Compounds as DNA probes, methods and applications thereof
Inventors
Thimmaiah, Govindaraju • Narayanaswamy, Nagarjun
Assignees
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Publication Number
US-10683273-B2
Publication Date
2020-06-16
Expiration Date
Abstract
The present disclosure relates to the field of pharmaceutical chemistry and biotechnology. The present disclosure also relates to a compound of Formula I and a process of preparation thereof. The disclosure furthermore relates to methods/use of Formula I compounds as DNA probes. Said Formula I compounds are employed for recognizing specific DNA sequences by near Infra-red (NIR)-Fluorescence Switch-on mechanism, and have related applications including but not limited to cell imaging. Also, method of treating parasitic infections by employing the present compound of Formula I is provided.
Core Innovation
The invention relates to Formula I DNA-probe compounds, optionally derivatives, tautomers, polymorphs, and solvates, characterized by a ring heteroatom X selected from oxygen, sulphur, and selenium, and by substituent constraints in which R1, R2, R3, R4, R5, R6, R7, and R8 are hydrogen. The compounds include a substituent R9 selected from H and —(CH2)n— with n being 1-6, and a substituent R10 selected from hydrogen, —OH, methyl, amine, terminal alkyne, alkene, alkyl acid, amine acid, and sulfonates (SO3−). An exemplary embodiment is a quinone cyanine-dithiazole compound (QCy-DT).
The invention addresses recognition of AT-rich DNA minor-groove sequences and near-infrared fluorescence detection via a fluorescence switch-on mechanism associated with hybridization to DNA sequences. The probe compounds bind DNA and produce a fluorescence change, thereby allowing detection and quantification based on a fluorescence readout. Specific DNA minor-groove targets include 5′-AAATTT-3′ and 5′-AATTT-3′ sequences.
In addition to sequence recognition, the disclosed probes are used for live and fixed mammalian cell imaging for nuclear DNA staining. The described imaging context includes low-toxicity, permeability, photostability, and a near-infrared fluorescence signal suitable for imaging. The invention further provides therapeutic and diagnostic use cases, including inhibiting cell growth and treating parasitic infections, and diagnosing diseases by contacting biological samples and removing unhybridized probe.
Claims Coverage
The claims cover the Formula I compound defined by heteroatom and substituent constraints, and multiple independent method claims directed to DNA-sequence recognition and fluorescence-based detection or quantification, including minor-groove binding mechanisms and biological application contexts.
Formula I compound with defined heteroatom and substituents
A compound of Formula I in which X is selected from oxygen, sulphur, and selenium; R1, R2, R3, R4, R5, R6, R7, and R8 are hydrogen; R9 is selected from H and —(CH2)n— where n is 1-6; and R10 is selected from hydrogen, —OH, methyl, amine, terminal alkyne, alkene, alkyl acid, amine acid, and sulfonates (SO3−).
Binding to AT-rich DNA sequences
A compound of claim 1 characterized by binding to AT-rich DNA sequences.
Fluorescence-change detection or quantification by hybridization
A method that detects or quantifies a DNA sequence by hybridizing a compound of Formula I with the DNA sequence and measuring a fluorescence change to determine binding intensity.
Minor-groove binding producing fluorescence via pi-electron delocalization
A method in which the compound binds in the minor groove of a DNA sequence via base pairing and/or covalent or non-covalent interactions, delocalizing pi electrons between donor and acceptor atoms upon binding and producing fluorescence.
Treatment and diagnosis in eukaryotic cell, infected cell, or parasitic contexts
A method characterized by using a eukaryotic cell selected as a cancerous cell, a microorganism-infected cell, a parasite or unicellular protozoan, or another abnormal cell, where the parasite is Plasmodium selected from Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, or Plasmodium malariae.
Overall, the independent claim coverage centers on Formula I compounds with specified heteroatoms and substituents, and extends to fluorescence switch-on DNA probing methods that rely on hybridization and fluorescence change, including minor-groove binding with pi-electron delocalization, plus biological use contexts involving AT-rich DNA recognition and Plasmodium-related applications.
Stated Advantages
Enables DNA sequence detection/quantification by fluorescence change based on binding intensity.
Provides NIR fluorescence switch-on upon interaction with AT-rich DNA.
Supports staining of nuclear DNA in live/fixed cells.
Inhibits cell growth, including parasite growth inhibition against Plasmodium spp.
Enables near-infrared fluorescence switch-on for recognizing AT-rich DNA minor-groove sequences.
Allows detection and quantification through a fluorescence-change readout.
Supports live and fixed mammalian cell imaging for nuclear DNA staining.
Described as having low-toxicity, permeability, and photostability.
Enables therapeutic and diagnostic use including inhibiting cell growth and treating parasitic infections.
Enables diagnosis by contacting biological samples and removing unhybridized probe.
Documented Applications
Pharmaceutical formulations of the compounds using pharmaceutically acceptable excipients and various administration routes.
DNA sequence detection/quantification by hybridization with fluorescence switch-on for AT-rich DNA.
Staining of nuclear DNA in live or fixed cells.
Use for cell growth inhibition, including parasite growth inhibition against Plasmodium spp. (Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae).
Recognition of AT-rich DNA minor-groove sequences, including 5′-AAATTT-3′ and 5′-AATTT-3′, with near-infrared fluorescence switch-on.
Detection and quantification of a DNA sequence by hybridization and fluorescence change measurement.
Live and fixed mammalian cell imaging for nuclear DNA staining.
Inhibition of cell growth as a therapeutic application.
Treatment of parasitic infections, including malaria parasites (Plasmodium).
Diagnosis of diseases by contacting biological samples and removing unhybridized probe.
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