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(12) Patent Application Publication (10) Pub. No.: US 2013/0203610 A1 Meller Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2013/0203610 A1 Meller Et Al US 20130203610A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/0203610 A1 Meller et al. (43) Pub. Date: Aug. 8, 2013 (54) TOOLS AND METHOD FOR NANOPORES Related U.S. Application Data UNZIPPING-DEPENDENT NUCLECACID (60) Provisional application No. 61/318,872, filed on Mar. SEQUENCING 30, 2010. (75) Inventors: Amit Meller, Brookline, MA (US); Alon Publication Classification Singer, Brighton, MA (US) (51) Int. Cl. (73) Assignee: TRUSTEES OF BOSTON CI2O I/68 (2006.01) UNIVERSITY, Boston, MA (US) (52) U.S. Cl. CPC .................................... CI2O I/6874 (2013.01) (21) Appl. No.: 13/638,455 USPC ................................................. 506/6:506/16 (57) ABSTRACT (22) PCT Filed: Mar. 30, 2011 Provided herein is a library that comprises a plurality of molecular beacons (MBs), each MB having a detectable (86). PCT No.: PCT/US2O11AO3O430 label, a detectable label blocker and a modifier group. The S371 (c)(1), library is used in conjunction with nanopore unzipping-de (2), (4) Date: Apr. 17, 2013 pendent sequencing of nucleic acids. Patent Application Publication Aug. 8, 2013 Sheet 1 of 18 US 2013/020361.0 A1 . N s Patent Application Publication Aug. 8, 2013 Sheet 2 of 18 US 2013/020361.0 A1 I’9IAI ::::::::::: Dº3.modoueN Patent Application Publication Aug. 8, 2013 Sheet 3 of 18 US 2013/020361.0 A1 Z’9IAI ~~~~~~~~~);........ Patent Application Publication Aug. 8, 2013 Sheet 4 of 18 US 2013/020361.0 A1 s :·.{-zzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzz iii. 9 iii.338 lii &S Patent Application Publication Aug. 8, 2013 Sheet 5 of 18 US 2013/020361.0 A1 s r Patent Application Publication Aug. 8, 2013 Sheet 6 of 18 US 2013/020361.0 A1 se xf t (a) Sat 8.8 is 38 is s S. 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C Sis S N S N 9 O O N-- Y 9 O O 8 5 Of) Patent Application Publication Aug. 8, 2013 Sheet 18 of 18 US 2013/020361.0 A1 s US 2013/020361.0 A1 Aug. 8, 2013 TOOLS AND METHOD FOR NANOPORES oligonucleotide representing each of the different bases, e.g., UNZIPPING-DEPENDENT NUCLECACD A, T, U, G, or C. The converted DNA is hybridized with SEQUENCING complementary molecular beacons to form a double-stranded DNA. There are distinct species of molecular beacons CROSS REFERENCE TO RELATED complementary oligonucleotide representing each of the dif APPLICATION ferent bases, e.g., A, T, U, G, or C. These different species of 0001. This application claims benefit under 35 U.S.C. molecular beacons are distinctly labeled for identification S119(e) of the U.S. Provisional Application No. 61/318,872 purposes, e.g., four different fluorophores for four species of filed Mar. 30, 2010, the contents of which are incorporated molecular beacons. To detect the sequence of the DNA, nan herein by reference in its entirety. opores of less than 2 nm are then used to sequentially unzip the beacons from the double-stranded DNA (dsDNA) com prising molecular beacons. With each unzipping event a new GOVERNMENT SUPPORT fluorophore is un-quenched, giving rise to a series of photon 0002 This invention was made with Government support flashes in different colors, which are recorded by a CCD under contract No. RO1-HGOO4128 awarded by the National camera (FIG.2). The unzipping process slows down the trans Institutes of Health. The Government has certain rights in the location of the DNA through the pore in a voltage-dependent invention. manner, to a rate compatible with optical recording. 0007. One limiting factor of DNA sequencing that is BACKGROUND OF INVENTION dependent on nanopore unzipping of a labeled dsDNA is that 0003 Nanopore sequencing is a promising technology the pore of the nanopore has to be Small enough to pry open being developed as a cheap and fast alternative to the conven the double-stranded structure, usually less than 2 nm in diam tional Sanger sequencing method. Nanopore sequencing eter. Currently, there are two general approaches to prepare methods can provide several advantages over the conven nanopores for nucleic acid analysis: (1) Organic nanopores tional Sanger sequencing method; they permit single mol that are prepared from naturally occurring molecules, such as ecule analysis, are not enzyme dependent (e.g., polymerase alpha-hemolysin pores. Although organic nanopores are enzyme is not required for chain extension), and require sig commonly used for DNA analysis, organic nanopores are nificantly less reagents. great for single DNA sequencing and not easily adaptable for 0004. A number of nanopore based DNA sequencing high throughput DNA sequencing requiring numerous nan methods have recently been proposed' and highlight two opores at the same time. (2) Synthetic solid-state nanopores major challenges': 1) The ability to discriminate among that are made by various conventional and non-conventional individual nucleotides (nt), e.g., the system must be capable fabrication techniques. Synthetically fabricated nanopores of differentiating among the four bases at the single-molecule holds more potential for high throughput DNA sequencing level, and 2) the method must enable parallel readout. requiring numerous nanopores at the same time. 0005. In nanopore based DNA sequencing methods, it had 0008 Another limiting factor of DNA sequencing that is been previously difficult to scale down DNA analysis to the dependent on nanopore unzipping of a labeled dsDNA is that single molecule level, mainly due to the relatively small dif a single nanopore can probe only a single molecule at a time. ferences between the four nucleotides constituting DNA, and Development of fast, high throughput, genomic sequencing due to the inherent noise in single molecule probing. The using nanopore base sequencing methods would entail an approach taken by Some to circumvent these problems is to array of nanopores and the simultaneous monitoring the nan magnify each of the individual bases of a DNA to distinct opores. Although fabrication of nanopores can produces lots entities that produces measurable signals that are signifi of synthetic nanopores, uniform constant quality manufac cantly greater than the background noise level, thereby ture of nanopores with very small pore is difficult. Alternative increasing the signal-to-noise ratio. This is achieved by an strategies in nanopore based unzipping sequencing methods initial preparation step of converting the DNA molecules to that permit the use of nanopores with slightly larger pore size be analyzed into longer and periodically structured DNA are desirable. molecule, named “Design Polymers'''. 0006 Currently, there are two general approaches used in SUMMARY OF THE INVENTION nanopore based DNA sequencing methods for “detecting” or 0009 Embodiments of the present invention are based on measuring the individual bases of a DNA: 1) by monitoring a the discovery that linking a modifier group to a moiety such as change in the pore conductivity when the DNA enters and a molecular beacon (MB) used in nanoporeunzipping-depen passes through the pore, the change in the pore conductivity dent sequencing of nucleic acids enables the use of a nanop can be measured directly e.g., using an electrometer; and 2) ore with a larger pore than the width of a standard double by optical detection of distinct molecular beacons as they are Stranded (ds) nucleic acid, which is ~2.2 nm. For nanopore unzipped by a nanopore that must be Small enough to exclude unzipping-dependent sequencing, a pore size of ~1.5-2.0 nm a double-stranded DNA but yet will permit the entry and allows only a single stranded nucleic acid to translocate translocation of a single stranded DNA. In the first approach, through the opening of the pore in an electric field. This bulky groups are attached to the bases of nucleotide to essentially forces strand separation of the ds nucleic acid in increase and make distinct the electronic blockade signals contact with the nanopore, this process is commonly termed generated for detection when the double-stranded DNA “unzipping.
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