RNA SENSORS AND USES THEREOF

Abstract

The invention generally concerns a novel class of cyclopentane modified FIT-PNA (cpFIT-PNA) probes and uses thereof.

Claims

1-60. (canceled)

61. A cyclopentyl-modified PNA comprising a PNA backbone and a plurality of pendant nucleobases, at least one of said pendent bases is a surrogate base and wherein one or more cyclopentyl groups are provided in proximity to said surrogate base, wherein the surrogate base is a monomethine dye.

62. A cyclopentyl-modified forced-intercalation-peptide nucleic acid molecule (FIT-PNA) comprising a PNA backbone and a plurality of pendant nucleobases, at least one of said pendent bases is a surrogate base selected amongst monomethine dyes and wherein one or more cyclopentyl groups are provided in proximity to said surrogate base.

63. The PNA according to claim 61, wherein the monomethine dye is a cyanine dye being BisQ or a dye herein designated Dye 1 or Dye 2, wherein each X represents a halogen atom: ##STR00013##

64. The PNA according to claim 61, wherein the one or more cyclopentyl (cp) group is positioned at a distance from the surrogate base that is not greater than one nucleobase.

65. The PNA according to claim 64, wherein the cp group is positioned on the PNA backbone between the position of the surrogate base and a position of the next nucleotide base, across a nucleotide base or on the PNA unit carrying the surrogate base.

66. The PNA according to claim 64, wherein the cp group is on the 3 end of the PNA or on the 5 end of the PNA.

67. The PNA according to claim 61, further comprising a charged guanine (G+) and/or a charged adenine (A+) nucleobase.

68. The PNA according to claim 67, wherein the charged G+ and/or the charged A+ is an alkylation product of a free nucleobase G and/or A.

69. The PNA according to claim 67, wherein the charged nucleobases having the structures: ##STR00014## wherein each of Z, independently, is an alkyl having between 1 and 5 carbon atoms.

70. The PNA according to claim 61, wherein the PNA further comprises an oxetane-modified PNA unit of the structure ##STR00015##

71. The PNA according claim 61, the PNA comprising: (i) cp-modified PNA unit, said unit optionally being the surrogate base bearing unit; or (ii) cp-modified PNA unit, said unit being a BisQ-bearing unit; or (iii) cp-modified PNA unit, said unit being optionally a G+ or A+ nucelobase; or (iv) cp-modified PNA unit, said unit being a BisQ-bearing unit; the PNA further comprising a G+ or A+ nucleobase, being optionally cpG+ and cpA+ nucleobase; or (v) cp-modified PNA unit, said unit being a BisQ-bearing unit; the PNA further comprising an oxetane-modified PNA base; or (vi) cp-modified PNA unit, said unit being a BisQ-bearing unit; the PNA further comprising a G+ and/or A+ base, a cpG+ and/or cpA+ base, and/or an oxetane-modified PNA base.

72. A method for detecting or for determining presence of a sequence of interest (SOI) in a sample, the method comprising contacting said sample with a PNA according to claim 61, under conditions permitting hybridization of said PNA with the SOI and detecting emission of light in the red-to-NIR region upon exposure to red-to-NIR radiation.

73. The method according to claim 72, wherein said contacting comprises incubating the PNA and the sample for a period of time to allow for hybridization of the PNA with the SOI.

74. The method according to claim 72, wherein the sample is in vivo or ex vivo.

75. The method according to claim 72, for determining malignancies in a fluorescence guided surgery.

76. The method according to claim 72, wherein the SOI is an RNA or a DNA sequence associated with a disease or a condition, indicative of a genetic condition or a pathogenic RNA/DNA.

77. The method according to claim 76, wherein the SOI is an oncogenic RNA or a pathogenic RNA.

78. The method according to claim 77, wherein the SOI is a DNA.

79. The method according to claim 72, for detecting a single point mutation associated with a genetic disorder.

80. A method for determining presence, development or progression of a disease or a condition; or a genetic condition; or presence of a pathogen or a parasite in a sample, the method comprising contacting a sample containing or suspected of containing a sequence of interest (SOI) indicative of presence of a disease, condition or pathogen or parasite in a subject with a PNA according to claim 61 under conditions permitting hybridization of said PNA with the SOI and detecting emission of light in the red-to-NIR region upon exposure to red-to-NIR radiation.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0099] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0100] FIGS. 1A-C demonstrates how cpPNA enhances fluorescence in all FIT-PNAs. (A) set 1 showing fluorescence of FIT-PNA:RNA duplex for FIT-PNAs 1-4, (B) set 2 showing fluorescence of FIT-PNA:RNA duplex for FIT-PNAs 5-7 and, (C) enhanced fluorescence of bis-cpFIT-PNAs (4 and 7) after RNA hybridization. [FIT-PNA]=[cpFIT-PNA]=3 M, [RNA]=4.5 M.

[0101] FIGS. 2A-B demonstrate mismatch sensitivity of double substituted cpFIT-PNAs compared to FIT-PNA controls. (A) double cpFIT-PNA 4 compared to FIT-PNA 1 and (B) double cpFIT-PNA 7 compared to FIT-PNA 5. FIT-PNAs were annealed to all possible 11-mer RNAs and measured on a plate reader (ex=575 nm, Em=619 nm, n=3). [FIT-PNA]=[cpFIT-PNA]=0.5 M, [RNA]=0.75 M.

[0102] FIGS. 3A-B provide mismatch sensitivity of single substituted cpFIT-PNAs. (A) cpFIT-PNA 2 and 3 and (B) cpFIT-PNA 6. FIT-PNAs (0.5 M) were annealed to all possible 11-mer RNAs (0.75 M) and measured on a plate reader (ex=575 nm, em=619 nm, n=3).

[0103] FIG. 4 provides HOTAIR FIT-PNA fluorescence with complementary RNA. FIT-PNAs were conjugated to CLIP directly or via a PEG linker (where n=3, 8, or 12).

[0104] FIGS. 5A-B depict the added value of the PEG8 linker. Fluorescence spectra of CLIP6 FIT-PNA (5A, right) and, CLIP6-PEG8-FIT-PNA (5B, left) with fully matched RNA (mutant) and mis-match RNA (WT).

[0105] FIG. 6 depicts the increase in fluorescence for BRAF FIT-PNA substituted with cp. Fluorescence spectra of CLIP6-PEG8-cpFIT-PNA (1 M) with fully matched RNA (mutant, (1.5 M)) and mis-match RNA (WT, (1.5 M)). A two-fold increase in fluorescence is obtained in comparison to the non-modified FIT-PNA (CLIP6-PEG8-FIT-PNA). An 18-fold enhancement in fluorescence is observed for CLIP6-PEG8-cpFIT-PNA in duplex form (with complementary RNA) in comparison to single strand.

[0106] FIG. 7 depicts an increase in fluorescence for CCAT1 FIT-PNA substituted with G.sup.+ as a neighboring base to BisQ at its 3 position. A 7-fold increase in fluorescence is observed for G.sup.+ modified CCAT1 FIT-PNA in comparison to a ca. 3-fold increase for non-modified CCAT1 FIT-PNA ([RNA]=1.5 M and [FIT-PNA]=1 M).

[0107] FIG. 8 depicts the increase in fluorescence for CCAT1 FIT-PNA substituted with cpG.sup.+ as a neighboring base to BisQ at its 3 position. A 10-fold increase in fluorescence is observed for cpG.sup.+ modified CCAT1 FIT-PNA; much higher than that observed for other modifications (i.e. G.sup.+ and cpG). ([RNA]=1.5 M and [FIT-PNA]=1 M).

DETAILED DESCRIPTION OF EMBODIMENTS

General Procedures and Materials

[0108] Manual solid-phase synthesis was performed by using 5 mL polyethylene syringe reactors (Phenomenex) that are equipped with a fritted disk. All column chromatography was performed using 60A, 0.04-0.063 mm Silica gel (Biolab, Israel) and manual glass columns. TLC was performed using Merck Silica Gel 60 F254 plates. HPLC purifications and analysis were performed on a Shimadzu LC-1090 system using a semi-preparative C18 reversed-phase column (Jupiter C18, 5u, 300 , 25010 mm, Phenomenex) at 50 C. Eluents: A (0.1% TFA in water) and B (MeCN) were used in a linear gradient (11-40% B in 38 min) with a flow rate of 4 mL/min. NMR spectra were recorded on a 300 and 600 MHz Bruker NMR using deuterated solvents as internal standards. Mass analysis of PNAs was acquired on a TSQ Quantum Access Max (Thermo Fisher Scientifc, Basel, Switzerland) mass spectrometer. The analysis was performed by direct injection into the mass spectrometer using electrospray ionization (ESI) in positive mode and full scan analysis (range of 200-1500 m/z).

[0109] RNA oligos were purchased from IDT, USA. Fmoc/Bhoc protected PNA monomers from PolyOrg Inc. (USA). Fmoc-D-Lysine and reagents for solid phase synthesis were purchased from Merck (Germany) and Biolab (Israel). Fmoc-protected cyclopentane PNA monomers (C and T) and BisQ were synthesized as previously reported.

Solid phase synthesis of cpFIT-PNA and FIT-PNA

[0110] Coupling of D-Lysine onto Novasyn TGA Resin. The resin (250 mg, 0.2 mmol/g) was allowed to swell in 10 ml DMF for 30 min. For pre-activation, DIC (5 eq.) and DIMAP (0.1 eq.) were added to a solution of Fmoc-protected D-Lysine (10 eq.) in DCM (15 ml) in an ice bath. After 15 min, the mixture was evaporated, re-dissolved in dry DMF and added to the resin. After 2.5 h, the resin was washed with DMF (52 mL), CH.sub.2Cl.sub.2 (52 mL) and the procedure was repeated.

[0111] Fmoc Cleavage. A solution of DMF/piperidine (4:1, 1 ml) was added to the resin. After 2 min the procedure was repeated. Finally, the resin was washed with DMF (31 ml), DCM (31 ml).

[0112] Coupling of Fmoc-Bhoc-PNA-Monomers. 4 eq. of PNA monomer, 4 eq. HATU, 4 eq. HOBt and 8 eq. of dry DIPEA in DMF (to 0.1 M PNA) were mixed in a glass vial equipped with a screw cap. After 3 min of pre-activation, the solution was transferred to the resin. After 60 min, the reaction mixture was discarded and the resin was washed with DMF (21 ml) and DCM (21 ml).

[0113] Coupling of BisQ. 4 eq. of BisQ monomer, 4 eq. HATU, 4 eq. HOBt and 8 eq. of dry DIPEA in DMF (to 0.1 M BisQ monomer) were mixed in a glass vial equipped with screw cap. Following 3 min of pre-activation, the solution was transferred to the resin. After 60 min, the procedure was repeated and finally the resin was washed with DMF (21 ml) and DCM (21 ml).

[0114] Coupling of cyclopentane modified PNAs. 4 eq. of cpPNA monomer, 4 eq. HATU, 4 eq. HOBt and 8 eq. of dry DIPEA in DMF (to 0.1 M cpPNA) were mixed in a glass vial equipped with screw cap. Following 3 min of pre-activation, the solution was transferred to the resin and shaked for 135 min. Finally, the resin was washed with DMF (21 ml) and DCM (21 ml).

[0115] Cleavage of PNA from resin. 1 ml TFA was added to the dry resin. After 2h another portion of TFA was added. The combined TFA solutions were concentrated in vacuo.

[0116] PNA Purification. PNAs were precipitated from the concentrated TFA solution by addition of cold diethyl ether (10 ml). The precipitate was collected by centrifugation and decantation of the supernatant. The residue was dissolved in water and purified by semi preparative HPLC. The purified PNAs were analysed by ESI-MS.

Fluorescence Spectrometry

[0117] Fluorescence spectra were recorded by using a Jasco FT-6500 spectrometer. Measurements were carried out in fluorescence quartz cuvettes (10 mm) at a 3 M concentration of FIT-PNA in a PBS buffered solution (100 mM NaCl, 10 mM NaH.sub.2PO.sub.4, pH 7). Quantum yields were determined relative to Cresyl Violet in PBS.sup.3. PNAs were hybridized to complementary RNA using a 1:1.5 mixture of PNA:RNA at 37 C. for 1-2 hr. Samples were excited at 575 nm and emission spectra were recorded at 585-800 nm.

[0118] Fluorescence end points were recorded by using a Cytation 3 plate reader. Measurements were carried out in Greiner 96 well black plates with flat bottom, at 0.5 M concentration in a PBS buffered solution (100 mM NaCl, 10 mM NaH.sub.2PO.sub.4, pH 7). FIT-PNAs were hybridized to complementary RNA using a 1:1.5 mixture of PNA:RNA at 37 C. for 1-2 hr or by allowing overnight incubation at RT (for limit of detection (LOD) measurements). Samples were excited at 575 nm and measured at 615 nm.

Synthesis

[0119] I Exemplary synthesis of a hybrid cyclopentane-modified BisQ PNA monomer (cpBisQ) is shown in Scheme 6 (selective deprotection of tBOC is marked with a star).

[0120] The Synthesis is Based on:

[0121] H. Zheng et. al., Org. Lett. 2018, 20, 7637-7640; and

[0122] L. S. Lin et. al., Tetrahedron Letters 41 (2000) 7013-7016.

##STR00010##

[0123] II Exemplary synthesis of cpG+ is depicted in Scheme 7. The same approach may be implemented for the cpA monomer (cpA+).

[0124] The synthesis is based on M. Hibino et. al., Chem. Commun., 2020, 56, 25462549.

##STR00011##

[0125] III Exemplary synthesis of Ox-BisQ PNA monomer (Scheme 8).

[0126] The Synthesis is Based on:

[0127] S. Roesner et. al., Org. Biomol. Chem., 2020, 18, 5400-5405, and WO 2019/186174.

##STR00012##