In various embodiments, the present invention provides fluorescent dyes that are linked to another species through an adaptor moiety. In an exemplary embodiment, the dye is linked to a polyphosphate nucleic acid through an adaptor. An adaptor can be a component of a linker. These conjugates find use in single molecule DNA sequencing and other applications. In various embodiments, the dye moiety is a cyanine dye. Cyanine dyes that are highly charged, such as those including multiple sulfonate, alkylsulfonate, carboxylate and/or alkylcarboxylate moieties are examples of cyanine dyes of use in the compounds of the invention.

The invention provides a novel class of scaffold-based labeled polymerase enzyme substrates. The polymerase enzyme substrates have a multivalent core or scaffold to which is attached fluorescent dye moieties and nucleoside phosphate moities. The polymerase enzyme substrates have multiple fluorescent dye moities and/or multiple nucleoside phosphate moieties. Preferred multivalent cores comprise trifunctional six membered aromatic moities. The invention also provides for sequencing methods and kits with scaffold-based labeled polymerase enzyme substrates.

The invention provides a novel class of cyanine dyes that are functionalized with sulfonic acid groups and a linker moiety that facilitates their conjugation to other species and substituent groups which increase the water-solubility, and optimize the optical properties of the dyes. Also provided are conjugates of the dyes, methods of using the dyes and their conjugates and kits including the dyes and their conjugates.

In various embodiments a molecular circuit is disclosed. The circuit comprises a negative electrode, a positive electrode spaced apart from the negative electrode, and an enzyme molecule conductively attached to both the positive and negative electrodes to form a circuit having a conduction pathway through the enzyme. In various examples, the enzyme is a polymerase. The circuit may further comprise molecular arms used to wire the enzyme to the electrodes. In various embodiments, the circuit functions as a sensor, wherein electrical signals, such as changes to voltage, current, impedance, conductance, or resistance in the circuit, are measured as substrates interact with the enzyme.

The present invention provides improved DNA polymerases, in particular, type A DNA polymerases, that may be better suited for applications in recombinant DNA technologies. Among other things, the present invention provides modified DNA polymerases derived from directed evolution experiments designed to select mutations that confer advantageous phenotypes under conditions used in industrial or research applications.

The present disclosure describes various improvements for programmable nuclease-based detection assays. Also provided are compositions, methods, kits, systems, and devices for practicing the same. Such improvements include improved reporter designs to facilitate the cleavage of a reporter immobilized on a substrate by an activated programmable nuclease. Improved reporter designs may comprise various lengths and structures of the reporter. The substrate can also comprise immobilized guide nucleic acids and/or programmable nucleases.

The present invention relates to a kit comprising a DNA-dependant DNA polymerase and at least one natural deoxynucleoside and to a kit comprising a DNA-dependent DNA polymerase and a detection system comprising a DNA template molecule, a DNA primer molecule, and a fluorescent moiety capable of being displaced from, or bound to, dsDNA synthesized by said DNA-dependent polymerase. It further relates to A method for measuring deoxynucleoside kinase activity in a sample characterized by; (i) contacting the sample, in a container, with a reaction mix comprising a DNA-dependent DNA polymerase, at least one deoxynucleoside and a detection system comprising a DNA template molecule, a DNA primer molecule, and a fluorescent moiety capable of being incorporated in, displaced from, or bound to dsDNA synthesized by said DNA dependent polymerase; (ii) incubating said container; (iii) measuring the signal from the fluorescent moiety; and correlating the signal from the fluorescent moiety to the deoxynucleoside kinase activity in the sample.

A method for cleaving a target DNA to isolate a target DNA fragment sequence of interest, directed by a targeting oligonucleotide followed by enriching the target DNA fragment sequence is disclosed. The targeting oligonucleotide binds to the target DNA during DNA cleavage. After cleavage, the target DNA fragment sequence of interest is modified using either a ligation or a polymerase extension method. The resulting target DNA fragment sequence of interest is enriched by exonuclease treatment.

The present disclosure relates to enzymatic cascade reactions for achieving better sensitivity in detecting a target nucleic acid sequence. Several aspects of the disclosure relate to a reaction master mix comprising four major components: a CRISPR enzyme, a guide RNA (gRNA), an aptamer (e.g., an inhibiting aptamer or an activating aptamer) and a signaling (i.e., reporter) enzyme. The aptamer interacts with the signaling enzyme and forms a complex, resulting in, e.g., inhibited signaling enzyme activity. When a target is present in the reaction mix, the Cas/guideRNA system becomes activated and preferentially collaterally cleave(s) all nucleic acids in the solution, including the aptamer. Once aptamer gets cleaved, the signaling enzyme is free to produce a signal. In the presence of a substrate, such signaling enzyme activity produces a robust signal that significantly improves the limit-of-detection of other techniques in the art.

A method of selective DNA amplification of a DNA mixture comprising a first population of DNA and a second population of DNA, wherein: a) a catalytically dead Staphylococcus aureus Cas9 complex (dSaCas9) comprising a dSaCas9 protein or derivative thereof complexed with one or more guide RNAs having selective binding affinity for DNA sequences preferentially present in the first population of DNA compared to the second population of DNA is contacted with the DNA mixture under a first reaction condition, said first reaction condition being suitable for binding of the dSaCas9 complex to DNA sequences for which it has a binding affinity, and then b) a strand-displacing DNA polymerase is contacted with the DNA mixture under a second reaction condition, said second reaction condition being suitable for amplification being suitable for amplification activity of the strand-displacing DNA polymerase; and related kits and uses.