JPWO2020193765A5

JPWO2020193765A5 -

Info

Publication number: JPWO2020193765A5
Application number: JP2020572471A
Authority: JP
Publication date: 2023-01-19

Claims

A method of determining the nucleotide sequence of a target polynucleotide, comprising:
(a) performing a sequencing reaction comprising one or more cycles of:
(i) incorporating four different types of nucleotide conjugates into a plurality of polynucleotides complementary to a target polynucleotide to produce extended polynucleotides, wherein the first type of nucleotide conjugate wherein the second type of nucleotide conjugate comprises a second photoswitchable label and the third type of nucleotide conjugate comprises a third label, wherein ;
(ii) detecting the first collection of signals from the extended polynucleotides at each cycle via the first imaging event;
(iii) illuminating the elongated polynucleotide with a light source to produce a change in the emission signals of the first photoswitchable label and the second photoswitchable label;
(iv) detecting a second set of signals from the extended polynucleotides via a second imaging event;
(b) determining the sequence of the target polynucleotide based on the sequentially integrated nucleotide conjugates;

2. The method of claim 1, wherein the incorporation of the first type of nucleotide conjugate is determined from the signal state in the first imaging event and the dark state in the second imaging event.

3. The method of claim 1 or 2, wherein the incorporation of the second type of nucleotide conjugate is determined from the dark state in the first imaging event and the signal state in the second imaging event.

4. The method of any one of claims 1-3, wherein the incorporation of the third type of nucleotide conjugate is determined from the signal state in the first imaging event and the second imaging event.

5. The method of any one of claims 1-4, wherein the incorporation of the fourth type of nucleotide conjugate is determined from the dark state in the first imaging event and the second imaging event.

A method according to any one of claims 1 to 5, wherein four different types of nucleotide conjugates are present simultaneously and compete for incorporation during each cycle.

The method of any one of claims 1-6, wherein step (a) is repeated at least 50 times.

The method of any one of claims 1-7, wherein incorporation of the nucleotide conjugate is performed by a polymerase.

Different types of nucleotide conjugates contain reversible terminator moieties;
step (a) further comprises cleaving the reversible terminator moiety from the incorporated nucleotide conjugate prior to the next incorporation cycle;
The method according to any one of claims 1-8.

10. The method of any one of claims 1-9 , wherein the nucleotide conjugate comprises a nucleotide type selected from the group consisting of dATP, dTTP, dUTP, dCTP, dGTP, and non-natural nucleotide analogues thereof.

the labels of the first type of nucleotide conjugate and the third type of nucleotide conjugate comprise the same fluorescent moiety; or
the labels of the first type of nucleotide conjugate, the second type of nucleotide conjugate, and the third type of nucleotide conjugate comprise different fluorescent moieties; optionally,
different fluorescent moieties are detected using the same emission filter,
A method according to any one of claims 1-10 .

A method according to any one of claims 1 to 11 , wherein the fourth type of nucleotide conjugate is not labeled with a fluorescent moiety.

13. The method of any one of claims 1-12 , wherein the irradiation in step (a)(iii) does not substantially alter the signal detected from the third label of the third type of nucleotide conjugate. .

14. The method of any one of claims 1-13 , wherein the illumination source in step (a)(iii) comprises a laser, a light emitting diode (LED), or a combination thereof.

15. The method of any one of claims 1-14 , wherein the illumination source in step (a)(iii) has a wavelength different from the excitation wavelength used in the first imaging event.

16. The method of any one of claims 1-15 , wherein the illumination source in step (a)(iii) has a wavelength of about 350 nm to about 450 nm ; optionally about 405 nm .

the first imaging event and the second imaging event have substantially the same excitation wavelength ; optionally,
said first imaging event and said second imaging event have excitation wavelengths between about 550 nm and 650 nm, and optionally,
said first imaging event and said second imaging event having an excitation wavelength of about 633 nm;
A method according to any one of claims 1-16 .

the first photoswitchable label comprises a first fluorescent moiety covalently attached to the first photochromic moiety, optionally via a first linker;
wherein the first fluorescent moiety comprises a red light-emitting fluorescent moiety, optionally a liconerhodamine moiety and/or a spiropyrano or spirothiopyrano moiety;
A method according to any one of claims 1-17 .

19. The method of claim 18 , wherein the first photochromic moiety comprises the structure of Formula (I):

In the formula:
X is O (oxygen) or S (sulfur), optionally X is S;
R ^1a and R ^2a are each independently H, halo, optionally substituted C _1-6 alkyl, optionally substituted C _2-6 alkenyl, optionally substituted C _2-6 alkynyl, C selected from the group consisting of _1-6 haloalkyl, C _1-6 alkoxy, and C _1-6 haloalkoxy , further optionally each R ^1a and R ^2a is C _1-6 alkyl ;
R ³ is selected from the group consisting of H, C _1-6 alkyl, and —(CH ₂ ) _n —R ⁴ ;
R ⁴ is selected from the group consisting of C _6-10 aryl, 5-10 membered heteroaryl, 3-7 membered carbocyclyl, and 3-7 membered heterocyclyl, each optionally substituted;
Ring A is C _6-10 aryl or 5-10 membered heteroaryl, each substituted with at least one electron withdrawing group , optionally phenyl or naphthyl and optionally the electron withdrawing groups are nitro, cyano, fluoro, bromo, —S(O) ₂ OH, —S(O) ₂ CF ₃ , ammonium, alkylammonium, and one or more of fluoro or bromo; selected from the group consisting of substituted C _1-6 alkyl;
n is an integer from 1 to 6; and the irradiation step (a)(iii) causes cleavage of the spiro C—X bond;
Structures wherein the first photochromic moiety is of formula (Ia):

Including .

20. The method of claim 18 or 19 , wherein the first photoswitchable label comprises a structure:

, where L ¹ is the first linker.

the second photoswitchable label comprises a second fluorescent moiety covalently attached to the second photochromic moiety, optionally via a second linker;
the second fluorescent moiety comprises a coumarin moiety, and optionally,
the second photochromic moiety comprises an oxazine or thiazine moiety;
A method according to any one of claims 1-20 .

22. The method of claim 21 , wherein the second photochromic moiety comprises the structure of formula (II):

In the formula:
Y is O (oxygen) or S (sulfur), optionally Y is O;
R ^1b and R ^2b are each independently H, halo, optionally substituted C _1-6 alkyl, optionally substituted C _2-6 alkenyl, optionally substituted C _2-6 alkynyl, C selected from the group consisting of _1-6 haloalkyl, C _1-6 alkoxy, and C _1-6 haloalkoxy, and further optionally R ^1b and R ^2b are each C _1-6 alkyl ;
Ring B is C _6-10 aryl or 5- to 10-membered heteroaryl; each substituted with at least one electron withdrawing group ; optionally Ring B is phenyl substituted with at least one electron withdrawing group; naphthyl and further optionally the electron withdrawing groups are nitro, cyano, fluoro, bromo, —S(O) ₂ OH, —S(O) ₂ CF ₃ , ammonium, alkylammonium, and one or more of fluoro or bromo and * indicates the point of attachment to the fluorescent moiety, and the irradiation step (a)(iii) causes cleavage of the carbon * _-Y bond optionally,
The second photochromic moiety has the structure of Formula (IIa):

Including .

23. The method of claim 22 , wherein the second photoswitchable label comprises a structure:

, where L2 is the second linker.

a plurality of polynucleotides attached to a substrate ;
detecting the first set of signals and the second set of signals includes obtaining an image of the substrate;
A method according to any one of claims 1-23 .

A nucleotide conjugate comprising a photoswitchable label,
the photoswitchable label comprises a fluorescent moiety, optionally covalently attached to the photochromic moiety via a linker ;
the fluorescent moiety comprises a silicon rhodamine moiety, and
the photochromic moiety comprises the structure of formula (I),
Nucleotide conjugate :

In the formula:
X is O (oxygen) or S (sulfur), optionally S;
R ^1a and R ^2a are each independently H, halo, optionally substituted C _1-6 alkyl, optionally substituted C _2-6 alkenyl, optionally substituted C _2-6 alkynyl, C selected from the group consisting of _1-6 haloalkyl, C _1-6 alkoxy, and C _1-6 haloalkoxy, optionally each R ^1a and R ^2a is C _1-6 alkyl;
R ³ is selected from the group consisting of H, C _1-6 alkyl, and —(CH ₂ ) _n —R ⁴ ;
R ⁴ is selected from the group consisting of C _6-10 aryl, 5- to 10-membered heteroaryl, 3- to 7-membered carbocyclyl, and 3- to 7-membered heterocyclyl, each optionally substituted;
Ring A is C _6-10 aryl or 5- to 10-membered heteroaryl, each substituted with at least one electron-withdrawing group; with at least one electron withdrawing group selected from the group consisting of O ₂ )OH, —S(O) ₂ CF ₃ , ammonium, alkylammonium, and C _1-6 alkyl substituted with one or more fluoro or bromo; is substituted phenyl or naphthyl; and
n is an integer of 1-6 .

26. The nucleotide conjugate of claim 25 , wherein the photochromic moiety comprises the structure of Formula (Ia):

.

27. The nucleotide conjugate of claim 25 or 26 , wherein the photoswitchable label comprises a structure:

, where L ¹ is a linker.

A nucleotide conjugate comprising a photoswitchable label,
the photoswitchable label comprises a fluorescent moiety covalently attached, optionally via a linker, to a photochromic moiety;
the fluorescent moiety is a coumarin moiety ; optionally includes an oxazine or thiazine moiety ; and
A nucleotide conjugate , wherein the photochromic moiety comprises the structure of Formula (II):

In the formula:
Y is. O (oxygen) or S (sulphur) , optionally O ;
R ^1b and R ^2b are each independently H, halo, optionally substituted C _1-6 alkyl, optionally substituted C _2-6 alkenyl, optionally substituted C _2-6 alkynyl, C is selected from the group consisting of _1-6 haloalkyl, C _1-6 alkoxy, and C _1-6 haloalkoxy , further optionally each R ^1b and R ^2b is C _1-6 alkyl ; and Ring B is C _6-10 aryl or 5- to 10-membered heteroaryl; each substituted with at least one electron-withdrawing group, and optionally ring B substituted with one or more fluoro or bromo, nitro, cyano, fluoro, phenyl substituted with at least one electron withdrawing group selected from the group consisting of bromo, —S(O ₂ )OH, —S, (O) ₂ CF ₃ , ammonium, alkylammonium, and C _1-6 alkyl; or is naphthyl ; and * indicates the point of attachment to the fluorescent moiety.

29. The nucleotide conjugate of claim 28 , wherein the photochromic moiety comprises the structure of Formula (IIa):

.

30. The nucleotide conjugate of claim 28 or 29 , wherein the photoswitchable label comprises the structure:

, wherein L ² is a linker ;
Optionally, a photoswitchable label is attached to the C5 position of the pyrimidine base or the C7 position of the 7-deazapurine base .

A nucleotide conjugate according to any one of claims 25-30 , wherein the photoswitchable label is attached to the nucleobase via a cleavable linker.

An oligonucleotide comprising a nucleotide conjugate according to any one of claims 25-31 .

A kit comprising four types of nucleotides, a first type of nucleotide being a labeled nucleotide conjugate according to any one of claims 25 to 27 and a second type of nucleotide being a labeled nucleotide conjugate according to claim 28 31. The labeled nucleotide conjugate of any one of claims 1 to 30 , wherein the third type of nucleotide comprises a label that emits at the same wavelength as the first type of nucleotide and the fourth type of nucleotide is labeled not;
the first type of nucleotide, the second type of nucleotide, and the third type of nucleotide are detectable at the same emission wavelength;
kit.