WO2013012434A1 - Sondes tige-boucle bioluminescentes, compositions contenant celles-ci et procédés utilisant celles-ci - Google Patents

Sondes tige-boucle bioluminescentes, compositions contenant celles-ci et procédés utilisant celles-ci Download PDF

Info

Publication number
WO2013012434A1
WO2013012434A1 PCT/US2011/063444 US2011063444W WO2013012434A1 WO 2013012434 A1 WO2013012434 A1 WO 2013012434A1 US 2011063444 W US2011063444 W US 2011063444W WO 2013012434 A1 WO2013012434 A1 WO 2013012434A1
Authority
WO
WIPO (PCT)
Prior art keywords
bioluminescent
loop
stem
hairpin
sequence
Prior art date
Application number
PCT/US2011/063444
Other languages
English (en)
Inventor
Sapna K. DEO
Eric A. HUNT
Original Assignee
University Of Miami
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University Of Miami filed Critical University Of Miami
Publication of WO2013012434A1 publication Critical patent/WO2013012434A1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label

Definitions

  • the present invention relates generally to stem-loop probes, and more particularly to bioluminescent stem-loop probes, and compositions and method utilizing bioluminescent stem-loop probes.
  • SLPs Stem-loop probes
  • qPCR real-time/quantitative polymerase chain reaction
  • SNP single-nucleotide polymorphism
  • a SLP is typically assembled as a single-stranded, self-complementary oligonucleotide that includes a 15-30 nt region complementary to the target sequence (loop) flanked by two short, self-complementary 5-7 nt stem regions.
  • Exemplary SLPs are disclosed in U.S. Patent Nos. 5,925,517 and 6,103,476, and U.S. Patent Publication No. 2010/0248385.
  • the two stem regions are conjugated to a fluorescent reporter molecule and a quencher, respectively.
  • the SLP switches from a closed (intramolecular hybridization) conformation to an open (intermolecular hybridization) conformation upon formation of a duplex between the target and the corresponding sequence in the loop region.
  • the reporter signal is quenched by direct or resonance energy transfer.
  • stimulating radiation is applied such that, when the SLP is in the open conformation a signal emitted from the fluorescent reporter may be detected.
  • the SLP design offers superior specificity over linear probes and eliminates the need to remove excess/unhybridized probes prior to measurement.
  • a bioluminescent stem-loop probe that includes a hairpin-loop sequence comprising a sequence complementary to a biological target sequence; a bioluminescent marker coupled to the hairpin-loop sequence; and a quencher coupled to the hairpin-loop sequence.
  • the hairpin-loop sequence can be between the bioluminescent marker and the quencher and the bioluminescent stem-loop probe can be capable of switching between a closed configuration and an open configuration.
  • the quencher can reduce a bioluminescent output of said bioluminescent marker when said probe is in the closed configuration.
  • Any of the BSLPs disclosed herein can also include an affinity handle between the quencher and the hairpin-loop sequence.
  • the hairpin-loop sequence can comprise an aptamer.
  • the hairpin-loop sequence can comprise an aptamer selected from the group consisting of a DNA aptamer, an RNA aptamer, a peptide aptamer and a combination thereof.
  • any of the BSLPs disclosed herein can also include a first stem sequence and a complementary second stem sequence, wherein the first stem sequence is between the hairpin-loop sequence and the bioluminescent marker, and the second stem sequence is between the hairpin-loop sequence and the quencher.
  • the first and second stem sequences can form a double- stranded stem that will open when the hairpin-loop sequence hybridizes with a complementary target sequence.
  • the bioluminescent marker can include a bioluminescent protein, a truncated bioluminescent protein, a bioluminescent enzyme, a truncated bioluminescent enzyme, or a combination thereof.
  • the bioluminescent marker can include a luciferase, a truncated luciferase, a bioluminescent protein, a truncated bioluminescent protein, or combinations thereof.
  • the bioluminescent marker can include a luciferase selected from the group consisting of Renilla luciferase (RLuc), Gaussia luciferase (GLuc), Photonus pyralis luciferase (FLuc), Vargula luciferase (VLuc), truncated variants thereof, and combinations thereof.
  • RLuc Renilla luciferase
  • GLuc Gaussia luciferase
  • FLuc Photonus pyralis luciferase
  • VLuc Vargula luciferase
  • truncated has its standard meaning in the art.
  • a truncated variants of a bioluminescent proteins or luciferases can include 50-90% of the native bioluminescent protein or luciferase, respectively.
  • Truncated bioluminescent markers can be made using any technique known to those skilled in the art, so long as the truncated variant can still participate in the relevant bioluminescent reaction.
  • any of the BSLPs disclosed herein can also include a linker coupled to the hairpin-loop sequence and the quencher, where the linker is between the hairpin- loop sequence and the quencher.
  • the quencher can reduce a bioluminescent output of the bioluminescent marker by energy transfer, active-site inhibition, or both.
  • the quencher can include an active-site inhibitor formed from a compound selected from the following:
  • Bioluminescent stem-loop probes that include a bioluminescent marker, and at least two hairpin-loop constructs coupled to the bioluminescent marker are also described.
  • the bioluminescent stem-loop probe can include at least three of the hairpin-loop constructs.
  • each of the hairpin-loop constructs can include a hairpin-loop sequence comprising a sequence complementary to a biological target sequence, and a quencher coupled to the hairpin-loop sequence.
  • the hairpin-loop sequence can be between the bioluminescent marker and the quencher, and the hairpin-loop construct can be being capable of switching between a closed position and an open position.
  • the BSLPs that include multiple-hairpin-loop constructs can have all of the same features of other BSLPs described herein.
  • the BSLPs that include multiple-hairpin-loop constructs can include any specific bioluminescent markers, stems, hairpin-loops, linkers, affinity tags, quenchers, and combinations thereof that are described herein.
  • Each of the hairpin-loop constructs coupled to the bioluminescent marker can be the same or different.
  • bioluminescent composition that includes a carrier liquid, and a bioluminescent stem-loop probe selected from any of the bioluminescent stem-loop probes described herein.
  • any of the bioluminescent compositions described herein can also include a bioluminescent facilitator complementary to the bioluminescent marker.
  • a bioluminescent facilitator selected from the group consisting of a bioluminescent substrate, a molecular oxygen source, adenosine triphosphate (ATP), a cation, and combinations thereof.
  • the bioluminescent marker can be a luciferase and the bioluminescent facilitator comprises a luciferin.
  • An analytical method that includes mixing any of the bioluminescent compositions described herein with a target composition, and detecting
  • bioluminescence from the mixture is also described. Any of the analytical methods described herein can be performed with no stimulating radiation introduced during the detecting phase. In any of the analytical methods described herein, a bioluminescent facilitator can be introduced to the mixture prior to detecting phase.
  • the target composition can include biological material.
  • the target composition can include biological material selected from the group consisting of DNA, DNA fragments, RNA, RNA fragments, proteins, protein fragments, naturally- occurring molecules, and mixtures thereof.
  • fragment has its standard meaning n the art.
  • a fragments of DNA, RNA or proteins can include 5-100 base pairs in their native arrangement.
  • Fig. 1 is a schematic of a bioluminescent probe as described herein in a closed configuration.
  • Fig. 2 is a schematic of the bioluminescent probe from Fig. 1 (without the affinity handle) in an open configuration.
  • Fig. 3 is a schematic of a bioluminescent probe as described herein employing a linker between the stem sequence and the quencher.
  • Fig. 4 is a schematic showing a bioluminescent probe as described herein that includes multiple hairpin-loop constructs in both the closed configuration (left) and the open configuration (right).
  • Fig. 5 is a synthetic scheme for the conjugation of Rluc to a stem-loop probe.
  • Fig. 6 is a picture of SDS-PAGE results of BSLP conjugation.
  • Fig. 7(A) is a graph of baseline-subtracted fluorescent emission of various amounts of SLP in the presence of 10 pmol of a target, while 7(B) in the background normalized signal-to-noise (S/N) response of varying amounts of SLP in the presence of 10 pmol of a target.
  • S/N signal-to-noise
  • Fig. 8 is a graph showing a S/N comparison between a bioluminescent SLP and a fluorescent SLP.
  • Fig. 9 is a graph showing a calibration performed to determine optimal conditions for a bioluminescent SLP and a fluorescent SLP.
  • Fig. 10 is a graph showing the relative bioluminescence for a
  • bioluminescent stem-loop probe exposed to serum samples from patients with varying stages of breast cancer.
  • Fig. 11 is a graph showing specificity of a bioluminescent probe to distinguish between perfectly matched target sequences and targets that were mismatched to varying degrees.
  • Fig. 12 is a graph showing an emission scan comparison for Rluc and Glue using a spectrofluorometer.
  • Fig. 13 is a picture of Glue purified by IMAC.
  • Fig. 14 is a graph showing the decay kinetics, i.e., relative luminescence versus time, for Glue and Rluc.
  • Fig. 15 is a graph showing the relative bioluminescence of Glue during PCR cycling.
  • Fig. 16 is a schematic showing a synthetic scheme for the chemical conjugation of a linker-active-site inhibitor complex attached to a SLP.
  • Bioluminescent SLPs compositions containing bioluminescent SLPs and methods employing bioluminescent SLPs are described. It has been discovered that employing a bioluminescent marker in a SLP design provides for low background noise, signal amplification by enzymatic light generation, and highly efficient quenching, which provides a sensing tool with superior sensitivity, selectivity, and applicability. This can be further enhanced through the use of active-site inhibitor as quenchers, rather than the conventional energy transfer-based molecules.
  • the bioluminescent SLPs can include a hairpin- loop sequence comprising a sequence complementary to a biological target sequence.
  • a bioluminescent marker can be coupled to the hairpin-loop sequence and a quencher can be coupled to the hairpin-loop sequence.
  • the bioluminescent marker and the quencher can be coupled to opposite ends of the hairpin-loop sequence, such that the hairpin-loop sequence is between the bioluminescent marker and the quencher.
  • the quencher can be coupled to the 3' end of the hairpin-loop sequence, while the bioluminescent marker can be coupled to the 5' end of the hairpin-loop sequence or vice versa.
  • the bioluminescent stem-loop probe can be capable of switching between a closed configuration, as shown in Fig. 1 , and an open configuration, as shown in Fig. 2.
  • Coupled has its standard meaning and includes where components are directly bonded to one another and where there are intermediate sequences and/or moieties between the coupled components.
  • the second stem sequence can be coupled to a quencher where the second stem sequence is covalently bonded directly to the quencher or where, as in Fig. 3, there is a linker between second stem sequence and the quencher.
  • Figs. 1 stem
  • Fig. 2 hairpin-loop-to-target
  • Fig. 3 stem
  • the quencher can reduce the bioluminescent output of the bioluminescent marker when the stem-loop probe is in the closed configuration. In other words, when the quencher is proximate the bioluminescent marker, the quencher can interact with the bioluminescent marker to reduce the detectable bioluminescence. It should be noted that where the quencher relies on an energy transfer mechanism, the
  • bioluminescent marker may emit radiation that is not detectible because the radiation is immediately absorbed by the quencher.
  • bioluminescent output refers to the amount of detectable bioluminescence originating from a bioluminescent source.
  • the quenchers disclosed herein include both energy transfer quenchers and active-site inhibitors (e.g., substrate analogs), including substrate (e.g., bioluminescent pigment) inhibitors.
  • energy transfer quenchers absorb bioluminescent energy emitted by the source, whereas active-site or substrate inhibitors actually prevent the bioluminescent reactions from occurring by preventing the interactions between the active-sites (e.g., the active sites of an enzyme, such as luciferase) and the substrates (e.g., luciferin) necessary to produce the bioluminescent reaction.
  • the quencher can reduce a bioluminescent output of the bioluminescent marker by energy transfer, active-site inhibition, or both.
  • active site inhibition and related terms, such as active site quencher, refer to reductions of bioluminescent output that result because the active-site cannot properly interact with the substrate.
  • energy transfer inhibition and related terms, such as energy transfer quencher, refer to reductions in bioluminescent output that result because bioluminescence is absorbed, whether by direct energy transfer, resonance energy transfer, or some other mechanism.
  • energy transfer inhibitors include, but are not limited to (i) rhodamine dyes selected from the group consisting of tetramethyl-6-carboxyrhodamine (TAMRA), tetrapropano-6-carboxyrhodamine (ROX), and (ii) DABSYL, DABCYL, cyanine dyes including nitrothiazole blue (NTB), anthraquinone, malachite green, nitrothiazole, and nitroimidazole compounds and the like.
  • TAMRA tetramethyl-6-carboxyrhodamine
  • ROX tetrapropano-6-carboxyrhodamine
  • DABSYL DABCYL
  • cyanine dyes including nitrothiazole blue (
  • the quencher can also be an active-site inhibitor formed from active-site substrate analogs, such as the following:
  • Quenchers A-H are reacted so that they can be coupled to the stem-loop probe.
  • the actual chemical structure of the quenchers will be slightly different that that shown in the free-standing compounds shown above.
  • the reaction will occur at an amine of the above compounds.
  • the bioluminescent stem-loop probe can also include a linker coupled to the hairpin-loop sequence and the quencher.
  • the linker can be between the hairpin-loop sequence and the quencher.
  • the hairpin-loop sequence and quencher can be coupled to opposite ends of the linker.
  • the linker can be located between the second stem sequence and the quencher.
  • a "linker” is any sequence that allows the quencher additional mobility when the bioluminescent stem-loop probe is in the closed
  • a linker may be of sufficient length to (i) enable the quencher to interact with an active site and/or substrate that is not immediately adjacent to the closed stem, and (ii) allow the bioluminescent stem-loop probe to close when the hairpin-loop sequence is hot hybridized with the target sequence.
  • the bioluminescent stem-loop probe can include more than one quencher per stem-loop complex.
  • a dendrimer linkage - for example, a dendrimer phosphoramidite - can be used to couple the hairpin-loop sequence to multiple quenchers.
  • the hairpin-loop sequence can comprise an aptamer.
  • the aptamer can be selected from a DNA aptamer, a RNA aptamer, a peptide aptamer and combinations thereof.
  • the aptamer can be selected to be complementary to a target sequence, such as a DNA sequence, an RNA sequence, a peptide sequence and combinations thereof.
  • target sequence such as a DNA sequence, an RNA sequence, a peptide sequence and combinations thereof.
  • DNA sequence is intended to be broadly construed to include less commonly studied DNA sequences that include, but are not limited to, DNA binding domains.
  • the bioluminescent stem-loop probe can also include a first stem sequence and a complementary second stem sequence.
  • the first stem sequence can be between the hairpin-loop sequence and the bioluminescent marker
  • the second stem sequence can be between the hairpin-loop sequence and the quencher.
  • the hairpin-loop and bioluminescent marker can be coupled at opposite ends of the first stem sequence and the hairpin-loop and quencher can be coupled at opposite ends of the second stem sequence.
  • the first and second stem sequences can form at least 3 G-C base pairs, at least 4 G-C base pairs, at least 5 G-C base pairs, or at least 6 G-C base pairs.
  • the first and second stem sequences can form a G-C analog.
  • the first and second stem sequences should be selected such that, under analytical conditions, the bioluminescent stem-loop probe will remain in a closed configuration until the hairpin-loop sequence hybridizes with the target sequence.
  • first and second stem sequences will form a double-stranded stem, but will open when the hairpin-loop sequence hybridizes with a complementary target sequence.
  • the bioluminescent marker can include a bioluminescent protein, a truncated bioluminescent protein, a bioluminescent enzyme, a truncated bioluminescent enzyme, or a combination thereof.
  • the bioluminescent marker can include a luciferase, a truncated luciferase, or a combination thereof.
  • the bioluminescent marker can include a luciferase selected from the group consisting of Renilla luciferase (RLuc), Gaussia luciferase (GLuc), Photonus pyralis luciferase (FLuc), Vargula luciferase (VLuc), truncated variants thereof, and combinations thereof.
  • bioluminescent substrates ⁇ i.e., bioluminescent pigments
  • bioluminescent pigments include, but are not limited to, coelenterazine, coelentrazine variants (e, f, fcp, h, hep, etc.), Vargulin, (4S)-2-(6-hydroxy-1 ,3-benzothiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid (“firefly luciferin”), and (£)-2-methyl-4-(2,6,6-trimethyl-1-cyclohex-1-yl)-1-buten-1-ol formate ( af/a luciferin”).
  • the bioluminescent enzyme and/or bioluminescent protein can catalyze the bioluminescent reaction of the substrate.
  • the bioluminescent enzyme can catalyze the reaction of coelenterazine into its two main metabolites coelenteramide and coelenteramine.
  • the bioluminescent marker can include a bioluminescent protein selected from the group consisting of aequorin, obelin, mitrocomin, clytin, and truncated variants of these proteins.
  • the bioluminescent proteins or truncated bioluminescent proteins can include a bioluminescent enzyme, a bioluminescent substrate, or both.
  • the bioluminescent stem-loop probe can also include an affinity handle between the quencher and the hairpin-loop sequence.
  • an affinity handle between the quencher and the hairpin-loop sequence.
  • one end of the affinity handle can be coupled to or proximate an end of the quencher or the hairpin- loop, while the opposite end of the affinity handle can be free (e.g., as shown in Fig. 1).
  • a biotin-modified thymidine such as that shown in Fig. 1 , can be used as an exemplary affinity handle.
  • the affinity handle can be used for isolating the bioluminescent SLPs from unreacted bioluminescent enzymes and/or bioluminescent proteins.
  • the bioluminescent stem-loop probe can be isolated using affinity chromatography.
  • the bioluminescent stem-loop probe can include multiple hairpin-loop constructs.
  • at least two hairpin-loop constructs can be coupled to the bioluminescent marker.
  • Each of the hairpin-loop constructs can include (i) a hairpin-loop sequence comprising a sequence complementary to a biological target sequence, and (ii) a quencher coupled to the hairpin-loop sequence, where the hairpin-loop sequence is between the bioluminescent marker and the quencher.
  • each of the hairpin-loop constructs can be capable of switching between a closed position and an open position.
  • any and all bioluminescent stem-loop probe configurations described herein can be incorporated into bioluminescent probes that include multiple hairpin-loop constructs.
  • the bioluminescent stem-loop probe can include at least three of the hairpin-loop constructs, at least four hairpin-loop constructs, at least five hairpin-loop constructs, or at least six hairpin-loop constructs.
  • the bioluminescent markers include a number of active-sites and/or substrates distributed in various locations, it can be beneficial to have multiple quenchers per bioluminescent stem-loop probe.
  • Another useful approach is to include a dendrimer linkage - for example, dendrimer phosphoramidite - to couple the hairpin- loop sequence to multiple quenchers.
  • the dendrimer linkage can be coupled to the quenchers directly or by way of individual linkers.
  • the quenchers can be any of the energy inhibitors or active-site inhibitors described herein, including any of the following active-site inhibitors:
  • a bioluminescent composition comprising a carrier liquid and one or more of the BSLPs described herein is also described.
  • the bioluminescent composition can include at least two different bioluminescent stem-loop probes, at least three different bioluminescent stem-loop probes, at least four different bioluminescent stem-loop probes, or at least five different bioluminescent stem-loop probes.
  • the first bioluminescent stem-loop probe can include a first bioluminescent marker and a first hairpin-loop sequence and the second bioluminescent probe can include a different bioluminescent marker and a different hairpin-loop sequence.
  • bioluminescent stem-loop probe can have a bioluminescent marker and hairpin-loop sequence that is different from the other bioluminescent stem-loop probes in the bioluminescent composition.
  • the bioluminescent composition can also include fluorescent stem-loop probes and other image enhancement compounds.
  • composition can also include a bioluminescent facilitator
  • bioluminescent facilitators include, but are not limited to a substrate, e.g., luciferin, a molecular oxygen source, adenosine triphosphate (ATP), and a cation, e.g., divalent cations, Ca 2+ and Mg 2+ . More than one bioluminescent facilitator may be included in the composition to facilitate bioluminescence.
  • the bioluminescent facilitator can be a bioluminescent substrate, e.g., a luciferin or coelenterazine.
  • the bioluminescent stem-loop probe in the composition can include a bioluminescent marker comprising a luciferase and the bioluminescent facilitator can include a luciferin and/or molecular oxygen.
  • An average ratio of hairpin-loop constructs-to-bioluminescent markers in the composition can be at least 2:1 , at least 3:1 , at least 4:1 , at least 5:1, at least 6:1 , or at least 10:1.
  • the average ratio of hairpin-loop constructs-to-bioluminescent markers can be calculated using a Bradford protein assay and the molecular extinction coefficient of the quencher.
  • the analytical method can include mixing a bioluminescent composition as described herein with a target composition, and then detecting bioluminescence from the mixture.
  • the method can include detecting bioluminescence and/or fluorescence from each of the different bioluminescent probes (and fluorescent probes, when applicable) present in the mixture sequentially, simultaneously or a combination of both.
  • the method can include introducing a bioluminescent facilitator to the mixture prior to detecting.
  • the bioluminescent facilitator can be introduced after the bioluminescent composition has been mixed with the target composition to form the mixture.
  • the bioluminescent facilitator can be introduced after sufficient time has elapsed for the hairpin-loop sequence of the bioluminescent stem-loop probes to hybridize with any target sequences present in the target composition.
  • the bioluminescent facilitator can be introduced less than two minutes prior to detection, less than one minute prior to detection, or less than 30 second prior to detection/ or less than 10 seconds prior to detection, or less than 5 seconds prior to detection, or during detection.
  • a bioluminescent substrate can be added to the bioluminescent-target mixture during detection.
  • the mixture can include a bioluminescent facilitator complementary to the bioluminescent marker.
  • the bioluminescent facilitator can be added as part of the bioluminescent composition or added separately.
  • the mixing can occur in vitro, in vivo, ex vivo, or in situ.
  • mixing can include injecting the mixture into a living organism, a petri dish, or a cuvette or similar laboratory container. This can be followed by injecting the bioluminescent facilitator into the mixture prior to or during detection.
  • the methods described herein can be used for any medical, analytical, environmental, forensic, or diagnostic purpose. However, the methods described herein can also be used for non-invasive, non-therapeutic, diagnostic purposes, i.e., only for data gathering.
  • the target composition can include biological material.
  • the target can include biological material selected from the group consisting of DNA, DNA fragments, RNA, RNA fragments, proteins, protein fragments, naturally-occurring molecules, and mixtures thereof.
  • the biological target sequence can be a nucleotide or peptide sequence that may be found in the biological, environmental, or food material.
  • the biological target sequence can be a portion of a nucleotide sequence known or believed to be associated with a particular condition (e.g., cancer, heart disease, etc.), associated with a predisposition to a disease state, an environmental contaminant, a food contaminant, etc.
  • FSLPs fluorescent stem-loop probes
  • This need for external irradiation reduces the signal-to-noise ratio and limits the usefulness of FSLPs.
  • a particular benefit of the bioluminescent stem-loop probes described herein is that no stimulating radiation is necessary to generate the bioluminescence.
  • stimulating radiation refers to any radiation emitted by an external radiation source in order to stimulate a fluorescent response from an electromagnetic radiation emitting marker, whether the marker is bioluminescent or fluorescent.
  • bioluminescent stem-loop probes that provide unprecedented sensitivity and selectivity for in vivo imaging and real-time in situ detection of biomarkers.
  • bioluminescent reporters as used herein “markers” and “reporters” are used interchangeably
  • SLPs stem-loop probes
  • BSLPs can provide a sensing tool with superior sensitivity, selectivity, and applicability. This is demonstrated by the data generated using a BSLP utilizing Renilla luciferase (Rluc) as the bioluminescent marker and DABCYL as the quencher.
  • Rluc Renilla luciferase
  • Example 1 The BSLPs described in Example 1 have demonstrated excellent sensitivity and a high signal-to-noise ratio, achieving one of the lowest detection limits (0.4 nM) reported so far with SLPs of any kind. Furthermore, the incorporation of a photoprotein as the bioluminescent marker in the BSLP did not impact the stability or ability to undergo conformational change upon hybridization with the target. [0076] Additional strategies for further enhancing the BSLP design include employing a more robust bioluminescent photoprotein.
  • Gaussia luciferase (Glue), which is (i) the smallest coelenterazine-dependent bioluminescent protein (185 aa, 19.9 kDa, -half the size of Rluc), (ii) thermostable up to 90 °C, and (iii) has been shown to yield significantly higher bioluminescence than Rluc. This should improve the sensitivity of the BSLP and make it robust enough for application in qPCR-based detection systems.
  • Another strategy is to incorporate a bioluminescent enzyme with high substrate turnover, such as Photinus pyralis "firefly" luciferase (Flue), to provide signal amplification, thus eliminating the need to perform PCR-based target amplification.
  • a bioluminescent enzyme with high substrate turnover such as Photinus pyralis "firefly" luciferase (Flue)
  • Flue Photinus pyralis "firefly" luciferase
  • bioluminescent stem-loop probes are quench the bioluminescent reporter signal using a substrate analog or other active- site inhibitor rather than an energy-transfer based quencher. This should help overcome the problem of inefficient quenching typical of FSLP systems and provide a unique control over the reporter signal as opposed to distance-dependent, energy- transfer systems.
  • Coelenterazine-based bioluminescent proteins have been shown to be non-toxic for in vivo applications and are thus aptly suited for live cell imaging
  • probes should be useful for using sBSLPs for in vivo imaging of breast tumors and circulating miRNA.
  • the embodiments described herein are unique with respect to at least the following features: • The design of BSLPs utilizing high-output, thermostable bioluminescent proteins as reporters.
  • miR-21 is a 22 nucleotide (nt) miRNA target implicated as a biomarker in several cancer types. Renilla luciferase (Rluc) was expressed in and purified from Escherichia coli (E. coli) using methods previously established in the published literature.
  • the 5' amino modification of the hairpin-loop construct was chemically converted to a benzaldehyde moiety by succinimidyl 4-formylbenzoate (SFB, Pierce - Thermo Fisher Scientific Inc.) in 50 x excess in a 100 mM sodium phosphate, 150 mM sodium chloride, pH 7.2 buffer.
  • Amine residues of purified Rluc were modified with succinimidyl 4-hydrazinonicotinate acetone hydrazone (SANH, Pierce - Thermo Fisher Scientific Inc.) in 10 x excess in a 100 mM sodium phosphate, 150 mM sodium chloride, pH 7.2 buffer.
  • the alkyl hydrazone was hydrolyzed by buffer exchanging into a 100 mM sodium citrate, 150 mM sodium chloride, pH 6.0 buffer, which yields the aldehyde-reactive hydrazine moiety.
  • SFB-modified oligonucleotide was added in 1.5 x excess to form a stable hydrazone conjugate (Fig. 5).
  • biotin affinity-handle on the probe stem was used to separate the bioluminescent stem-loop probes from the excess Rluc remaining in solution after conjugation.
  • the thymidine in the stem of the probe was replaced with a biotin-modified thymidine (Bio-dT). This provided a convenient affinity handle for purification without detriment to the structural integrity of the SLP sequence needed for proper stem-loop formation.
  • a monomeric avidin-immobilized agarose (Pierce Thermo Scientific) purification column was used for separation. The purification protocol suggested by the manufacturer was followed and elution of the BSLP was performed using D-biotin.
  • FSLP fluorescent stem-loop probe
  • the FSLP was synthesized (Eurofins MWG Operon) with the same sequence as the BSLP, utilizing 6-carboxyfluorescein (6FAM) as the fluorescent reporter (ex. 495 nm, em. 524 nm) and Black Hole Quencher 1 (BHQ1) as the quencher (ex. 534 nm).
  • 6FAM 6-carboxyfluorescein
  • BHQ1 Black Hole Quencher 1
  • the FSLP was characterized by fixing the target concentration at a point in the middle of the linear portion of the initial calibration curve and varying the concentration of FSLP used to achieve the best S/N ratio (Fig. 7).
  • the BSLP was also evaluated using the same procedure.
  • the optimal concentration for each SLP was chosen by varying the amount of SLP while holding the target concentration constant.
  • An optimal S/N ratio of 1.6 was achieved using 10 pmol of FSLP, whereas 0.5 pmol of BSLP gave an optimal S/N ratio of 9 ( Figure 8).
  • the FSLP signal was too weak to be distinguished from background noise. This drastic improvement in signal amplitude achieved with the BSLP appears to be characteristic of bioluminescent reporters because of the absence of external excitation. This increased signal amplitude allows the BSLP to generate a reliable quantitative response even when added at a low concentration.
  • the BSLP signal is 50 times more sensitive than the FSLP signal. Consequently, the BSLP was able to achieve a limit of detection (LOD) of 0.4 nM, whereas the FSLP exhibited a LOD of 10 nM (Fig. 9). This 25 times lower LOD is a significant improvement over conventional FSLPs, as well as many of the advancements made to improve the traditional fluorescence method (Table 1 ).
  • Micro-RNA detection in a Human Serum Matrix A mouse serum spiked with miRNA target miR21 was used to prepare a calibration curve. All serum samples were diluted to 25% initial concentration with 100 mM sodium phosphate, 1 mM EDTA pH 7.4 buffer and boiled for 5 min to remove any nuclease. Once cooled, 50 ⁇ _ of the serum was added to each well before the addition of synthetic target and BSLP. The total well volume before coelenterazine injection was held at 100 ⁇ _. Serum samples from breast cancer patients were employed in place of synthetic target and the mouse serum in the assay set-up to detect the levels of miR21 in these samples (Fig. 10). The concentrations of miR21 detected in serum samples were validated using a solid-phase miRNA detection method developed in the laboratory.
  • the mismatch probes contained the following mutations to the wild- type miR21 sequence:
  • TM triple mismatch
  • thermostable, high-output bioluminescent proteins Stem- loop probes are commonly employed in the quantitative/real-time detection of PCR products.
  • a thermostable bioluminescent protein such as Gaussia princeps luciferase (Glue).
  • Glue is derived from the mesopelagic (350-1000 m) marine copepod Gaussia princeps.
  • Glue is the smallest coelenterazine-dependent bioluminescent protein among the luciferases isolated so far.
  • Glue is a 185 amino acid, 19.9 kDa bioluminescent photoprotein capable of generating light through the enzyme-assisted oxidative decarboxylation of coelenterazine to coeleteramide. This reaction gives off light at approximately 487 nm (Fig. 12).
  • Fig. 12 shows emission scans for Rluc and Glue monitored using Varian Cary Eclipse spectrofluorometer. The Glue emission is red-shifted ⁇ 10nm from the Rluc and exhibits almost a 10-fold higher relative luminescent output. The dotted curves correspond to normalized emissions.
  • Glue contains 10 cysteines, making proper folding in non-native, prokaryotic expression systems extremely difficult due to the lack of chaperones.
  • the Glue gene was codon-optimized for Escherichia coli and inserted as a synthetic construct containing Nde ⁇ and Xba ⁇ restriction sites at the EcoRV site in the general vector pUC57 (Genscript). It was subsequently cloned using the Nde ⁇ and Xba ⁇ restriction sites into the cold shock expression vector pCold I (Takara Bio, Japan / Clontech, US).
  • the pCold I vector has an N-terminal 6x Histag with Xa cleavage site upstream of the MCS for convenient purification of the recombinant protein by IMAC.
  • Glue was expressed by growing E. coli ER2523 cultures transformed with the pCold::Gluc vector to an OD600 of approximately 0.5-0.7, cold shocking in an ice- bath for 1 h, inducing the lac operon with 1 mM IPTG, and expressing at 15 °C/150 rpm for 24 h.
  • the crude Glue was purified using a ⁇ agarose column.
  • the elution fractions were analyzed for bioluminescent activity using a PerkinElmer Victor X Light and visualized by SDS-PAGE (Fig. 13). The activity of Glue was compared with that of Rluc ( Figure 1 1 & 13).
  • Glue purified by IMAC typically runs at ⁇ 25kDa
  • thermostability of Glue was also verified by exposing Glue to several PCR cycles and monitoring its luminescence activity (Fig. 15). The bioluminescence readings were taken at room temperature. The PCR cycles were as follows, 1 minute at 90°C, 1 minute at 55°C and 1 minute at 65°C.
  • BSLPs utilizing Glue as the bioluminescent marker can be synthesized following the same protocol synthesis protocol utilized to produce the Rluc-based BSLPs described in Example 1.
  • the same validation studies described herein could be used to validate and calibrate a Gluc-based BSLP. After comparing the performance of Glue BSLP for in vitro miRNA detection, this probe could also be used in a PCR-based detection.
  • miR21 could be used as the target miRNA and its serum detection can be performed using RT-PCR method employing Glue BSLP. Standard RT-PCR protocol will be followed by an end-point PCR detection.
  • the target sample will be prepped using a small RNA extraction kit (Ambion). Small RNAs will be modified with a self-annealing hairpin primer.
  • the next preparation will be an RT reaction containing dNTP mix, nuclease-free water, reverse transcriptase, and primer- modified miRNA template.
  • Pulsed RT will be performed using the thermal cycler with conditions of 30 min at 16°C, followed by pulsed RT of 60 cycles at 30°C for 30 s, 42°C for 30 s and 50°C for 1 s.
  • the PCR reaction mix will be incubated at 85°C for ' 5 min to inactivate the reverse transcriptase. This will be followed by an end-point PCR detection using the Glue SLP.
  • a mixture will be formed from the RT-PCR product with nuclease-free water, PCR buffer, dNTP mix, forward and reverse primer, polymerase, and the Glue SLP.
  • Reactions will be placed in a preheated (94°C) thermal cycler and incubated at 94°C for 2 min, followed by 20-40 cycles of 50°C for 15 s and 60°C for 1 min. Reaction products will be analyzed by measuring the bioluminescence signal using the luminometer. RT PCR and end-point PCR reactions will be also performed using a control RNA. The sensitivity and specificity of the PCR product detection using the Gluc-based BSLP will be evaluated.
  • SLPs with these different quenchers can be obtained from commercial sources (Molecular Probes) and can be chemically conjugated to SLPs.
  • succinimidyl ester derivatives of these quenchers can be directly conjugated to amine- modified SLPs using well-established conjugation protocols.
  • molecular assemblies of multiple (usually three) quenchers can be attached using a linker with a dendritic modification.
  • the use of multi-quencher dendritic assemblies with FSLPs has shown improved quenching efficiency giving a 20- fold improvement in S/N by statistically improving the likelihood of proper dipole alignment and coupling between the fluorophore and quencher.
  • bioluminescent proteins described herein may have an additional advantage in that their glow-type signal emission can be integrated over longer time periods. This can overcome the limitation of one SLP signal per target molecule. Additionally, incorporation of bioluminescent enzymes such as Flue may further enhance sensitivity, because the enzymatic turnover of the substrate is very high.
  • BSLPs could eliminate the need for PCR target amplification, which is generally coupled to the SLP for detection of low concentration targets.
  • Bioluminescent proteins that utilize coelenterazine as the substrate emit luminescence based on flash or in glow-type kinetics.
  • the half-life of Glue luminescence is 1.1 s, which is significant compared to the 0.05 sec half life of Rluc.
  • the bioluminescence signal generated from the Glue can be integrated for about 3 min ( ⁇ 20 % maintenance of activity at 3 min) allowing for signal enhancement.
  • integration of the signal from the Gluc-based BSLP may provide improved performance for some techniques.
  • Flue is a 61 kDa protein cloned from beetles (Photinus pyralis), while Rluc is a 39 kDa protein from sea pansies ⁇ Renilla reniformis). Flue requires luciferin, ATP, magnesium, and molecular oxygen to produce bioluminescence, while Rluc requires only coelenterazine and molecular oxygen.
  • the bioluminescence light generated by Flue is greenish yellow with an emission maximum around 560 nm, whereas Rluc emits blue light with an emission maximum around 480 nm. Due to these differences Flue and Rluc are often used together in dual-reporter assays. The quantum yield of the Flue bioluminescence reaction is 0.88, which is very high.
  • the Flue can be obtained using conventional techniques and can be conjugated to the SLP using amine chemistry, as described above.
  • Flue has been well characterized and has been cloned and expressed in E. coli (see, e.g., J. de Wet, et al., "Cloning of Firefly Liciferase cDNA and the Expression of Active
  • LNA locked nucleic acid
  • SLPs SLPs conjugated to photoproteins, such as Flue.
  • LNAs are bicyclic high affinity RNA analogs. These nucleotides are synthesized by chemically locking the furanose ring of the sugar phosphate backbone in an N-type conformation, by the introduction of a 2'-0,4'-C methylene bridge.
  • LNA base pair This confers higher structural rigidity to the LNA base pair, thus providing for higher stability to the stem when incorporated in place of DNA bases.
  • Vargula luciferase is a 555 amino acid protein, however, a truncated Vargula luciferase (Pro28-Cys 3 2) has been shown to retain about 38 % of the bioluminescence activity of the full length protein. This 248 amino acid, 38.7 kDa protein is comparable in size to Rluc, which was used in the BSLP construct of Example 1. Therefore, the small size of the bioluminescent protein, combined with the enzymatic turnover property of Vargula luciferase, could prove advantageous in the design of highly sensitive BSLP. [00112] Vargula luciferase genes can be obtained commercially, such as from
  • the gene corresponding to the truncated protein (amino acid 28-312) can be cloned in the pCold vector by following the same protocol as the
  • the BSLPs described herein can include bioluminescent markers that are quenched by an active-site inhibitor when the BSLP is in the closed conformation. Opening of the hairpin-loop will separate the inhibitor from the active-site, allowing the bioluminescent substrate to access the active-site to produce bioluminescence.
  • bioluminescence should be effectively quenched through disruption of the enzymatic catalysis of the substrate. Tethering the active-site inhibitor to the BSLP will bring the active-site inhibitor close to the binding site on the protein in the closed conformation. However, when the stem opens in the presence of the target sequence the inhibitor is moved away from the binding site allowing for the substrate to bind to the
  • bioluminescent protein releasing radiation.
  • the active site inhibitor can be chosen in such a way that its binding constant is considerably weaker than the substrate (e.g., coelenterazine, which has a K D ⁇ 30 nM 40 ).
  • a weak inhibitor binding constant is beneficial because it will help ensure that the binding affinity of the inhibitor does not pose a significant contribution to the stem melting temperature (T M ) and prevent the SLP from undergoing conformational opening upon binding target.
  • bioluminescent marker is Glue or any other coelenterazine-dependent protein or enzyme.
  • linker between the SLP and the inhibitor may be particularly useful for BSLP designs incorporating active-site inhibitors.
  • the linker used must be sufficiently soluble in the surrounding solution and provide enough flexibility that the linker does not prevent proper binding in the active-site.
  • one approach for synthesizing a BSLP with a linker- quencher complex is to couple a carboxyl containing linker to the active-site inhibitor compounds through the aliphatic and aromatic primary amines present in these structures.
  • the linker can be attached through the formation of an amide bond via reaction with 2,4 > 6-triacyloxy-1 ,3,5-triazine.
  • a polyethylene glycol (PEG) linker containing a carboxylic acid (COOH) and i-butyl carbamate protected amine (NBoc) can be used.
  • PEG polyethylene glycol
  • COOH carboxylic acid
  • NBoc i-butyl carbamate protected amine
  • reaction can proceed through the following steps:
  • step 1) the carboxyl group replaces the chlorine in cyanuric chloride to yield amine-reactive 2,4,6-triacyloxy-1 ,3,5-triazine;
  • step 2) the amide bond is formed through the nucleophilic attack on the carbonyl of 2,4,6-triacyloxy-1 ,3,5-triazine, resulting 3 equivalents of PEG-linker modified inhibitor;
  • step 3) the boc-protecting group is removed from the linker exposing the amine for conjugation to SLP through bifunctional linkers.
  • the linker can be attached to the custom-modified hairpin-loop sequence using a cross-linker such as SANH, SMCC, DSS, etc. (all of which are available from Pierce - Thermo Fisher Scientific Inc.). Rluc can be conjugated to the hairpin-loop construct using SANH as shown in Figure 5.
  • the current method used to conjugate SLP(s) to Rluc attaches aldehyde- modified SLPs to primary amines (Lys residues) present on the Rluc surface.
  • the SLP stem duplex is B-form DNA and is approximately 20 A in diameter. Assuming that Rluc and the inhibitor are both attached with similarly sized flexible linkers, the stem duplex distance provides a good starting distance for the selection of potential attachment sites.
  • Controlled conjugation of the SLP to the bioluminescent marker can also be achieved using maleimide chemistry.
  • surface-accessible cysteines can be introduced near the active site of the bioluminescent reporter using point mutations produced by site-directed mutagenesis. This will allow for very controlled conjugation of the SLP to the bioluminescent reporter using maleimide chemistry, such as the heteroblfunctional crosslinker succinimidyl-4-(A/-maleimidomethyl)cyclohexane-1- carboxylate (SMCC) available from Pierce-Thermo Fisher Scientific Inc.
  • SMCC heteroblfunctional crosslinker succinimidyl-4-(A/-maleimidomethyl)cyclohexane-1- carboxylate
  • BSLPs as an imaging technique to detect circulating miRNAs released by a primary tumor and metastases, as well as, imaging of the primary tumor and metastases themselves.
  • miRNAs being secreted by a triple negative breast cancer cell line MDA231 will be identified by expression profiling.
  • BSLPs specific to the miRNAs expressed by MDA231 can be further screened to exclude BSLPs that are cross-reacting with rodent miRNAs. Further screening of BSLPs specific to human tumor derived miRNAs lacking cross-reactivity with rodent forms can be tested in mouse serum in vitro.
  • BSLPs targeting specific miRNAs that were identified in vitro to be suitable to detect circulating and tumor-derived miRNAs can be tested in mice using in vivo small animal
  • MDA231 cells, exosomes, and cell culture supernatant will be subjected to miRNA profiling by real-time PCR arrays for both human miRNA expression as well as cross-reactivity to rodent miRNAs.
  • the detected miRNAs will be categorized in high, medium and low expressing species. miRNAs that were detected with the rodent panel will be compared by sequence alignment to human miRNAs and possible cross-reacting species will be excluded from further use for downstream applications.
  • Bioluminescent stem-loop probe design The probes will be designed based on the miRNA target sequences. Either Gluc-based BSLPs, Fluc- based BSLPs, or both can be used to look at a single target or dual targets.
  • Exiqon LNA based miRNA inhibitors could also be used as target sequences to target specific human miRNAs. This can be used to identify the best approach for using BSLPs in miRNA imaging.
  • MDA231 cells stably expressing red fluorescent protein (RFP) will be implanted in mammary fat pads of NOG (NOD/Shi-sc/d/IL-2Rynull) mice to form a primary tumor.
  • RFP red fluorescent protein
  • MDA231 cells were chosen because, in NOG mice, these cells reliably form tumors that metastasize to lung, liver and bone tissue.
  • Primary tumors are typically formed after approximately 30 days and can be assessed by palpitation at the injection site and detection of RFP using in vivo imaging.
  • a miRNA profile against both human and rodent miRNAs can be created by miRNA qPCR arrays.
  • the profile can be compared to profiles from parental control mice not containing tumors and miRNA profiles from Part I.
  • the miRNAs for imaging can be selected by being detected with qPCR arrays in MDA231 cell supernatant, serum of mice implanted with MDA231 and the lack of cross reactivity with rodent miRNAs based on expression profiling and sequence comparison.
  • Candidate miRNAs can also be tested in mouse serum with BSLPs specific to these miRNAs to determine suitability for downstream applications.
  • NOG mice can be injected with luciferin and increasing amounts of a Fluc-based BSLP that can yield bioluminescence over a long time period allowing for sensitivity evaluations, such as specificity and tolerable dose.
  • Synthetic miRNAs injected into mice and BSLPs specifically designed to detect the synthetic miRNAs can be used to assess the sensitivity of the system for detecting circulating miRNAs.
  • NOG mice containing MDA231 derived tumors can be injected with a dose of BSLPs reacting with a specific miRNA (determined in partsl of this aim) and imaged at 10, 20 and 30 days to detect primary tumor. Once the primary tumor has been detected, the mice can be further injected and imaged after 60, 90 and 120 days to detect metastasis. The presence of primary tumor and larger metastasis will be verified by imaging of RFP.

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

La présente invention concerne une sonde tige-boucle bioluminescente, des compositions contenant des sondes tige-boucle bioluminescentes et des procédés d'utilisation de celles-ci. La sonde tige-boucle bioluminescente peut comprendre une séquence de boucle en épingle à cheveux comprenant une séquence complémentaire d'une séquence cible biologique ; un marqueur bioluminescent couplé à la séquence de boucle en épingle à cheveux ; et un inactiveur couplé à la séquence de boucle en épingle à cheveux. La séquence de boucle en épingle à cheveux peut être entre le marqueur bioluminescent et l'inactiveur. La sonde tige-boucle bioluminescente peut être capable de basculer entre une configuration fermée et une configuration ouverte, et l'inactiveur peut réduire une sortie bioluminescente du marqueur bioluminescent lorsque la sonde est dans la configuration fermée. La présente invention concerne en outre des sondes tige-boucle bioluminescentes dans lesquelles plus d'un complexe séquence de boucle en épingle à cheveux-inactiveur est couplé à chaque marqueur bioluminescent.
PCT/US2011/063444 2011-07-15 2011-12-06 Sondes tige-boucle bioluminescentes, compositions contenant celles-ci et procédés utilisant celles-ci WO2013012434A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201161508246P 2011-07-15 2011-07-15
US61/508,246 2011-07-15

Publications (1)

Publication Number Publication Date
WO2013012434A1 true WO2013012434A1 (fr) 2013-01-24

Family

ID=47558384

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2011/063444 WO2013012434A1 (fr) 2011-07-15 2011-12-06 Sondes tige-boucle bioluminescentes, compositions contenant celles-ci et procédés utilisant celles-ci

Country Status (1)

Country Link
WO (1) WO2013012434A1 (fr)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107208160A (zh) * 2015-01-30 2017-09-26 哈佛学院院长及董事 无显微镜成像
CN110191953A (zh) * 2016-11-21 2019-08-30 特里比奥迪卡有限责任公司 通过接近增强的反应性实现分裂蛋白模板组装的方法
US10876971B2 (en) 2010-10-29 2020-12-29 President And Fellows Of Harvard College Nucleic acid nanostructure barcode probes
US11186839B2 (en) 2016-11-21 2021-11-30 Tribiotica Llc Methods for preventing titration of bimolecular templated assembly reactions by structurally-determined differential hybridizations
US11253536B2 (en) 2016-11-21 2022-02-22 Tribiotica Llc Methods for directed folding assembly or dimerization of proteins by templated assembly reactions
US11286517B2 (en) 2016-02-17 2022-03-29 President And Fellows Of Harvard College Molecular programming tools
US11359229B2 (en) 2016-09-20 2022-06-14 President And Fellows Of Harvard College Molecular verification systems
CN115112902A (zh) * 2022-06-23 2022-09-27 南京浦光生物科技有限公司 检测目标蛋白的试剂组合、试剂盒、检测系统及检测方法
US11492661B2 (en) 2017-01-10 2022-11-08 President And Fellows Of Harvard College Multiplexed signal amplification
US11981956B2 (en) 2018-01-26 2024-05-14 President And Fellows Of Harvard College Proximity detection methods and compositions

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5925517A (en) * 1993-11-12 1999-07-20 The Public Health Research Institute Of The City Of New York, Inc. Detectably labeled dual conformation oligonucleotide probes, assays and kits
US20020064791A1 (en) * 2000-09-19 2002-05-30 Whitaker Tom J. Detection of unlabeled hybridized DNA and RNA using restriction enzyme digestion
US20100248385A1 (en) * 2004-06-17 2010-09-30 University Of Florida Research Foundation, Inc. Multi-acceptor molecular probes and applications thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5925517A (en) * 1993-11-12 1999-07-20 The Public Health Research Institute Of The City Of New York, Inc. Detectably labeled dual conformation oligonucleotide probes, assays and kits
US20020064791A1 (en) * 2000-09-19 2002-05-30 Whitaker Tom J. Detection of unlabeled hybridized DNA and RNA using restriction enzyme digestion
US20100248385A1 (en) * 2004-06-17 2010-09-30 University Of Florida Research Foundation, Inc. Multi-acceptor molecular probes and applications thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ERIC A. HUNT ET AL.: "BIOLUMINESCENT STEM-LOOP PROBES FOR HIGHLY SENSITIVE NUCLEIC ACID DETECTION", CHEM. COMMUN., vol. 47, 19 July 2011 (2011-07-19), pages 9393 - 9395 *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10876971B2 (en) 2010-10-29 2020-12-29 President And Fellows Of Harvard College Nucleic acid nanostructure barcode probes
CN107208160A (zh) * 2015-01-30 2017-09-26 哈佛学院院长及董事 无显微镜成像
US11639522B2 (en) 2015-01-30 2023-05-02 President And Fellows Of Harvard College Microscope-free imaging
CN107208160B (zh) * 2015-01-30 2023-02-17 哈佛学院院长及董事 无显微镜成像
US11286517B2 (en) 2016-02-17 2022-03-29 President And Fellows Of Harvard College Molecular programming tools
US11359229B2 (en) 2016-09-20 2022-06-14 President And Fellows Of Harvard College Molecular verification systems
US11253536B2 (en) 2016-11-21 2022-02-22 Tribiotica Llc Methods for directed folding assembly or dimerization of proteins by templated assembly reactions
US11186839B2 (en) 2016-11-21 2021-11-30 Tribiotica Llc Methods for preventing titration of bimolecular templated assembly reactions by structurally-determined differential hybridizations
EP3541415A4 (fr) * 2016-11-21 2020-10-14 Tribiotica Llc Procédés d'assemblage de matrice de protéine fragmentée par réactivité améliorée par la proximité
JP2020501530A (ja) * 2016-11-21 2020-01-23 トリビオティカ・エルエルシー 近接増強反応性による分割タンパク質の鋳型アセンブリ方法
CN110191953A (zh) * 2016-11-21 2019-08-30 特里比奥迪卡有限责任公司 通过接近增强的反应性实现分裂蛋白模板组装的方法
US11492661B2 (en) 2017-01-10 2022-11-08 President And Fellows Of Harvard College Multiplexed signal amplification
US11981956B2 (en) 2018-01-26 2024-05-14 President And Fellows Of Harvard College Proximity detection methods and compositions
CN115112902A (zh) * 2022-06-23 2022-09-27 南京浦光生物科技有限公司 检测目标蛋白的试剂组合、试剂盒、检测系统及检测方法
CN115112902B (zh) * 2022-06-23 2024-05-24 南京浦光生物科技有限公司 检测目标蛋白的试剂组合、试剂盒、检测系统及检测方法

Similar Documents

Publication Publication Date Title
WO2013012434A1 (fr) Sondes tige-boucle bioluminescentes, compositions contenant celles-ci et procédés utilisant celles-ci
Branchini et al. Red-emitting luciferases for bioluminescence reporter and imaging applications
US9757478B2 (en) Mutant protease biosensors with enhanced detection characteristics
EP3303613B1 (fr) Procédé pour détecter l'activité d'une polymérase
JPS61146198A (ja) 触媒作用されたルミネセンスを用いるポリヌクレオチドハイブリツド形成分析
Tian et al. dsDNA/ssDNA-switchable isothermal colorimetric biosensor based on a universal primer and λ exonuclease
JP4481656B2 (ja) ３’→５’エキソヌクレアーゼ耐性クエンチャードメインを含むｆｅｔ標識オリゴヌクレオチドを使用する方法およびそれを実施するための組成物
Lu et al. Rapid and highly specific detection of communicable pathogens using one-pot loop probe-mediated isothermal amplification (oLAMP)
KR101825117B1 (ko) Ｐｎａ 기반의 실시간 ｐｃｒ 클램핑을 이용한 ｂｒａｆ 돌연변이 검출 방법 및 키트
CN113881650B (zh) 用于探针法qPCR的Taq DNA聚合酶突变体
JP5207355B2 (ja) 標的ヌクレオチド配列を有する核酸の検出方法、プローブセット、及び核酸の識別方法
KR20110130638A (ko) Ｐｎａ 기반의 실시간 ｐｃｒ 클램핑을 이용한 유전자의 메틸화 분석 방법 및 키트
JP6006070B2 (ja) ホタル由来ルシフェラーゼ
KR102525012B1 (ko) 근접 단백질가수분해 반응에 기반한 표적 핵산 검출방법
TW201833328A (zh) 核酸分子檢測方法及檢測套組
CN106868111A (zh) 利用通用TaqMan探针检测SNP的方法和试剂盒
US20150152395A1 (en) Chimeric luciferases
Lamas et al. Evaluation of the effect of outer primer structure, and inner primer linker sequences, in the performance of Loop-mediated isothermal amplification
Liu et al. One-step determination of deletion mutation based on loop-mediated isothermal amplification
Liu et al. Review of characteristics of G-quadruplex and development of its nucleic acid assisted detection method.
KR20120127679A (ko) Ｐｎａ 기반의 실시간 ｐｃｒ 클램핑을 이용한 ｉｄｈ1, ｉｄｈ2 돌연변이 검출 방법 및 키트
CN114250275B (zh) 一种荧光定量pcr反应系统、pcr反应试剂盒及核酸定量检测方法
WO2012124822A1 (fr) Luciférase issue de lucidina accensa
KR102575618B1 (ko) 가이드 프로브 및 클램핑 프로브를 이용한 표적핵산 증폭방법 및 이를 포함하는 표적핵산 증폭용 조성물
WO2016051517A1 (fr) Luciférase émettant de la lumière orange

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11869554

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11869554

Country of ref document: EP

Kind code of ref document: A1