Biological nanopores are capable of resolving small analytes down to a monoatomic ion. In this research, tetrachloroaurate(III), a polyatomic ion, is discovered to bind to the methionine residue (M113) of a wild-type α-hemolysin by reversible Au(III)-thioether coordination. However, the cylindrical pore geometry of α-hemolysin generates shallow ionic binding events (~5–6 pA) and may have introduced other undesired interactions. Inspired by nanopore sequencing, a Mycobacterium smegmatis porin A (MspA) nanopore, which possesses a conical pore geometry, is mutated to bind tetrachloroaurate(III). Subsequently, further amplified blockage events (up to ~55 pA) are observed, which report the largest single ion binding event from a nanopore measurement. By taking the embedded Au(III) as an atomic bridge, the MspA nanopore is enabled to discriminate between different biothiols from single molecule readouts. These phenomena suggest that MspA is advantageous for single molecule chemistry investigations and has applications as a hybrid biological nanopore with atomic adaptors.
Diverse functions of proteins, including
synthesis, catalysis,
and signaling, result from their highly variable amino acid sequences.
The technology allowing for direct analysis of protein sequences,
however, is still unsatisfactory. Recent developments of nanopore
sequencing of DNA or RNA have motivated attempts to realize nanopore
sequencing of peptides in a similar manner. The core challenge has
been to achieve a controlled ratcheting motion of the target peptide,
which is currently restricted to a limited choice of compatible enzymes.
By constructing peptide–oligonucleotide conjugates (POCs) and
measurements with nanopore-induced phase-shift sequencing (NIPSS),
direct observation of the ratcheting motion of peptides has been successfully
achieved. The generated events show a clear sequence dependence on
the peptide that is being tested. The method is compatible with peptides
with either a conjugated N- or C-terminus. The demonstrated results
suggest a proof of concept of nanopore sequencing of peptide and can
be useful for peptide fingerprinting.
MicroRNAs (miRNAs) are a class of short non-coding RNAs that function in RNA silencing and post-transcriptional gene regulation. However, direct characterization of miRNA is challenging due to its unique properties such as its low abundance, sequence similarities, and short length. Although urgently needed, single molecule sequencing of miRNA has never been demonstrated, to the best of our knowledge. Nanopore-induced phase-shift sequencing (NIPSS), which is a variant form of nanopore sequencing, could directly sequence any short analytes including miRNA. In practice, NIPSS clearly discriminates between different identities, isoforms, and epigenetic variants of model miRNA sequences. This work thus demonstrates direct sequencing of miRNA, which serves as a complement to existing miRNA sensing routines by the introduction of the single molecule resolution. Future engineering of this technique may assist miRNA-based early stage diagnosis or inspire novel cancer therapeutics.
Cisplatin, which selectively binds
to N7 atoms of purines
to inhibit normal replication and transcription, is a widely applied
chemotherapeutic drug in the treatment of cancer. Though direct identification
of cisplatin lesions on DNA is of great significance, existing sequencing
methods have issues such as complications of preamplification or enrichment-induced
false-positive reports. Direct identification of cisplatin lesions
by nanopore sequencing (NPS) is in principle feasible. However, relevant
investigations have never been reported. By constructing model sequences
(83 nucleotides in length) containing a sole cisplatin lesion, identification
of corresponding lesions by NPS is achieved with <10 ng of input
sequencing library. Moreover, characteristic high-frequency noises
caused by cisplatin lesions are consistently observed during NPS,
clearly identifiable in corresponding high-pass filtered traces. This
feature is, however, never observed in any other combinations of natural
DNA bases and could be taken as a reference to identify cisplatin
lesions on DNA. Further investigations demonstrate that cisplatin
stalls the replication of phi29 DNA polymerase, which appears as a
∼5 pA level fluctuation in the single-molecule resolution.
These results have confirmed the feasibility of NPS to identify cisplatin
lesions at the genomic level and may provide new insights into understanding
the molecular mechanism of platinum-based drugs.
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