NZ758304B2

NZ758304B2 - Fluorogen-binding rna aptamers

Info

Publication number: NZ758304B2
Application number: NZ758304A
Authority: NZ
Inventors: Amir Abdolahzadeh; Alexis Autour; Razvan Cojocaru; Elena Dolgosheina; D Amare Adrian Ferre; Sunny Chiu Yuk Jeng; Shanker Shyam Sundhar Panchapakesan; Michael Ryckelynck; Robert Trachman; Peter Unrau
Original assignee: Centre National De La Recherche Scientifique; Simon Fraser University; The United States Of America As Represented By The Secretary Department Of Health And Human Services; Université De Strasbourg
Filing date: 2018-04-23
Publication date: 2025-06-04

Abstract

RNA aptamers are disclosed with distinct fluorescent properties, fluorophore binding affinities, and salt dependence. Also disclosed are corresponding fluorophores, with selected fluorophores evidencing high cellular permeability. The aptamer's high fluorophore affinities, the high brightness of the bound complexes, and their thermal and salt stability, provide distinct aspects of the disclosed aptamers.

Claims

1. A RNA aptamer comprising an active core sequence as set forth in : 5'- GG@ (T1/WGW)GG(#1H/WG) WGGN@ (#2/-)G(T2 /H)GNH(AN@T3/G)-3' (SEQ ID NO : 2) wherein, within the active core sequence - represents no nucleotide; W ents A or U; H represents A, C or U; N represents A, C, G or U; and @ represents N or a deletion; and wherein T1 represents any nucleotide, T2 and T3 being defined as follows : when T1 is A, T2 can be either A,G or U; and when T1 is A and T2 is A, then T3 is U; when T1 is A and T2 is G, then T3 is U; and when T1 is A and T2 is U, then T3 is A or U; or when T1 is C, T2 can be either G or U; and when T1 is C and T2 is G, then T3 is C or G; and when T1 is C and T2 is U, then T3 is G; or when T1 is G, T2 can be either G or C, and T3 is C; or when T1 is U, T2 can be either A or C; and when T1 is U and T2 is A, then T3 is A or U; and when T1 is U and T2 is C, then T3 is A; wherein #1 and #2 ents any nucleotide pair such that when #1 is A, then #2 represents A, C, G or U; or when #1 is C, then #2 is C; or when #1 is G, then #2 is G; or when #1 is U, then #2 represents A, G or U. wherein the aptamer adopts a determined tridimensional conformation which is a fluorophore binding mation, said aptamer when it adopts the fluorophore binding conformation being liable interact with a fluorophore; wherein the r further comprises, contiguous with the active core ce, a 5' leader sequence attached, or operably linked to (by covalent bound, i.e. phosphodiester bound), to the 5' terminus of the active core and a 3' tail sequence attached, or operably linked to (by nt bridge mentioned below), to the 3' us of the active core, wherein the 5' leader sequence and the 3' tail ce together mediate the juxtaposition of the 5' terminus of the active core and the 3' terminus of the active core when the aptamer is the fluorophore binding conformation.

2. The aptamer according to claim 1, wherein said aptamer can interact with a fluorophore, said fluorophore being selected from group consisting of the following compounds of the following Formula I and Formula II : wherein : X represents O, S, Se, or C(CH3)n; R1 represents an alkyl having from 1-6 carbons; R2 represents an alkyl having from 1-6 carbons; R3 is either a fused benzene, an alkyl having 1-6 carbons, a methoxy or R4 is an alkyl having 1-6 carbons, a methoxy or H; and n = zero or an integer from 1-6; R8 R7 R5 R4 R9 R6 R3 R10 N N R2 R11 R12 O R1 (II) R1 represents any substituent; R2 through R5 represent H, F, Cl, Br, I, CH3, linear polymers, or extended heterocycles R6-R9 represent H, F, Cl, Br, I, CH3, linear polymers, or extended heterocycles R10 represents H, F, Cl, Br, I, CH3, linear polymers, or extended heterocycles. R11 through R12 represent H, F, Cl, Br, I, CH3, linear polymers, or extended heterocycles X represents the ing atoms O, S and Se; and n is 1 or 3 or 5.

3. The aptamer according to claim 1 or 2, wherein the core active sequence comprises the following sequence 5'-$1GGNT1GG#1HUGGHA#2GT2GNHAN@T3$2-3' (SEQ ID NO : 5) wherein : H ents A, C or U N represents A, C, G or U @ represents any nucleotide or no nucleotide #1 and #2 represents any nucleotide pair such that when #1 is A, then #2 represents A,C, G or U; or when #1 is C, then #2 is C; or When #1 is G, then #2 is G; or when #1 is U, then #2 represents A, G or U. $1 and $2 represent any pair of nucleotides such that when $1 is A, then $2 is A, G or U; or when $1 is C, then $2 is A, G or U; or when $1 is G, then $2 is A, C, G or U; or when $1 is U, then $2 is A, G.

4. The RNA aptamer according to claim 1 or 2, wherein the core active sequence comprises the following sequence : GG@WGWGGWGWGGN@GHGGHG (SEQ ID NO : 6) wherein: W ents A or U; H represents A, C or U; N represents A, C, G or U; and @ represents N or a no nucleotide.

5. The RNA aptamer according to anyone of claims 1 to 3, wherein the core active sequence comprises the following sequence : GAA(GG)1AA(GG)2NUU(GG)3UAN'G4iUG4iiGUAUAUUC (SEQ ID NO : 7) N is any nucleotide, N' the anti-watson crick partner to N wherein (GG)1, (GG)2, (GG)3 and G4i and G4ii form a quadruplex structure when the active core is in the fluorophore binding conformation, with the 5’ terminus of the active core being juxtaposed to the 3’ us of the active core.

6. The RNA r according to anyone of claims 1 to 2, or 4, wherein the core active sequence comprises the following sequence : [G/C]NR(GG)1[R/D]AG[A/U](GG)2NGN(GG)3 A*[A/U/D]GA*(GG)4 [A/C]R[A/D] (SEQ ID NO : 8) wherein: A* is A or N, N is any nucleotide R is a purine, D denotes the e of nucleotide, wherein (GG)1, (GG)2, (GG)3 and (GG)4 form a quadruplex structure when the active core is in the fluorophore binding conformation, with the 5’ terminus of the active core being juxtaposed to the 3’ terminus of the active core.

7. The RNA aptamer according to anyone of claims 1 to 6, wherein the core active sequences ses one of the sequences as set forth in SEQ ID NO : 7 to SEQ ID NO : 6887.

8. The RNA aptamer comprising a core active sequence as defined in anyone of claims 1 to 7, with the proviso that said aptamer active core does not have the core sequence: GNR(GG)1GNN(GG)2NGN(GG)3AGN(GG)4AGA SEQ ID NO : 6888, wherein R is a purine, and N is any nucleotide.

9. The RNA r according to anyone of claims 1 to 8, wherein the 5' leader sequence and the 3' tail sequence are complementary, so that g of the 5' leader sequence to the 3' tail sequence mediates the juxtaposition of the 5' terminus of the active core and the 3' terminus of the active core when the aptamer is the fluorophore g conformation.

10. The RNA aptamer according to anyone of claims 1 to 9, wherein the 5' leader sequence and the 3' tail sequence are mentary to a target sequence, the target sequence comprising: a leader bait ce that is complementary to the 3' tail sequence of the aptamer; and, a tail bait sequence that is mentary to the 5' leader sequence of the aptamer; the leader and tail bait sequences being juxtaposed in the target sequence, so that binding of: the 5' leader sequence to the tail bait sequence; combined with, binding of the 3' tail sequence to the leader bait sequence; mediates the juxtaposition of the 5' us of the active core and the 3' terminus of the active core when the aptamer is the fluorophore binding conformation.

11. The RNA aptamer according to anyone of claims 1 to 10, wherein the leader and tail bait sequences being contiguous in the target sequence.

12. The RNA aptamer according to anyone of claims 1 to 11, wherein juxtaposition of the 5' terminus of the active core and the 3' terminus of the active core when the aptamer is the fluorophore binding conformation brings a phosphate group from the 5’ terminus to within 10 Å, or to within 9 Å or to within 8 Å, of a phosphate group from the 3' terminus.

13. The RNA aptamer according anyone of claims 1 to 12, wherein the aptamer has a fluorophore binding affinity of at least 400nM, 300nM, 200nM, 100nM, 50nM, 40nM, 30nM, 20nM, 10nM, 5nM, 1nM or 0.5nM when the core is in a phore binding conformation.

14. The RNA aptamer according anyone of claims 1 to 13, wherein the fluorophore-aptamer complex has a brightness of at least 7,000 1, 8,000 M-1cm-1, 9,000 M-1cm-1, 10,000 M-1cm-1, 43,000 M-1cm-1.

15. The RNA r according anyone of claims 1 to 14, wherein the fluorophore-aptamer complex has a fluorescent lifetime of at least 1ns, or at least 2ns, or at least 3ns, or at least 4ns or at least 5ns, or at least 6ns, or in the range of 1-6 ns.

16. The RNA aptamer according anyone of claims 1, 2, 4 or 6 to 15, wherein the fluorophore-aptamer complex is scent in a dehyde solution.

17. The RNA aptamer according anyone of claims 1 to 16, comprising a ary aptamer sequence having affinity for a secondary target moiety.

18. An RNA aptamer as claimed in any one of claims 1 to 17, substantially as described herein and with reference to any example thereof. Gene Fluorescence- Amplification Droplet fusion IVT dualization activated sorting 1? Collection and Ns# ^ Collection and thermocycling * o ? incubation cf t t t * Emulsification +T01-Biotin +competitor b c d 8000 3.5 9 7- 280 k. ? Mango I O 7000 1 1 IS R12 library 8 w s 3.0 t 6- 9 o 6000 §25 } I 7 o i) CT * S 5- 210 | 5000 V) 8 2 2.0 R ^t> O 2 4- o 4000 O O 140 = 1.5 4 3= 3- | 3000 0) 5 £ 3 § 1.0 2- ' 4 ? t £ 2000 2 o 70 1000 -0.5 1 - 1- 0 0 0 0 0 10 20 30 40 R12 1 2 3 4 5 R12 1 2 3 4 5 6 7 8 9 10 Green fluorescence (RFU) Round Round