here, room-temperature phenol is employed to release biotin-tagged DNA constructs from streptavidin rapidly and efficiently. It is demonstrated that synthetic biotinylated DNA can be recovered at yields approaching 100% from both solution-phase and bead-bound streptavidin with as little as 12% (v/v) phenol, leaving the biotin tag active and reusable after extraction. As an application of this recovery method, biotinylated DNA fragments are isolated from a mixed solution to provide selectivity for solid-state nanopore detection.

To maintain stereospecific biochemistry in cells, living organisms have evolved mechanisms to exclude D-amino acids ( D AA) in their protein synthesis machinery, which also limits our exploration of the realm of mirror-image molecules. Here, we show that high affinity between EF-Tu and aminoacyl-tRNA promotes D-amino acid incorporation. More strikingly, Elongation Factor P efficiently resolves peptidyl transferase stalling between two consecutive D-amino acids, and hence enables the translation of D-peptides.Life is an anti-entropic phenomenon with two mutually-reinforcing characters: homochirality and stereospecific catalysis. The exclusive presence of L-amino acids in proteins of the living world is a prominent example of this. However, D-amino acid containing peptides (DAACP) are still present in microbial, fungal and amphibian secretions, and often carry interesting bioactivities 1 . In nature, these molecules are made through non-ribosomal pathways, such as non-ribosomal peptide synthesis or post-translational modification, e.g. epimerization. DAACPs have been shown to have prolonged half lives in serum and resistance toward proteases without immunogenicity 2 , which is a desirable property in therapeutic reagents. Therefore, by gradually rewiring the present bio-machineries to overcome natural D AA barrier, we aim to build a bridge leading us to the space of mirror-image biomolecules, where they can serve as new tools for the biotech and pharmaceutical industry.Core protein translation machinery exhibits significant discrimination of D AAs from L-amino acids ( L AAs) at three steps 3 : aminoacylation of tRNAs by aminoacyl-tRNA synthetases (aaRSes), formation of ternary complexes with EF-Tu-GTP, and peptide-bond formation catalyzed by the ribosome (Figure 1a). In order to study these discriminations without the interference from D-amino acid oxidase and D-aminoacyl-tRNA deacylase 4,5 , we use a purified E. coli protein synthesis system (PURE) 6 , along with chemically acylated D-aminoacyl-tRNA 7 , as a model system for engineering D AA-tolerant translation machinery. We adapted the amber codon read through assay described in Fujino et al. 8 with slight modifications to assess D AA incorporation during elongation (Figure 1b). In brief, mRNA templates encoding an N-terminus FLAG epitope and a C-terminus stretch of artificial peptide composed of six different amino peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission. Incorporation could possibly be improved by additional EF-Tu and higher concentrations of charged tRNAs in the in vitro translation system, but we had earlier found that this only achieved a moderate improvement in L Ala 11 . We therefore thought to compensate for the disadvantage of low-incorporating L/D AAs by using a tRNA body that binds very efficiently to EF-Tu, noting that the K D s of tRNA bodies to EF-Tu-GTP span 600-fold from the weakest to the strongest 9 . Therefore, we chose four tRNA bodies (Glu2, Thr2, Gly2 and Ala2, sequences provided in Sup...