Methods and compositions for performing highly sensitive in vitro assays to define substrate preferences and off-target sites of nucleic-acid binding, modifying, and cleaving agents.

Provided are methods for detecting a target molecule or particle suspected to be present in a sample, comprising (a) contacting the sample with (i) a fusion molecule comprising a ligand capable of binding to the target molecule or particle and a binding domain, and (ii) a polymer scaffold comprising at least one binding motif to which the binding domain is capable of binding, under conditions that allow the target molecule or particle to bind to the ligand and the binding domain to bind to the binding motif; (b) loading the polymer into a device comprising a pore that separates an interior space of the device into two volumes, and configuring the device to pass the polymer through the pore from one volume to the other volume, wherein the device further comprises a sensor adjacent to the pore configured to identify objects passing through the pore; and (c) determining, with the sensor, whether the fusion molecule or particle bound to the binding motif is bound to the target molecule or particle, thereby detecting the presence of the target molecule or particle in the sample.

Provided are methods for detecting a target molecule or particle suspected to be present in a sample, comprising (a) contacting the sample with (i) a fusion molecule comprising a ligand capable of binding to the target molecule or particle and a binding domain, and (ii) a polymer scaffold comprising at least one binding motif to which the binding domain is capable of binding, under conditions that allow the target molecule or particle to bind to the ligand and the binding domain to bind to the binding motif; (b) loading the polymer into a device comprising a pore that separates an interior space of the device into two volumes, and configuring the device to pass the polymer through the pore from one volume to the other volume, wherein the device further comprises a sensor adjacent to the pore configured to identify objects passing through the pore; and (c) determining, with the sensor, whether the fusion molecule or particle bound to the binding motif is bound to the target molecule or particle, thereby detecting the presence of the target molecule or particle in the sample.

Methods and related products are disclosed that improve the probability of interaction between a target molecule and a nanopore by capturing the target molecule on a surface comprising the nanopore. The captured target molecule, the nanopore, or both, are able to move relative to each other along the surface. When the leader of the target molecule is in proximity with the nanopore, interaction of the target portion of the target molecule with the nanopore occurs, thereby permitting sensing of the target portion. Confining the target molecule and nanopore in this manner leads to significantly enhanced interaction with the nanopore.

Methods and related products are disclosed that improve the probability of interaction between a target molecule and a nanopore by capturing the target molecule on a surface comprising the nanopore. The captured target molecule, the nanopore, or both, are able to move relative to each other along the surface. When the leader of the target molecule is in proximity with the nanopore, interaction of the target portion of the target molecule with the nanopore occurs, thereby permitting sensing of the target portion. Confining the target molecule and nanopore in this manner leads to significantly enhanced interaction with the nanopore.

The present invention relates to the field of nucleic acid purification. In particular, it relates to methods and tools for purifying nucleic acids in a sample; which are compatible with high-throughput sequencing and diagnosis. The inventors have shown that nucleic acid binding proteins recruited to polymerized tubulin (i.e. microtubules) could, subsequently, be isolated from cell lysates. Surprisingly, it has now been found that the amount of recovered nucleic acid found in these microtubule pellets increases dramatically in the presence of nucleic acid-trapping proteins comprising a nucleic acid-binding moiety and a polymerized tubulin-binding moiety, by comparison to proteins devoid of the nucleic acid-binding moiety; and that the recovery of the purified nucleic acids was itself particularly efficient. This purification method is particularly amenable to high-throughput sequencing and/or in the context of a diagnosis method for identifying or comparing the amount of nucleic acids in a set of samples.

The present invention relates to the field of nucleic acid purification. In particular, it relates to methods and tools for purifying nucleic acids in a sample; which are compatible with high-throughput sequencing and diagnosis. The inventors have shown that nucleic acid binding proteins recruited to polymerized tubulin (i.e. microtubules) could, subsequently, be isolated from cell lysates. Surprisingly, it has now been found that the amount of recovered nucleic acid found in these microtubule pellets increases dramatically in the presence of nucleic acid-trapping proteins comprising a nucleic acid-binding moiety and a polymerized tubulin-binding moiety, by comparison to proteins devoid of the nucleic acid-binding moiety; and that the recovery of the purified nucleic acids was itself particularly efficient. This purification method is particularly amenable to high-throughput sequencing and/or in the context of a diagnosis method for identifying or comparing the amount of nucleic acids in a set of samples.

The present invention relates to a composition for DNA sequence analysis and a method for DNA sequence analysis, the method comprising treating a sample with the composition. The composition of the present invention can attain efficient optical identification at a single-DNA molecule level by linking both an A/T-specific DNA-binder agent and an A/T-non-specific complementary DNA-binder agent to DNA, and thus can be helpfully used in studying chromosomal organization of genomes, protein immunolocalization, and the like.

The present invention relates to a composition for DNA sequence analysis and a method for DNA sequence analysis, the method comprising treating a sample with the composition. The composition of the present invention can attain efficient optical identification at a single-DNA molecule level by linking both an A/T-specific DNA-binder agent and an A/T-non-specific complementary DNA-binder agent to DNA, and thus can be helpfully used in studying chromosomal organization of genomes, protein immunolocalization, and the like.

In an aspect, a method for evaluating the partitioning of nucleic acid molecules in a sample of polynucleotides based on epigenetic state, comprising: (a) adding a set of epigenetic-control nucleic acid molecules to the nucleic acid molecules in the sample of polynucleotides, whereby producing a spiked-in sample; (b) partitioning nucleic acid molecules of the spiked-in sample into plurality of partitioned sets; (c) enriching a subset of molecules from the plurality of partitioned sets to generate enriched molecules, wherein the enriched molecules comprises a group of epigenetic-control nucleic acid molecules and a group of nucleic acid molecules from the sample of polynucleotides; (d) sequencing the enriched molecules to produce sequencing reads; (e) analyzing the sequencing reads to generate one or more epigenetic partition scores of the epigenetic-control nucleic acid molecules; and (f) comparing the one or more epigenetic partition scores with one or more epigenetic partition cut-offs.