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EP-4735622-A1 - PROTECTING FULLY FUNCTIONAL NUCLEOTIDES FROM DEGRADATION

EP4735622A1EP 4735622 A1EP4735622 A1EP 4735622A1EP-4735622-A1

Abstract

This application relates to methods and compositions for protecting fully functional nucleotides (ffNs) against degradation. In some examples, micelles or surfactants are used to protect ffNs. In some examples, bulky cation compounds are used to protect ffNs. In some examples, the compositions that protect ffNs are included in lyophilized material.

Inventors

  • LAUW, Sherman
  • TEO, Yin Nah
  • LEE, Mei Xian Agnes
  • NG, Denise
  • MARIANI, Angelica
  • OSOTHPRAROP, Trina
  • DAY, HENRY
  • BASUKI, Johan
  • BEVIS-MOTT, CLAIRE
  • BEARD, Hester
  • YUN, CHOL STEVEN
  • HUBER, Faye
  • IAVICOLI, Patrizia

Assignees

  • Illumina, Inc.

Dates

Publication Date
20260506
Application Date
20240628

Claims (20)

  1. 1. A method of stabilizing fully functional nucleotides (ffNs), the method comprising: including the ffNs and at least one surfactant into lyophilized material.
  2. 2. The method of claim 1, wherein a concentration of the ffNs is between about 10 pM and about 15,000 pM.
  3. 3. The method of claim 1 or claim 2, wherein a concentration of the at least one surfactant is between about 0.5% and about 2.5%.
  4. 4. The method of any one of claims 1-3, wherein including the ffNs and the at least one surfactant into the lyophilized material comprises freeze-drying the ffNs.
  5. 5. The method of any one of claims 1-4, wherein the ffNs are encapsulated in a hydrophobic core of the surfactant, which inhibits or prevents hydrolysis of the ffNs.
  6. 6. The method of any one of claims 1-5, wherein the at least one surfactant comprises any one or more of tricosaethylene glycol dodecyl ether, poloxamer 407, polyethylene glycol (PEG) stearate, SB- 12, lauramidopropyl betaine, n-dodecyl-P-D-maltoside, CHAPS, polyoxyethylene sorbitan monolaurate, and polyoxyethylene sorbitan monopalmitate.
  7. 7. A method of stabilizing a fully functional nucleotides (ffNs), the method comprising: including the ffNs and at least one bulky cation compound into lyophilized material.
  8. 8. The method of claim 7, wherein a concentration of the ffNs is between about 10 pM and about 15,000 pM.
  9. 9. The method of claim 7 or claim 8, wherein the concentration of the at least one bulky cation compound is between about 1 mM and about 40 mM.
  10. 10. The method of any one of claims 7-9, wherein including the ffNs and the at least one bulky cation compound into the lyophilized material comprises freeze-drying the ffNs.
  11. 11. The method of any one of claims 7-10, wherein an ionic interaction is produced between a positive charge on the bulky cation compound and a negative charge on one or more phosphate groups of the ffNs.
  12. 12. The method of claim 11, wherein the ionic interaction stabilizes the ffNs through steric hindrance, which inhibits or prevents hydrolysis of the ffNs.
  13. 13. The method of claim 11 or claim 12, wherein the ionic interaction stabilizes the ffNs through charge neutralization that reduces the ffNs electrophilicity to water, which inhibits or prevents hydrolysis of the ffNs.
  14. 14. The method of any one of claims 7-13, wherein the at least one bulky cation compound comprises any one or more of an imidazolium compound, a pyridinium compound, and a quaternary ammonium compound.
  15. 15. The method of claim 14, wherein the imidazolium compound comprises l-ethyl-2- methylimidazolium chloride (EMICL), l-butyl-3-methylimidazolium chloride (BUMICL), 1- butyl-3-methylimidazolium bromide (BUMIBR), or l-benzyl-3-methylimidazolium chloride (BZMICL).
  16. 16. The method of claim 14, wherein the pyridinium compound comprises 1- butylpyridinium bromide (BPB), 2-chloro-l-methlypyridinium iodide (CMPI), or l-ethyl-4- (methoxycarbonyl)pyridinium iodide (EMPI).
  17. 17. The method of claim 14, wherein the quaternary ammonium compound comprises ammonium chloride (ACL), choline chloride (CCL), tetrabutylammonium chloride (TBACL), tetramethylammonium chloride (TMACL), bis(2- hydroxyethyl)dimethylammonium chloride (DMACL), tris(2- hydroxyethyl)methylammonium methylsulfate (THEMAMS), tributylmethylammonium chloride (TBMACL), trimethylphenylammonium chloride (TMPACL), or diallyldimethylammonium chloride (DADMAC).
  18. 18. A method of preparing fully functional nucleotides (ffNs) for sequencing, the method comprising: mixing the ffNs with at least one surfactant in a solution; diluting a concentration of the ffNs in a sequencing mix relative to a concentration of the ffNs in the solution by a factor of between about lOx and about 200x; and diluting a concentration of the at least one surfactant in the sequencing mix, relative to a concentration of the at least one surfactant in the solution, by a factor of between about lOx and about l,000x.
  19. 19. The method of claim 18, wherein the concentration of the ffNs in the solution is between about 10 qM and about 15,000 qM, and the concentration of the ffNs in the sequencing mix is between about 5 qM and about 9 qM.
  20. 20. The method of claim 18 or claim 19, wherein the concentration of the at least one surfactant in the solution is between about 1.5% and about 2.5%, and the concentration of the at least one surfactant in the sequencing mix is between about 0.05% and about 0.25%.

Description

PROTECTING FULLY FUNCTIONAL NUCLEOTIDES FROM DEGRADATION CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/511,391, filed on June 30, 2023 and entitled “Protecting Fully Functional Nucleotides from Degradation,” the entire contents of which are incorporated by reference herein. FIELD [0002] This application relates to methods and compositions for protecting fully functional nucleotides against degradation. BACKGROUND [0003] Many sequencing reagents, in particular fully functional nucleotides (ffNs), suffer from poor stability and therefore need to be handled and stored at lowered temperatures. This incurs additional energy, equipment and material costs, and/or time due to the need for refrigeration, dry ice and/or insulation packaging. One method to improve stability is through lyophilization. However, the improved stability that is obtained through lyophilization is dependent on the lyophilized material being maintained in a dry state. SUMMARY [0004] Some examples herein provide a method of stabilizing fully functional nucleotides (ffNs), the method including a step of including the ffNs and at least one surfactant into lyophilized material. [0005] In some examples, the concentration of the ffNs is between about 10 pM and about 15,000 pM. In some examples, the concentration of the at least one surfactant is between about 0.5% and about 2.5%. [0006] In some examples, including the ffNs and the at least one surfactant into the lyophilized material includes freeze-drying the ffNs. In some examples, the ffNs are encapsulated in a hydrophobic core of the surfactant, which inhibits or prevents hydrolysis the ffNs. [0007] In some examples, the at least one surfactant includes any one or more of tricosaethylene glycol dodecyl ether, poloxamer 407, CHAPS, polyoxyethylene sorbitan monolaurate, and polyoxyethylene sorbitan monopalmitate. [0008] Some examples herein provide a method of stabilizing a fully functional nucleotides (ffNs), the method including a step of including the ffNs and at least one bulky cation compound into lyophilized material. [0009] In some examples, the concentration of the ffNs is between about 10 pM and about 15,000 pM. In some examples, the concentration of the at least one bulky cation compound is between about 1 mM and about 40 mM. In other examples, the concentration of the at least one bulky cation compound is between about 40 mM and about 200 mM. Illustratively, the concentration of the at least one bulky cation compound may be between about 90 mM and about 200 mM. [0010] In some examples, including the ffNs and the at least one bulky cation compound into the lyophilized material includes freeze-drying the ffNs. [0011] In some examples, an ionic interaction is produced between a positive charge on the bulky cation compound and a negative charge on one or more phosphate groups of the ffNs. In some examples, the ionic interaction stabilizes the ffNs through steric hindrance, which inhibits or prevents hydrolysis of the ffNs. In some examples, the ionic interaction stabilizes the ffNs through charge neutralization that reduces the ffNs electrophilicity to water, which inhibits or prevents hydrolysis of the ffNs. [0012] In some examples, the at least one bulky cation compound comprises any one or more of an imidazolium compound, a pyridinium compound, and a quaternary ammonium compound. In some examples, the imidazolium compound includes l-ethyl-2- methylimidazolium chloride (EMICL), l-butyl-3-methylimidazolium chloride (BUMICL), 1- butyl-3-methylimidazolium bromide (BUMIBR), or l-benzyl-3-methylimidazolium chloride (BZMICL). In some examples, the pyridinium compound includes 1 -butylpyridinium bromide (BPB), 2-chloro-l-methlypyridinium iodide (CMPI), or l-ethyl-4- (methoxycarbonyl)pyridinium iodide (EMPI). In some examples, the quaternary ammonium compound includes ammonium chloride (ACL), choline chloride (CCL), tetrabutylammonium chloride (TBACL), tetramethylammonium chloride (TMACL), bis(2- hydroxyethyl)dimethylammonium chloride (DMACL), tris(2- hydroxy ethyl)m ethylammonium methyl sulfate (THEMAMS) tributylmethylammonium chloride (TBMACL), trimethylphenylammonium chloride (TMPACL), or diallyldimethylammonium chloride (DADMAC). In one nonlimiting example, the bulky cation compound is or includes TBMACL. [0013] Some examples herein provide a method of preparing ffNs for sequencing, the method including mixing the ffNs with at least one surfactant in a solution; diluting a concentration of the ffNs in a sequencing mix relative to a concentration of the ffNs in the solution by a factor of between about lOx and about 200x; and diluting the concentration of the at least one surfactant in the sequencing mix relative to the concentration of the at least one surfactant in the solution, by a factor of between about lOx and about l,000x. [0014] In some examples, the concentration of the ffNs in the solution is bet