Methods and Tools for Purifying Nucleic Acids Using Polymerized Tubulin

Content Provider	The Lens
Description	La présente invention concerne le domaine de la purification d'acides nucléiques. En particulier, l'invention concerne des procédés et des outils destinés à purifier des acides nucléiques dans un échantillon, qui sont compatibles avec un séquençage et un diagnostic à haut débit. Selon l'invention, des protéines de liaison à l'acide nucléique recrutées dans de la tubuline polymérisée (c'est-à-dire des microtubules) pourraient, par la suite, être isolées à partir de lysats cellulaires. De manière inattendue, il a été découvert que la quantité d'acide nucléique récupéré trouvé dans les pastilles de microtubules augmente considérablement en présence de protéines de piégeage d'acide nucléique comprenant une fraction de liaison à l'acide nucléique et une fraction de liaison à la tubuline polymérisée, par comparaison avec des protéines dépourvues de la fraction de liaison à l'acide nucléique, et que la récupération des acides nucléiques purifiés est elle-même particulièrement efficace. Le procédé de purification est particulièrement adapté au séquençage à haut débit et/ou dans le contexte d'un procédé de diagnostic destiné à identifier ou à comparer la quantité d'acides nucléiques dans un ensemble d'échantillons.
Abstract	The present invention relates to the field of nucleic acid purification. In particular, it relates to methods and tools for purifying nucleic acids in a sample; which are compatible with high-throughput sequencing and diagnosis. The inventors have shown that nucleic acid binding proteins recruited to polymerized tubulin (i.e. microtubules) could, subsequently, be isolated from cell lysates. Surpisingly, it has now been found that the amount of recovered nucleic acid found in these microtubule pellets increases dramatically in the presence of nucleic acid-trapping proteins comprising a nucleic acid-binding moiety and a polymerized tubulin-binding moiety, by comparison to proteins devoid of the nucleic acid-binding moiety; and that the recovery of the purified nucleic acids was itself particularly efficient. This purification method is particularly amenable to high-throughput sequencing and/or in the context of a diagnosis method for identifying or comparing the amount of nucleic acids in a set of samples.
Related Links	https://www.lens.org/lens/patent/010-962-833-720-011/frontpage
Language	English
Publisher Date	2019-01-31
Access Restriction	Open
Alternative Title	Procédés Et Outils De Purification D'acides Nucléiques À L'aide De Tubuline Polymérisée
Content Type	Text
Resource Type	Patent
Date Applied	2018-07-27
Agent	Jupin, Claude
Applicant	Inst Nat Sante Rech Med Univ Devry Val Dessonne
Application No.	2018070433
Claim	CLAIMS A method for purifying nucleic acid molecules in a sample, comprising at least the step of : a) contacting said sample with at least one tubulin and one or more nucleic acid-trapping proteins comprising a nucleic acid-binding moiety and a polymerized tubulin-binding moiety; in efficient conditions for forming polymerized tubulin in complex with the said nucleic acid-trapping proteins bound to the said nucleic acids to be purified; and b) recovering the nucleic acid-trapping protein(s) which is/are bound to the nucleic acid molecules, thereby purifying said nucleic acid molecules. The method according to claim 1, wherein the nucleic acid molecule(s) is/are R A molecule(s) and the nucleic acid-trapping protein(s) is/are RNA-trapping protein(s). The method for purifying nucleic acid molecules according to claim 1 or 2, wherein in step a): - the said nucleic acid-trapping proteins are added in vitro to the said sample prior to immobilization on polymerized tubulin; or - the sample comprises cells expressing said nucleic acid-trapping proteins or a cell lysate thereof. The method according to any one of the preceding claims, wherein the nucleic acid-trapping proteins are immobilized on tubulin under conditions suitable for tubulin polymerization. The method according to any one of the preceding claims, wherein the polymerized tubulin-binding moiety comprised in a nucleic acid-trapping protein comprises one or more Microtubule Binding Domains (MBDs) present in proteins selected from the group consisting of: Tau, MAPI A, MAP2, MAP4, MAP6 and EB1. The method according to any one of the preceding claims, wherein the nucleic acid-binding moiety comprised in a nucleic acid-trapping protein comprises one or more nucleic acid-binding domains selected in a group consisting of: TDP43, FUS, TAF15, NF45/NF90, DDX6, hnNRP Al, DHX36, FMRP, HuD, hnR P L, HUR, G3BP1, Lin28A, Lin28B, AGO, HuR, METTL3, METTL14, FTO, ALKBH, YTHDF1-3, PABP1 and YBX1. The method according to any one of the preceding claims, wherein a nucleic acid-trapping protein comprises a linker region located between the nucleic acid- binding moiety and the polymerized tubulin-binding moiety. The method according to any one of the preceding claims, wherein step b) consists in expressing one or more nucleic acid-trapping proteins in eukaryotic cells The method according to any one of the preceding claims, further comprising a step of collecting the nucleic acid molecules which are complexed with the nucleic acid-trapping protein(s) . A method for characterizing, preferably sequencing, nucleic acid molecules in a sample, comprising at least the steps of: a) contacting said sample with at least one tubulin and or more nucleic acid- trapping proteins comprising a nucleic acid-binding moiety and a polymerized tubulin- binding moiety, in efficient conditions for forming polymerized tubulin in complex with the said nucleic acid-trapping proteins bound to the said nucleic acids to be purified; b) recovering the nucleic acid-trapping protein(s) which is/are bound to the nucleic acid molecules, thereby purifying said nucleic acid molecules; c) characterizing, preferably sequencing, the purified nucleic acid molecules. 11. The method according to any one of the preceding claims, further comprising a step of collecting the nucleic acid molecules which are bound to the nucleic acid-trapping protein(s). A method for comparing the amounts of target nucleic acid molecules between two samples comprising at least the steps of: a) performing a method for purifying nucleic acid molecules according to any one of the preceding claims on a first sample by using a selected nucleic acid-trapping protein, so as to obtain a first collection of purified target nucleic acid molecules, b) performing a method for purifying nucleic acid molecules according to any one of the preceding claims on a second sample by using the same selected nucleic acid- trapping molecule as that used at step a), so as to obtain a second collection of purified target nucleic acid molecules, and c) determining the amount of target nucleic acid molecules comprised in the first collection of purified target nucleic acid molecules and in the second collection of purified target nucleic acid molecules, respectively. A method for preparing an affinity support for purifying nucleic acid molecules contained in a sample, comprising an in vitro step of immobilizing one or more nucleic acid-trapping proteins on polymerized tubulin; wherein the nucleic acid-trapping protein comprises a nucleic acid-binding moiety and a polymerized tubulin-binding moiety, thereby providing said affinity support for purifying nucleic acid molecules. A kit for purifying nucleic acid molecules and/or for preparing an affinity support for purifying nucleic acid molecules, comprising : a) at least one nucleic acid-trapping protein, and/or a vector comprising an expression cassette for expressing a nucleic acid-trapping protein, and/or a cell expressing a nucleic acid-trapping protein; and b) lyophilized or purified tubulin; An affinity support for purifying nucleic acid molecules comprising nucleic acid-trapping proteins immobilized on recombinant or synthetic polymerized tubulin, wherein the nucleic acid-trapping protein comprises a nucleic acid-binding moiety and a polymerized tubulin-binding moiety.
CPC Classification	MEASURING OR TESTING PROCESSES INVOLVING ENZYMES; NUCLEIC ACIDS OR MICROORGANISMS ;COMPOSITIONS OR TEST PAPERS THEREFOR;PROCESSES OF PREPARING SUCH COMPOSITIONS;CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES PEPTIDES MICROORGANISMS OR ENZYMES;COMPOSITIONS THEREOF;PROPAGATING; PRESERVING; OR MAINTAINING MICROORGANISMS;MUTATION OR GENETIC ENGINEERING;CULTURE MEDIA
Extended Family	057-798-757-624-311 126-312-591-887-100 096-004-619-852-890 163-999-622-095-954 048-953-267-488-012 010-962-833-720-011 160-393-011-014-638
Patent ID	2019020798
Inventor/Author	Pastre David Desforges Bénédicte Maucuer Alexandre
IPC	C12Q1/6806 C12N15/10
Status	Pending
Simple Family	057-798-757-624-311 126-312-591-887-100 096-004-619-852-890 163-999-622-095-954 048-953-267-488-012 010-962-833-720-011 160-393-011-014-638
CPC (with Group)	C12Q1/6806 C07K2319/70 C07K2319/735 C07K2319/85 C12N15/1003
Issuing Authority	United States Patent and Trademark Office (USPTO)
Kind	Patent Application Publication