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CN-122029203-A - Novel enzymes

CN122029203ACN 122029203 ACN122029203 ACN 122029203ACN-122029203-A

Abstract

The present invention relates to modified Dda helicases that can be used to control the movement of analytes such as polynucleotides. The modified Dda helicase is used for analyte detection and characterization.

Inventors

  • R.V. Bowen
  • E. E. Magill
  • DIEFENDERFER, RANDALL, R.
  • M. J. Bruce
  • E J Wallace
  • P. R. Moody
  • D. C. Peggy
  • F. Bosa
  • M. Mossaebi
  • C. P. Youde

Assignees

  • 牛津纳米孔科技公开有限公司

Dates

Publication Date
20260512
Application Date
20241018
Priority Date
20231018

Claims (20)

  1. 1. A modified DNA-dependent atpase (Dda) helicase, wherein the helicase (a) is a helicase ：F3、D4、I16、K24、K25、H26、H27、V28、P33、T42、F44、I46、A48、A60、P62、A65、K67、K68、I69、K72、K76、S79、I84、P104、K108、V117、Y120、R122、L124、I132、P134、W135、T137、N144、K145、E154、A157、Y158、P161、K166、Q170、T174、V176、N180、A181、V190、Y197、V200、V201、R207、T213、A214、L215、M219、V220、I225、K227、L229、V237、M238、K247、L248、I251、R253、K254、F257、D262、V265、D282、I289、Q295、Y304、S306、V309、V314、G316、L319、R321、T329、G331、D333、L349、F352、T362、N365、N367、G369、G370、P373、A380、Q383、S385、P391、S393、F395、M401、S402、V403、D404、R405、A406、I413、H414、V418、E419、A421、Q422、V427、V429、G432、Y434、D435 and F437 wherein one or more of the positions corresponding to the following amino acid positions in Dda 1993 are modified or substituted and/or (b) comprises one or more of the following substitutions or one or more of the following substitutions at the positions corresponding to the following amino acid positions in Dda 1993: T2N, T2K, T2S, T2D, T2I, T2L, T2E, T2F, T2G, T2H, T2Q, T2R, T2A, T2M, T P or T2V, E47D, E47G, E47H, E47N, E47P, E47Q, E47S, E47T, E47A or E47R, E54I, E54V, E54K, E54Y, E T, E54L, E N, E54A, E54F, E M, E54D, E54H, E54P, E54Q, E R or E54S, T55F, T55V, T55H, T55I, T55L, T55M, T55W, T55Y, T55A, T55P, T55Q or T55R, S83E, S83D, S83A, S83P, S G or S83M, K86N, K86G, K86D, K86E, K86H, K86Q, K86R, K86S, K86T, K M or K86P, N88K, N88E, N88T, N88S, N88V, N88Q, N88A, N88L, N88D, N G, N88M, N88I or N88P, P89G, P89K, P89M, P89Q, P R or P89D, V90E, V90D, V90K, V90Q, V90 3496 90 3790I, V90L, V90M, V90P, V90R, V90F, V90Y, V90H or V90G, T91E, T91D, T R, T91K, T91Y, T91H, T91P, T91A, T91V, T91L, T91I, T91S or T91M, Y92K, Y92E, Y92R, Y P, Y I or Y92M, E93P, E93D, E G, E93 3493 5293K, E93Q, E S, E93 3493H, E N or E93R, V96K, V96 5292 96P, V96D, V96G, V96S, V96 34924 96 5293 96A, V H or V96M, -F98M or F98I, K101R, K101T, K101S, K101V, K101E, K101I, K101G, K101L, K101M, K101P, K101Q, K101D, K H or K101N, V103K, V103N, V103L, V103I, V T, V103R, V103 37103S, V103D, V103A, V103E, V H, V103M, V103P, V103Q or V103F, -A C114K or a C114R, D151G, D151V, D N, D151K, D151T, D151A, D151H, D151P, D151Q, D R or D151S, G153E, G153S, G D, G153T, G153N, G153K, G153A, G153Q, G153R, G153L, G H or G153P, N155K, N155P, N155S, N155E, N155D, N155G, N155T, N155A, N155V, N155L, N155I, N155H, N155M, N Q or N155R, T156H, T156Q, T R or T156W, Either K177A or K177P, -W195F, W195Y, W195P, W195K, W195E, W195I, W195L, W195D, W195S, W195G, W195T, W195V, W195Q, W195N, W195R, W195M or W195H, The I196P, I196L, I196D, I196A, I196E, I196K, I196Q, I196S, I196T, I196F, I196M, I V or I196Y, D198N, D198, P, D198E, D198G, D198H, D198K, D198Q, D198R, D S or D198T, T210A, T210M, T P, T210Q, T210I, T210L, T V or T210F, N221P, N S or N221G, K243R, K243E, K243D, K243A, K243S, K243M, K243N, K243P, K243T or K243Y, E258G, E258K, E N, E258P, E D, E S, E35258A, E H, E Q, E R or E258T, F276T, F276,276, 276S, F276, 276L, F276, 276P, F276, 276E, F276,276, 276D, F276N or F276Q, I281E, I281K, I A, I281D, I281H, I281P, I281Q, I281R, I281S, I T, I281F, I L, I281M or I281Y, N292E, N292A, N292S, N D, N292K, N292H, N R, N292Q or N292T, E301K, E301D, E301N, E301S, E301A, E301H, E301M, E301P, E301Q, E R or E301T, F308 42308P, F R, F308 32308G, F34308 308 37308 308A, F308 68308N, F M, F D, F H, F Q or F308W, Y335P, Y335K, Y335E, Y F, Y335T, Y335Q, Y335S, Y335R, Y335D, Y335V, Y335N, Y335G, Y335H, Y M or Y335W, -Y336E, Y336H, Y336K, Y336I, Y336G, Y336 56336T, Y336D, Y336V, Y336P, Y336 38338 336R, Y336Q, Y336A, Y336F, Y M or Y336W, R337E, R337K, R T, R Q, R337P, R337V, R F, R S, R337D, R337G, R337Y, R A, R337H or R337N, -Y350P, L354K, L354E, L354D, L354V, L354Y, L354R, L354S, L354N, L354H, L M, L354P, L354T, L F or L354I, Either K358G or K358P, T359V, T359M, T359A, T359 32359Y, T F, T359 3434 359K, T359 37359N, T359D, T359P, T359Q, T359S, T359W or T359G, The process is carried out by applying K364N, K Q, K D, K E, K364G, K H, K S, K T, K M, K364P, K364Y, K I, K L or K364V, -W366D, W366N, W366K, W366A, W366S, W366I, W366E, W366R, W366M, W366T, W366Q, W366Y, W366G, W366F, W366V, W366P or W366H, -K368A, K368E, K368S, K368G, K368D, K368F, K368M, K368T, K368V, K368Y, K368H, K368N, K368P, K368Q or K368R, and -S382R, S382N, S382K, S E, S382Q, S382A, S382H, S382M, S382T, S382D, S G or S382P.
  2. 2. The modified DNA-dependent atpase (Dda) helicase according to claim 1, wherein A position corresponding to amino acid position F3 in Dda 1993 is substituted by K, Y, I, E, G, L, A, S, D, R, V, T, N, H, M, P, Q or W, A position corresponding to amino acid position D4 in Dda 1993 is substituted by I, L, K, P, S, E, Y, T, F, M, V, A, G, H, N or Q, A position corresponding to amino acid position I16 in Dda 1993 is substituted by G, A, E, L, K, D, N, S, F, M, T, V or Y, A position corresponding to amino acid position K24 in Dda 1993 is substituted by G, P, A, D, N, S, E, H, M, Q, R or T, A position corresponding to amino acid position K25 in Dda 1993 is substituted with A, N, G, D, E, I, L, M, P, Q, R, S, T, V or H, A position corresponding to amino acid position H26 in Dda 1993 is substituted by L, K, R, Q, S, M, A, T, N, P, G, E, I, V, F, Y or D, A position corresponding to amino acid position H27 in Dda 1993 is substituted by F, I, V, L, Y, A, T, S, M, W, D, E, K, N, Q or R, A position corresponding to amino acid position V28 in Dda 1993 is substituted by I, F, A, L, M, T or Y, A position corresponding to amino acid position P33 in Dda 1993 is substituted by A, Y, F, G, E, I, K, L, M, Q, R, S, T, V, D, H or W, A position corresponding to amino acid position T42 in Dda 1993 is substituted by I, L, A, F, M, V, Y, D, E, K, N, P, Q, R or S, A position corresponding to amino acid position F44 in Dda 1993 is substituted by K, A, R, Y, G, H, T, S, Q, L, N, E, M, V, D, P, I or W, A position corresponding to amino acid position I46 in Dda 1993 is substituted by V, A, F, L, M, T or Y, A position corresponding to amino acid position A48 in Dda 1993 is substituted by K, H, Y, R, E, N, Q, S, T, D, M, P, G, I, L, V, F or W, A position corresponding to amino acid position A60 in Dda 1993 is substituted by T, D, E, I, K, L, M, N, P, Q, R, S, V or G, A position corresponding to amino acid position P62 in Dda 1993 is substituted by S, A, D, E, G, H, K, M, N, Q, R or T, A position corresponding to amino acid position A65 in Dda 1993 is substituted by V, Q, K, G, R, Y, L, I, F, T, M, H, E, P, S, D or N, A position corresponding to amino acid position K67 in Dda 1993 is substituted by A, V, T, S, R, M, E, G, I, L, P, Q, D, H, N, F or Y, A position corresponding to amino acid position K68 in Dda 1993 is substituted with A, N, E, G, I, L, M, P, Q, R, S, T, V, D or H, A position corresponding to amino acid position I69 in Dda 1993 is substituted by E, V, R, Q, N, A, K, T, S, D, L, H, M, P, F or Y, A position corresponding to amino acid position K72 in Dda 1993 is substituted by A, E, G, I, L, M, P, Q, R, S, T, V, D, H or N, A position corresponding to amino acid position K76 in Dda 1993 is substituted by M, Y, F, I, V, R, A, L, Q, S, T, W, D, E, H, N or P, A position corresponding to amino acid position S79 in Dda 1993 is substituted with T, K, R, V, E, A, D, I, L, M, N, P, Q, G or H, A position corresponding to amino acid position I84 in Dda 1993 is substituted by L, V, F, A, M, T or Y, A position corresponding to amino acid position P104 in Dda 1993 is substituted by Y, K, E, F, H, I, L, M, Q, V, W, A, D, S or T, A position corresponding to amino acid position K108 in Dda 1993 is substituted by Y, E, D, H, N, S, G, F, I, L, M, Q, V, W, A, P, R or T, A position corresponding to amino acid position V117 in Dda 1993 is substituted by A, S, E, G, I, K, L, M, P, Q, R, T, F or Y, A position corresponding to amino acid position Y120 in Dda 1993 is substituted by V, L, I, M, F, A, T, S, H, Q or W, A position corresponding to amino acid position R122 in Dda 1993 is substituted by E, K, N, D, L, T, S, A, V, I, P, Q, H or M, A position corresponding to amino acid position L124 in Dda 1993 is substituted by T, A, D, E, I, K, M, N, P, Q, R, S, V, F or Y, A position corresponding to amino acid position I132 in Dda 1993 is substituted by T, V, L, A, D, E, K, M, N, P, Q, R, S, F or Y, A position corresponding to amino acid position P134 in Dda 1993 is substituted by D, E, K, S, L, A, R, I, N, V, T, G, H or Q, A position corresponding to amino acid position W135 in Dda 1993 is substituted by D, H, T, N, E, G, S, K, Y, Q, R, F, A, P or M, A position corresponding to amino acid position T137 in Dda 1993 is substituted by K, R, I, V, A, D, E, H, M, N, P, Q, S or L, A position corresponding to amino acid position N144 in Dda 1993 is substituted by S, P, K, T, R, E, A, D, I, L, Q, Y, H, V, M or G, A position corresponding to amino acid position K145 in Dda 1993 is substituted by D, A, H, R, G, N, E, P, Q, S, T or M, A position corresponding to amino acid position E154 in Dda 1993 is substituted by N, S, V, I, T, D, G, H, K, Q, R, A or P, A position corresponding to amino acid position A157 in Dda 1993 is substituted with E, D, S, K, P, N, H, Q, R, T, G, I, L, M or V, A position corresponding to amino acid position Y158 in Dda 1993 is substituted by Q, S, E, L, D, A, I, K, F, N, G, R, V, T, H, P, M or W, A position corresponding to amino acid position P161 in Dda 1993 is substituted with E, D, K, S, N, R, Q, T, A or H, A position corresponding to amino acid position K166 in Dda 1993 is substituted by N, P, D, E, G, H, Q, R, S, T, A or M, A position corresponding to amino acid position Q170 in Dda 1993 is substituted by K, I, V, T, S, E, R, N, H, Y, F, A, D, M or P, A position corresponding to amino acid position T174 in Dda 1993 is substituted by S, A, D, E, G, H, K, M, N, P, Q, R, I, L or V, A position corresponding to amino acid position V176 in Dda 1993 is substituted by I, G, P, A, T, R, S, Q, K, L, E, F, M or Y, A position corresponding to amino acid position N180 in Dda 1993 is substituted by D, E, K, A, G, H, P, Q, S, T or R, A position corresponding to amino acid position A181 in Dda 1993 is substituted with G, N, L, S, R, T, D, K, Q, H, E, I, M, P or V, A position corresponding to amino acid position V190 in Dda 1993 is substituted by I, A, F, L, M, T or Y, A position corresponding to amino acid position Y197 in Dda 1993 is substituted by K, P, R, S, E, G, D, T, L, N, I, V, F, A, H, M, Q or W, A position corresponding to amino acid position V200 in Dda 1993 is substituted by I, T, F, K, D, L, N, E, S, Y, P, A or M, A position corresponding to amino acid position V201 in Dda 1993 is substituted by F, Y, I, K, L, H, M, W, A or T, A position corresponding to amino acid position R207 in Dda 1993 is substituted by I, H, K, V, F, Y, L, T, E, A, M, N, Q, S or D, A position corresponding to amino acid position T213 in Dda 1993 is substituted by D, N, E, K, S, G, A, Q, H, P, I, L, M, R or V, A position corresponding to amino acid position A214 in Dda 1993 is substituted by G, S, E, K, D, L, N, I, T, V, M, P, Q, R or H, A position corresponding to amino acid position L215 in Dda 1993 is substituted by Y, I, E, F, D, H, M, Q, V, W, A, T, K, N, P, R or S, A position corresponding to amino acid position M219 in Dda 1993 is substituted by L, I, V, A, F, Y, K, D, E, S, T, Q, R or W, A position corresponding to amino acid position V220 in Dda 1993 is substituted by K, E, N, A, D, R, S, T, Q, L, I, G, H, M, P, F or Y, A position corresponding to amino acid position I225 in Dda 1993 is substituted by V, K, T, D, N, S, E, G, R, Q, P, A, H, L, Y, F or M, A position corresponding to amino acid position K227 in Dda 1993 is substituted by G, A, D, N, S, E, H, M, P, Q, R or T, A position corresponding to amino acid position L229 in Dda 1993 is substituted by S, K, V, I, N, E, D, T, G, A, R, Q, H, M, P, F or Y, A position corresponding to amino acid position V237 in Dda 1993 is substituted by M, I, A, L, F, K, Q, R, S, T, W or Y, A position corresponding to amino acid position M238 in Dda 1993 is substituted by I, L, V, F, A, T, Y, K, Q, R, S or W, A position corresponding to amino acid position K247 in Dda 1993 is substituted by N, E, R, D, G, H, Q, S, T, A, M or P, A position corresponding to amino acid position L248 in Dda 1993 is substituted by F, I, H, M, V, W, Y, A or T, A position corresponding to amino acid position I251 in Dda 1993 is substituted by F, L, E, Y, A, T, H, M, V or W, A position corresponding to amino acid position R253 in Dda 1993 is substituted by Q, A, D, E, H, K, M, N, P, S, T or Y, A position corresponding to amino acid position K254 in Dda 1993 is substituted by N, S, D, E, G, H, Q, R, T, A, M or P, A position corresponding to amino acid position F257 in Dda 1993 is substituted by Y, H, I, L, M, Q, V or W, A position corresponding to amino acid position D262 in Dda 1993 is substituted by P, E, A, K, Q, S, T, G, H or N, A position corresponding to amino acid position V265 in Dda 1993 is substituted by E, P, A, K, S, G, D, N, T, H, Q, R, F, I, L, M or Y, A position corresponding to amino acid position D282 in Dda 1993 is substituted by P, A, E, K, Q, S, T, G, H or N, A position corresponding to amino acid position I289 in Dda 1993 is substituted by L, V, M, K, E, G, A, F, T or Y, A position corresponding to amino acid position Q295 in Dda 1993 is substituted by D, E, M, S, G, H, K, N, P, T, A, R or Y, A position corresponding to amino acid position Y304 in Dda 1993 is substituted by K, E, L, P, D, N, S, G, A, R, T, Q, H, V, I, M, F or W, A position corresponding to amino acid position S306 in Dda 1993 is substituted by E, Y, D, K, T, G, F, V, N, A, H, P, Q, R or M, A position corresponding to amino acid position V309 in Dda 1993 is substituted by F, K, I, H, L, M, W, Y, A or T, A position corresponding to amino acid position V314 in Dda 1993 is substituted by I, K, E, L, A, F, M, T or Y, A position corresponding to amino acid position G316 in Dda 1993 is substituted by E, K, D, V, I, N, L, A, H, P, Q, R, S or T, A position corresponding to amino acid position L319 in Dda 1993 is substituted by E, K, I, V, T, N, D, G, A, H, P, Q, R, S, F, M or Y, A position corresponding to amino acid position R321 in Dda 1993 is substituted by K, N, E, V, I, D, L, A, H, M, P, Q, S or T, A position corresponding to amino acid position T329 in Dda 1993 is substituted by P, S, G, K, A, E, D, I, V, L, N, R, Y, F, Q or M, A position corresponding to amino acid position G331 of Dda 1993 is substituted by D, E, K, L, S, I, N, T, V, H, P, Q or A, A position corresponding to amino acid position D333 in Dda 1993 is substituted with G, K, E, I, A, N, S, H, P, Q or T, A position corresponding to amino acid position L349 in Dda 1993 is substituted by E, N, K, Q, A, T, S, D, V, R, I, H, P, F, M or Y, A position corresponding to amino acid position F352 in Dda 1993 is substituted by Y, L, K, E, R, I, A, N, D, H, M, Q, V or W, A position corresponding to amino acid position T362 in Dda 1993 is substituted by A, I, V, L, E, K, M, D, R, Y, N, Q, S, F, G or P, A position corresponding to amino acid position N365 in Dda 1993 is substituted by K, E, A, S, R, D, H, M, P, Q, T or G, A position corresponding to amino acid position N367 in Dda 1993 is substituted by S, K, L, E, A, T, I, V, Q, R, Y, F, M, D, G, H or P, A position corresponding to amino acid position G369 in Dda 1993 is substituted by E, K, D, N, S, T, R, L, I, A, Y, H, P or Q, A position corresponding to amino acid position G370 in Dda 1993 is substituted by K, V, E, I, S, A, N, D, H, M, P, Q, R or T, A position corresponding to amino acid position P373 in Dda 1993 is substituted with H, L, N, K, M, Y, F, I, V, S, E, T, A, R, D, G, Q or W, A position corresponding to amino acid position A380 in Dda 1993 is substituted by V, L, T, I, K, M, F, Y, E, G, P, Q, R or S, A position corresponding to amino acid position Q383 in Dda 1993 is substituted by L, T, M, Y, K, F, I, V, R, A, E, S, N, D, G, H or P, A position corresponding to amino acid position S385 in Dda 1993 is substituted with I, T, A, Y, K, H, R, F, L, M, V, D, E, G, N, P or Q, A position corresponding to amino acid position P391 in Dda 1993 is substituted with G, Y, F, H, A, V, D, N, S, E, K, Q or T, A position corresponding to amino acid position S393 of Dda 1993 is substituted with I, V, L, T, E, D, M, F, A, Y, G, H, K, N, P, Q or R, A position corresponding to amino acid position F395 in Dda 1993 being substituted by I, V, Y, L, T, A, M, H or W, A position corresponding to amino acid position M401 in Dda 1993 is substituted by L, S, V, I, G, D, E, R, Q, A, W, N, T, F, Y, K, H or P, A position corresponding to amino acid position S402 in Dda 1993 is substituted by T, V, R, I, K, L, E, H, Q, M, A, D, N, P or G, A position corresponding to amino acid position V403 in Dda 1993 is substituted with F, Y, I, H, L, M, W, A or T, A position corresponding to amino acid position D404 in Dda 1993 is substituted by N, K, R, E, G, H, Q, S, T or P, A position corresponding to amino acid position R405 in Dda 1993 is substituted by N, K, D, E, G, H, Q, S, T, A, M or P, A position corresponding to amino acid position A406 in Dda 1993 is substituted by T, V, I, D, E, K, L, M, N, P, Q, R, S, G, F or Y, A position corresponding to amino acid position I413 in Dda 1993 is substituted by E, L, K, S, D, G, N, P, Y, F, T, R, A, H, Q, M or V, A position corresponding to amino acid position H414 in Dda 1993 is substituted by Y, D, F, Q, K, N, S, E, G, P, R, T, L, I, A, M, V or W, A position corresponding to amino acid position V418 in Dda 1993 is substituted by K, E, P, R, D, T, A, S, N, L, I, Y, G, H, M, Q or F, A position corresponding to amino acid position E419 in Dda 1993 is substituted by Q, D, A, H, K, M, N, P, R, S, T or Y, A position corresponding to amino acid position A421 in Dda 1993 is substituted by Q, L, R, N, S, K, D, E, H, M, P, T, Y, G, I, V or F, A position corresponding to amino acid position Q422 in Dda 1993 is substituted by K, R, L, N, A, D, E, H, M, P, S, T or Y, A position corresponding to amino acid position V427 in Dda 1993 is substituted by T, E, D, A, I, K, L, M, N, P, Q, R, S, F or Y, A position corresponding to amino acid position V429 in Dda 1993 is substituted by I, L, A, F, M, T or Y, A position corresponding to amino acid position G432 in Dda 1993 is substituted by P, A, S, T, D, E, K, Q or N, A position corresponding to amino acid position Y434 in Dda 1993 is substituted by K, I, V, R, E, T, N, D, L, S, Q, H, G, A, M, P, F or W, -The position corresponding to amino acid position D435 in Dda 1993 is substituted by E, Q, A, H, K, N, P, R, S, T or G, and/or -The position corresponding to amino acid position F437 in Dda 1993 is substituted with Y, I, H, L, M, Q, V or W.
  3. 3. A construct comprising a helicase according to claim 1 or 2 and an additional polynucleotide binding moiety, wherein the helicase is linked to the polynucleotide binding moiety and the construct has the ability to control the movement of an analyte.
  4. 4. A construct according to claim 3, wherein the construct comprises two or more helicases according to claim 1 or 2.
  5. 5. A polynucleotide comprising a sequence encoding a helicase according to claim 1 or 2 or a construct according to claim 3 or 4.
  6. 6. A vector comprising the polynucleotide of claim 5 operably linked to a promoter.
  7. 7. A host cell comprising the vector of claim 6.
  8. 8. A method of making a helicase according to claim 1 or 2 or a construct according to claim 3 or 4, comprising expressing a polynucleotide according to claim 5, transfecting a cell with a vector according to claim 6, or culturing a host cell according to claim 7.
  9. 9. A method of controlling movement of an analyte comprising contacting the analyte with a helicase according to claim 1 or 2 or a construct according to claim 3 or 4, thereby controlling the movement of the analyte.
  10. 10. The method of claim 9, wherein the method is used to control movement of analytes through a transmembrane pore.
  11. 11. A method of characterizing an analyte of interest, comprising: (a) Contacting the target analyte with a transmembrane pore and a helicase according to claim 1 or 2 or a construct according to claim 3 or 4 such that the helicase or construct controls movement of the target analyte through the pore, and (B) One or more measurements are made as the target analyte moves relative to the well, wherein the measurements are indicative of one or more characteristics of the target analyte, thereby characterizing the target analyte.
  12. 12. The method of claim 11, wherein the method comprises: (a) Contacting the target analyte with a transmembrane pore and a helicase according to claim 1 or 2 or a construct according to claim 3 or 4 such that the helicase or the construct controls movement of the target analyte through the pore, and (B) Measuring a current through the well as the target analyte moves relative to the well, wherein the current is indicative of one or more characteristics of the target analyte, thereby characterizing the target analyte.
  13. 13. The method of claim 11 or 12, wherein the target analyte is a polynucleotide.
  14. 14. The method of any one of claims 11 to 23, wherein the pore is a transmembrane protein pore or a solid state pore.
  15. 15. A method of forming a sensor for characterizing an analyte of interest, comprising forming a complex between (a) a well and (b) a helicase according to claim 1 or 2 or a construct according to claim 3 or 4, thereby forming a sensor for characterizing the analyte of interest.
  16. 16. The method of claim 15, wherein the complex is formed by covalently linking the pore to the helicase or construct.
  17. 17. A sensor for characterizing an analyte of interest comprising a complex between (a) a pore and (b) a helicase according to claim 1 or 2 or a construct according to claim 3 or 4.
  18. 18. Use of a helicase according to claim 1 or 2 or a construct according to claim 3 or 4 to control the movement of a target analyte through a pore.
  19. 19. A kit for characterizing an analyte of interest, comprising (A) A well and a helicase according to claim 1 or 2 or a construct according to claim 3 or 4, or (B) A helicase according to claim 1 or 2 or a construct according to claim 3 or 4 and one or more loading moieties.
  20. 20. An apparatus for characterizing an analyte of interest in a sample comprising (a) a plurality of wells and (b) a plurality of helicases according to claim 1 or 2 or a plurality of constructs according to claim 3 or 4.

Description

Novel enzymes Technical Field The present invention relates to modified Dda helicases that can be used to control the movement of analytes such as polynucleotides. The modified Dda helicase is used for analyte detection and characterization. Background The use of nanopore sensing to characterize two of the basic components of an analyte (especially a polymer) is (1) to control movement of the polymer through the pore and (2) to group building blocks as the polymer moves through Kong Shiou. During nanopore sensing, the narrowest portion of the pore generally corresponds to the most discernable portion of the nanopore relative to the change in measurement signal, which changes as the analyte moves relative to the nanopore. WO2015/055981, WO2015/166276, WO2016/055777 and PCT/EP2023/059821 describe polynucleotide binding proteins, particularly Dda helicase, which can be used to control movement of an analyte relative to a transmembrane protein pore (such as the CsgG pore described herein), which are incorporated herein by reference in their entirety. Disclosure of Invention The inventors have surprisingly identified specific Dda mutants (herein referred to as modified Dda helicases or modified helicases) having improved ability to control the movement of analytes through the pore. When using a well to sequence a polynucleotide, the system estimates the number and type of bases/nucleotides passing through the well in combination. Better control of the variability of the movement speed may reduce one of the sources of statistical noise and simplify the estimation task. Continuous short residence of the polynucleotide in the well may trigger failure to recognize the underlying nucleotide/base, resulting in deletion errors. An abnormally long dwell may lead to an insertion error. Ensuring that each nucleotide/base spends a sufficient time interval in the well helps to resolve the statistical uncertainty of the nucleotide/base species from noise signal levels. Further information can be extracted from the dependence of the residence time on nucleotide/base type, for example via interaction with a motor enzyme. Reducing the overall variability of residence time helps to extract more accurate information through this channel. During regions where signal levels provide limited information about movement (e.g., long homopolymer regions), polynucleotide/base residence time can be used to infer the number of bases that pass through the pore. Reducing the variability of residence time may make these inferences more accurate. In some embodiments, the modified helicases of the invention exhibit increased rates when used in methods for controlling movement of analytes through a transmembrane pore and in methods for characterizing analytes using a transmembrane pore. When Dda helicases are used that do not comprise a mutation of the invention, the analyte passage/velocity relative to the pore may be increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500% or more relative to the velocity of the analyte movement relative to the pore. The inventors have surprisingly found that these changes in speed (such as increases in speed) have minimal or no effect on accuracy readings. This is particularly advantageous in methods of characterizing analytes, wherein the analyte is contacted with a well and a helicase of the invention such that the polynucleotide binding protein controls movement of the analyte of interest through/relative to the well. In some embodiments, the modified helicases of the invention exhibit improved accuracy when used in methods for controlling movement of analytes through a transmembrane pore and in methods for characterizing analytes using a transmembrane pore. In the context of analyte characterization (particularly polynucleotides), accuracy is interpreted to mean single-pass accuracy of the original read, i.e., a single pass of a single molecule through a transmembrane pore. Accuracy is a useful indicator of platform improvement in tracking sequencing devices. Accuracy may also refer to consistency accuracy or accuracy in detecting a particular thing (e.g., a mutation in a polynucleotide analyte). Additionally or alternatively, accuracy is interpreted to mean the percentage of bases above a certain confidence level, where the confidence level has been pre-calibrated. In some embodiments, the modified helicases of the invention exhibit improved accuracy with minimal to no change in speed. In some embodiments, accuracy is improved to give less than 10% error, less than 5% error