DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Priority
Acknowledgment is made of applicant's claim for foreign priority based on an application filed in EP on 5/18/2021. It is noted, however, that applicant has not filed a certified copy of the EP21174437.0 application as required by 37 CFR 1.55.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 4/12/2024, 11/15/2024, 12/11/2024, 8/25/2025, 3/27/2026, 6/30/2026 has been considered by the examiner.
Election/Restrictions
Applicant's election of Group II, Claims 36-50, without traverse in the reply filed on 06/30/2026 is acknowledged.
Specification
Applicant is reminded of the proper language and format for an abstract of the disclosure.
The abstract should be in narrative form and generally limited to a single paragraph on a separate sheet preferably within the range of 50 to 150 words in length. The abstract should describe the disclosure sufficiently to assist readers in deciding whether there is a need for consulting the full patent text for details.
The language should be clear and concise and should not repeat information given in the title. It should avoid using phrases which can be implied, such as, "The disclosure concerns," "The disclosure defined by this invention," "The disclosure describes," etc. In addition, the form and legal phraseology often used in patent claims, such as "means" and "said," should be avoided.
The abstract of the disclosure is objected to because the sheet of the abstract also contains “Title: Nanopore proteomics.” Correction is required. See MPEP § 608.01(b).
Claim Objection
Claim 49 is objected to because of the following informalities:
Claim 49: please amend “a lumen-facing non-aromatic amino acid(s)” to – [[a]] one or more lumen-facing non-aromatic amino acid(s)--.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 49 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as failing to set forth the subject matter which the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the applicant regards as the invention.
Regarding claim 49, claim 49 recites “wherein (a) further comprises substituting a lumen-facing non-aromatic amino acid(s) with one or more natural or non-natural aromatic amino acid(s) on every monomeric unit of the nanopore”, which is unclear if a lumen-facing non-aromatic amino acid(s) is the same as or different than the one or more lumen facing amino acids recited in claim 36, and it is unclear if one or more natural or non-natural aromatic amino acid(s) is the same as or different than the one or more natural or non-natural aromatic amino acid(s) recited in claim 36. Therefore, the scope of claim 49 is indefinite.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 36-47 and 49-50 are rejected under 35 U.S.C. 103 as being unpatentable over Huang et al. (WO2020055246A1), and in view of Maglia et al. (US20150008126A1).
Regarding claim 36, Huang teaches a method (a method for providing a system according to the invention [claim 11]), comprising:
(a) providing a system comprising (i) a fluidic chamber; (ii) a membrane that separates the fluidic chamber into a first side and a second side; and (iii) a nanopore disposed in the membrane (providing a system as shown in Fig.1a comprising a fluidic chamber separated by a membrane [lipid bilayer] into a first side and a second side [cis chamber and trans chamber, Ln 25-31 on pg.22], a FraC nanopore disposed in the membrane [abstract]), wherein the first side or the second side of the fluidic chamber comprises a pH of less than 4.5 (pH 3.8 and 3.0 as shown in Fig.5A [Ln 28 on pg. 15 to Ln 7 on pg. 16]),
wherein the nanopore comprises (1) at least a portion of an alpha helical pore forming protein or peptide (FraC nanopore [abstract]); and
(b) bringing the nanopore in contact with an analyte ( the nanopore is typically positioned between a first liquid medium and a second liquid medium, wherein at least one liquid medium comprises an analyte, and wherein the system is operative to detect a property of the analyte. In one embodiment, the system is operative to detect a property of the analyte comprises subjecting the nanopore to an electric field such that the analyte electrophoretically and/or electroosmotically translocates through the nanopore [Ln 4-16 on pg. 9]).
Huang further teaches wherein the at least the portion of the alpha helical pore forming protein or peptide comprises a modification of one or more lumen facing amino acids (The mutant FraC polypeptide comprises a mutation at position D109, W112 and/or W116; any one of these mutations may be supplemented with one or more unnatural amino acids comprising a moiety that holds a negative charge at low pH [Ln 1-17 on pg. 10]).
Huang does not explicitly teach wherein the one or more lumen facing amino acids is modified into one or more natural or non-natural “aromatic” amino acids.
Maglia teaches methods of enhancing translocation of charged analytes through transmembrane protein pores (title and Fig.1). The method of the invention comprises increasing the net opposing charge of the barrel or channel and/or entrance of the pore. The opposing charge is the opposite charge to the charge of the analyte. If the analyte is negatively charged, the method involves increasing the net positive charge of the barrel or channel and/or entrance of the pore [para. 0097]. The net positive charge is preferably increased by introducing one or more positively charged amino acids into the barrel or channel and/or entrance of the pore. The one or more positively charged amino acids may be introduced by addition. The one or more positively charged amino acids are preferably introduced by substitution [para. 0099]. The positively charged amino acid(s) can be naturally-occurring or non-naturally-occurring [para. 0100]. Wherein the net positive charge is increased substituting one or more negatively charged amino acids in the barrel or channel and/or entrance of the pore with one or more uncharged amino acids, non-polar amino acids and/or aromatic amino acids (Claim 9). Aromatic amino acids have an aromatic side chain. Suitable aromatic amino acids include, but are not limited to, histidine (H), phenylalanine (F), tryptophan (W) and tyrosine (Y) [para. 0104]. Thus, Maglia teaches the one or more lumen facing amino acids is modified into one or more natural or non-natural “aromatic” amino acids for increasing the net positive charge of the nanopore for enhancing the translocation (abstract and [para. 0097]).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the one or more lumen facing amino acids into one or more natural or non-natural aromatic amino acids, as taught by Maglia, since it would enhance the translocation of the analyte (abstract, [para. 0099-0100, 0104] and claim 9 in Maglia).
Regarding claim 37, modified Huang teaches the method of claim 36, and Huang teaches further comprising translocating the analyte through the nanopore (the system is operative to detect a property of the analyte comprises subjecting the nanopore to an electric field such that the analyte electrophoretically and/or electroosmotically translocates through the nanopore [the 2nd paragraph on pg. 9]).
Regarding claim 38, modified Huang teaches the method of claim 37, and Huang teaches further comprising measuring a signal generated by translocating the analyte through the nanopore (Figs.3A, 3B and 6B show the measured current signals generated by translocating the analyte through the nanopore) .
Regarding claim 39, modified Huang teaches the method of claim 36, and Huang teaches wherein the nanopore is brought in contact with the analyte using an electro-osmotic force (the system is operative to detect a property of the analyte comprises subjecting the nanopore to an electric field such that the analyte electrophoretically and/or electroosmotically translocates through the nanopore [the 2nd paragraph on pg. 9]).
Regarding claim 40, modified Huang teaches the method of claim 36, and Huang teaches wherein the nanopore comprises the at least the portion of the alpha helical pore forming protein or peptide (FraC nanopore [abstract]).
Regarding claim 41, modified Huang teaches the method of claim 36, and Huang teaches wherein the at least the portion of the alpha helical pore forming protein or peptide comprises an actinoporin (FraC nanopore [abstract]; FraC comprises actinoporin).
Regarding claim 42, modified Huang teaches the method of claim 36, and Huang is silent to wherein the nanopore comprises the at least the portion of the beta barrel pore forming protein or peptide.
Maglia teaches pores for use in accordance with the invention can be β-barrel pores or α-helix bundle pores. β-barrel pores comprise a barrel or channel that is formed from β-sheets. Suitable β-barrel pores include, but are not limited to, β-toxins, such as α-hemolysin and leukocidins, and outer membrane proteins/porins of bacteria, such as Mycobacterium smegmatis porin A (MspA), outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A and Neisseria autotransporter lipoprotein (NalP) [para. 0075]. Thus, Maglia teaches pores can be β-barrel pores or α-helix bundle pores.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the α-helix pore to β-barrel pore including such as MspA, as taught by Maglia, since Maglia teaches β-barrel pore would be suitable alternative to the α-helix pore for detecting analytes translocating through the transmembrane protein pore (abstract and [para. 0075]).
Regarding claim 43, modified Huang teaches the method of claim 36, and Huang is silent to wherein the at least the portion of the beta barrel pore forming protein or peptide comprises a cytolysin, leukocidin, bacterial outer membrane porin, or ade novo designed pore peptide.
Maglia teaches pores for use in accordance with the invention can be β-barrel pores or α-helix bundle pores. β-barrel pores comprise a barrel or channel that is formed from β-sheets. Suitable β-barrel pores include, but are not limited to, β-toxins, such as α-hemolysin and leukocidins, and outer membrane proteins/porins of bacteria, such as Mycobacterium smegmatis porin A (MspA), outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A and Neisseria autotransporter lipoprotein (NalP) [para. 0075]. Thus, Maglia teaches pores can be β-barrel pores or α-helix bundle pores, wherein the at least the portion of the beta barrel pore forming protein or peptide comprises leukocidin, bacterial outer membrane porin such as MspA.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the α-helix pore to β-barrel pore, wherein the at least the portion of the beta barrel pore forming protein or peptide comprises leucocidin or bacterial outer membrane porin such as MspA, as taught by Maglia, since Maglia teaches β-barrel pore comprising leucocidin or bacterial outer membrane porin such as MspA would be suitable alternative to the α-helix pore for detecting analytes translocating through the transmembrane protein pore (abstract and [para. 0075]).
Regarding claim 44, modified Huang teaches the method of claim 36, and Huang teaches wherein the analyte comprises a non-nucleic acid analyte (the analyte is a proteinaceous substance, preferably a peptide [claim 10]).
Regarding claim 45, modified Huang teaches the method of claim 36, and Huang teaches wherein the analyte comprises a peptide or a protein (the analyte is a proteinaceous substance, preferably a peptide [claim 10]).
Regarding claim 46, modified Huang teaches the method of claim 36, and Huang is silent to wherein the at least the portion of the beta barrel pore forming protein or peptide comprises a Cytolysin K, lysenin, Anthrax toxin, Mycobacterium smegmatis porin A (MspA), Mycobacterium smegmatis porin B (MspB), Mycobacterium smegmatis porin C (MspC), Mycobacterium smegmatis porin D (MspD), outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A (OMPLA), ferric hydroxamate uptake component A (FhuA), Curli production transport component CsgG, or a Neisseria autotransporter lipoprotein (NalP).
Maglia teaches pores for use in accordance with the invention can be β-barrel pores or α-helix bundle pores. β-barrel pores comprise a barrel or channel that is formed from β-sheets. Suitable β-barrel pores include, but are not limited to, β-toxins, such as α-hemolysin and leukocidins, and outer membrane proteins/porins of bacteria, such as Mycobacterium smegmatis porin A (MspA), outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A and Neisseria autotransporter lipoprotein (NalP) [para. 0075]. Thus, Maglia teaches pores can be β-barrel pores or α-helix bundle pores, wherein the at least the portion of the beta barrel pore forming protein or peptide comprises comprises Mycobacterium smegmatis porin A (MspA), outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A (OMPLA), or a Neisseria autotransporter lipoprotein (NalP).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the α-helix pore to β-barrel pore, wherein the at least the portion of the beta barrel pore forming protein or peptide comprises Mycobacterium smegmatis porin A (MspA), outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A (OMPLA), or a Neisseria autotransporter lipoprotein (NalP), as taught by Maglia, since Maglia teaches β-barrel pore comprising Mycobacterium smegmatis porin A (MspA), outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A (OMPLA), or a Neisseria autotransporter lipoprotein (NalP) would be suitable alternative to the α-helix pore for detecting analytes translocating through the transmembrane protein pore (abstract and [para. 0075]).
Regarding claim 47, modified Huang teaches the method of claim 36, and Huang is silent to wherein the at least the portion of the alpha helical pore forming protein or peptide or the at least the portion of the beta barrel pore forming protein or peptide further comprises a second modification of one or more lumen facing amino acids into one or more negatively charged amino acids.
Huang further teaches the constriction of the nanopore still retains enough negative charge to recognize the peptide charge. Most likely, a negatively charged constriction is important for creating an electrophoretic environment for peptide-mass recognition (the 2nd paragraph on pg. 8). The mutant FraC polypeptide comprises a mutation at position D109, W112 and/or W116; any one of these mutations may be supplemented with one or more unnatural amino acids comprising a moiety that holds a negative charge at low pH (Ln 1-17 on pg. 10).
Maglia teaches any amino acid may be substituted with a negatively charged amino acid. One or more uncharged amino acids, non-polar amino acids and/or aromatic amino acids may be substituted with one or more negatively charged amino acids. Preferably, one or more positively charged amino acids are substituted with one or more negatively charged amino acids [para. 0111]. Pores for use in accordance with the invention can be β-barrel pores or α-helix bundle pores [para. 0075]. Thus, Maglia teaches wherein the at least the portion of the alpha helical pore forming protein or peptide further comprises a second modification of one or more lumen facing amino acids into one or more negatively charged amino acids (One or more uncharged amino acids may be substituted with one or more negatively charged amino acids; one or more positively charged amino acids are substituted with one or more negatively charged amino acids).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the at least the portion of the alpha helical pore forming protein to further comprise a second modification of one or more lumen facing amino acids into one or more negatively charged amino acids, since a negatively charged constriction would be important for creating an electrophoretic environment for peptide-mass recognition (the 2nd paragraph on pg. 8 in Huang).
Regarding claim 49, modified Huang teaches the method of claim 36, Huang is silent to wherein (a) further comprises substituting a lumen-facing non-aromatic amino acid(s) with one or more natural or non-natural aromatic amino acid(s) on every monomeric unit of the nanopore.
Maglia teaches wherein Table 1 shows non-aromatic amino acids and aromatic amino acids [para. 0079]. Any number of positively charged amino acids may be introduced. For instance, 1, 2, 5, 10, 15, 20, 25 or more positively charged amino acids may be introduced [para. 0106]. Wherein the net positive charge is increased substituting one or more negatively charged amino acids in the barrel or channel and/or entrance of the pore with one or more uncharged amino acids, non-polar amino acids and/or aromatic amino acids. The uncharged amino acids, non-polar amino acids and/or aromatic amino acids can be naturally-occurring or non-naturally-occurring. They may be synthetic or modified (claim 9 and [para. 0107]). Negatively charged amino acids may be substituted with (3) aromatic amino acids; (5) uncharged amino acids and aromatic amino acids; and (5) non-polar amino acids and aromatic amino acids; or (6) uncharged amino acids, non-polar amino acids and aromatic amino acids. [para. 0108].
Given the teachings of Maglia regarding any number positively charged amino acids may be introduced by substituting with natural or non-natural aromatic amino acids, It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substituting a lumen-facing non-aromatic amino acid(s) with one or more natural or non-natural aromatic amino acid(s) on every monomeric unit of the nanopore, since it would allow to increase the net opposing charge and accordingly enhance the translocation [para. 0097 and abstract in Maglia].
Regarding claim 50, modified Huang teaches the method of claim 36, and Huang teaches wherein a lumen-facing recognition region of the nanopore comprises a diameter of at most 2 nanometers (nm) (Fig.1a shows the narrowest constriction of the nanopore has a diameter of 1.6 nm. Type II FraC nanopores are furthermore characterized by an apparent pore size (at the narrowest constriction) of about 1.1 nm as calculated from homology modeling. Type III FraC nanopores are furthermore characterized by a pore size (at the narrowest constriction) of about 0.8 nm as shown by homology modeling [Ln 3-12 on pg. 4]).
Allowable Subject Matter
Claim 48 objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
The following is a statement of reasons for the indication of allowable subject matter.
Regarding claim 48, the prior art of the record does not teach and/or suggest wherein the one or more natural or non-natural aromatic amino acids and the one or more negatively charged amino acids are within 4 nanometers of each other, recited in claim 48.
As allowable subject matter has been indicated, applicant's reply must either comply with all formal requirements or specifically traverse each requirement not complied with. See 37 CFR 1.111(b) and MPEP § 707.07(a).
Conclusion
The prior arts made of record and not relied upon are considered pertinent to applicant's disclosure: Maglia et al. (US20190292235A1) teaches the mutant FraC comprises at least one substitution of a negatively charged amino acid residue in the narrow part of the pore into a neutral or positively charged amino acid residue, and/or at least one substitution of a neutral amino acid residue in the narrow part of the pore into a positively charged amino acid residue [para. 0010]. Huang et al. (Electro-osmotic capture and ionic discrimination of peptide and protein biomarkers with FraC nanopores, Nature Communications, 2017, 8, 935) teaches FraC nanopore for detecting peptide and protein biomarkers. Maglia et al. (Enhanced translocation of single DNA molecules through α-hemolysin nanopores by manipulation of internal charge, Biphysics and Computational Biology, 2008, 105, 19720-19725) teaches enhancing DNA translocation by augmenting the internal positive charge within the α-hemolysin pore and varying its distribution.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to SHIZHI QIAN whose telephone number is (571)272-3487. The examiner can normally be reached Monday-Thursday 8:00 am-5:00 pm.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Luan V. Van can be reached on (571) 272-8521. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/SHIZHI QIAN/Primary Examiner, Art Unit 1795
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