JPWO2021217146A5 - - Google Patents

Description

The invention also provides kits for use in the methods of the invention.
In an embodiment of the present invention, for example, the following items are provided:
(Item 1)
1. A method for determining the nucleotide sequence of a polynucleotide, comprising:
(a) a solid-state substrate comprising a cis side and a trans side, the substrate defining a reaction volume; and
(i) a proximal through-hole extending between the cis side and the trans side of the substrate;
(ii) one or more sidewalls;
(iii) a distal opening; and
a solid-state substrate, the solid-state substrate further comprising an opaque metal layer that substantially blocks excitation light from penetrating into the reaction volume and onto the cis side of the substrate;
(b) a carrier particle comprising a fluorescently labeled polynucleotide strand bound to the carrier particle, the fluorescently labeled polynucleotide strand comprising:
(i) a proximal end attached to the carrier particle; and
(ii) an exonuclease-cleavable distal end; and
(iii) at least one fluorescently labeled nucleotide comprising a fluorescent label;
providing a carrier particle located on the cis side of the substrate but not passing through the through-hole, such that the fluorescently labeled polynucleotide strand protrudes through the through-hole, such that the distal end of the fluorescently labeled polynucleotide strand is within the reaction volume;
reacting the fluorescently labeled polynucleotide strand with an exonuclease such that mononucleotides are continuously released from the distal end of the strand and diffuse out of the reaction volume through the distal opening;
illuminating the trans side of the substrate with excitation light during the reaction to create a fluorescent excitation zone adjacent the distal opening of the reaction well such that fluorescently labeled mononucleotides within the excitation zone emit a fluorescent signal;
Detecting a fluorescent signal as a function of time.
Including,
A method whereby the nucleotide sequence is determined from the time sequence of fluorescent signals detected from the released fluorescently labeled mononucleotides.
(Item 2)
13. The method of any one of the preceding items, wherein the distal opening of the reaction well has a minimum diameter of at least 30 nm.
(Item 3)
13. The method of any one of the preceding items, wherein the distal opening of the reaction well has a minimum diameter of 50 to 150 nm.
(Item 4)
8. The method of any one of the preceding items, wherein the one or more walls of the reaction well are not tapered.
(Item 5)
13. The method of any one of the preceding items, wherein the one or more walls of the reaction well are substantially cylindrical.
(Item 6)
13. The method of any one of the preceding items, wherein the opaque metal layer comprises gold or aluminum.
(Item 7)
13. The method of any one of the preceding items, wherein the opaque metal layer has a thickness of 100 nm to 600 nm.
(Item 8)
13. The method of any one of the preceding items, wherein the reaction wells have a well depth of at least 200 nm.
(Item 9)
13. The method of any one of the preceding items, wherein the reaction wells have a well depth of 200 nm to 1000 nm.
(Item 10)
2. The method of any one of the preceding items, wherein the fluorescently labeled polynucleotide strand in the reaction volume comprises a fluorescently labeled polynucleotide segment containing at least 100 contiguous nucleotides.
(Item 11)
2. The method of claim 1, wherein the through-holes have a minimum diameter of at least 2 nm.
(Item 12)
2. The method of claim 1, wherein the through-holes have a minimum diameter of 2 nm to 50 nm.
(Item 13)
13. The method of any one of the preceding items, wherein the substrate comprises a thin film layer comprising the proximal through-hole and having a thickness of 20 nm to 50 nm.
(Item 14)
Item 14. The method of item 13, wherein the thin film layer comprises silicon nitride.
(Item 15)
13. The method of any one of the preceding items, wherein the excitation light has a wavelength of 380 nm or greater.
(Item 16)
13. The method of any one of the preceding items, wherein the solid-state substrate comprises a surface portion(s) defining the reaction volume, the surface portion(s) comprising at least one surface passivation coating.
(Item 17)
13. The method of any one of the preceding items, wherein one or more sidewalls of the reaction well comprise at least one of a silicon oxide coating and an aluminum oxide coating.
(Item 18)
13. The method of any one of the preceding items, wherein the fluorescently labeled polynucleotide strand comprises at least two different types of nucleotides, each type labeled with a distinct fluorescent label.
(Item 19)
13. The method of any one of the preceding items, wherein during the reaction, the carrier particles are maintained adjacent the proximal through-holes by a voltage bias.
(Item 20)
20. The method of claim 19, wherein after the reaction, the voltage bias is stopped to move the carrier particles away from the proximal through-holes, so that the remaining fluorescently labeled polynucleotide strands are removed from the reaction volume, and then a voltage bias is applied to move the same or different carrier particles toward the proximal through-holes, so that new fluorescently labeled polynucleotide strands are delivered to the reaction well for reaction with an exonuclease.
(Item 21)
13. The method of any one of the preceding items, wherein the carrier particles are non-magnetic.
(Item 22)
13. The method of any one of the preceding items, wherein the carrier particles are magnetic.
(Item 23)
2. The method of any one of the preceding items, wherein the fluorescently labeled polynucleotide strands in the reaction volume comprise double-stranded nucleic acids.
(Item 24)
23. The method of any one of items 1 to 22, wherein the fluorescently labeled polynucleotide strands in the reaction volume comprise single-stranded nucleic acids.
(Item 25)
13. The method of any one of the preceding items, wherein the carrier particles comprise a plurality of fluorescently labeled polynucleotide strands.
(Item 26)
13. The method of any one of the preceding items, wherein the carrier particles comprise a plurality of fluorescently labeled polynucleotide strands having different polynucleotide sequences from each other.
(Item 27)
2. The method of any one of the preceding items, wherein the solid-state substrate comprises a plurality of reaction wells.
(Item 28)
28. The method of claim 27, wherein the plurality of reaction wells are configured as a one- or two-dimensional array.
(Item 29)
29. The method of claim 27 or 28, wherein two or more of the plurality of reaction wells each contain a fluorescently labeled polynucleotide strand to be sequenced.

The depth of the reaction well is usually selected to be longer than the length of the segment of the fluorescently labeled polynucleotide strand that is cut by the exonuclease to generate sequence information. Double-stranded DNA is a relatively rigid rod-like structure with a unit length of about one base pair per 3.6 angstroms (0.36 nm). Thus, a dsDNA segment containing 1000 consecutive base pairs has a length of about 360 nm. Thus, a reaction well depth of 400 nm may be suitable for encapsulating an immobilized 1000 bp dsDNA without exposing the distal end of the dsDNA to undesired excitation light, and a well depth of 500 nm may provide even better protection from undesired excitation light. Alternatively, in the case of single-stranded DNA or RNA strands, since single-stranded nucleic acids are less rigid and have a smaller diameter than double-stranded nucleic acids, a reaction well depth of 400 nm may encapsulate an immobilized fluorescently labeled strand with more than 1000 consecutive bases without exposing the distal end of the ssDNA to undesired excitation light.

Substrates containing reaction wells for use in the present invention can be manufactured by any suitable _method in various forms of solid materials, including, but not limited to, silicon ( _e.g. , _Si3N4 , _SiO2 ), metals, metal oxides (e.g., _Al2O3 ), plastics, glass, semiconductor materials, and combinations thereof. Manufacturing techniques for making solid-state substrates can be found in the following exemplary references, which are incorporated by reference: Golovchenko et al., U.S. Pat. No. 6,464,842; Sauer et al., U.S. Pat. No. 7,001,792; Su et al., U.S. Pat. No. 7,744,816; Meller et al., International Patent Publication WO 2009/020682; Yan et al., Nano Letters, 5(6):1129-1134 (2005); Wanunu et al., Nano Letters, 7(6):1580-1585 (2007); Dekker, Nature Nanotechnology, 2:209-215 (2007); Storm et al., Nature
Materials, 2:537-540 (2003); Zhe et al., J. Micromech. Microeng., 17:304-313 (2007); and the like.

Impingement of excitation light on the trans side of substrate 202 , and in particular on the distal openings of one or more reaction wells of substrate 202 , generates a fluorescent excitation zone (see Figures 1B-1D) adjacent the distal openings of the reaction wells.

Each emitted fluorescently labeled mononucleotide can be identified by features of the measured fluorescent signal, such as (1) the specific emission wavelength or peak shape of the fluorescence of the fluorescent label associated with each different type of nucleotide, (2) the signal intensity, which can be measured as the sum of multiple photons from the same mononucleotide while passing through the excitation zone of the reaction well, and (3) the absence of fluorescent signal contributions from any other emitted mononucleotide (e.g., as A, C, G, or T). For example, a fluorescently labeled mononucleotide that diffuses from the excitation zone of a first reaction well to the excitation zone of a second reaction well can be excluded from the fluorescent signal detected for the second well based on the trajectory of the fluorescently labeled mononucleotide's movement toward the second well. Similarly, a fluorescently labeled mononucleotide that diffuses from the excitation zone of a first reaction well and then back to the excitation zone can be excluded from the fluorescent signal detected for the first well based on the trajectory of the fluorescently labeled mononucleotide's movement back toward the first well.

Example 1
Conjugation of Nucleic Acids to Carrier Particles A. Selection and Design of DNA for Attachment to Carrier Particles For direct conjugation of DNA to gold or silver nanoparticles or surfaces, single or multiple (2-6) thiol groups are attached to either the 3' or 5' end of the oligonucleotide (5'-GCTAGTGGCGCGGTATTAT-3') (SEQ ID NO:3). Single thiols are obtained from commercially available precursors, 3'- or 5'-disulfide (CH ₂ ) _n -S-S-(CH ₂ ) _n -OH (n=3 or 6) modified oligonucleotides (IDT, Iowa). Two thiol groups are introduced via conjugation of commercially available 3'- or 5'-amino functionalized oligonucleotides (IDT, Iowa or TriLink) with (±)-α-lipoic acid. Alternatively, multiple thiols are introduced with one, two or three consecutive DTPA phosphoramidites at either the 3' or 5' end of the oligonucleotide (IDT, Iowa). To conjugate DNA to functionalized nanoparticles, suitable complementary conjugation groups (amine, thiol, DBCO, BCN) are attached to either the 3' or 5' end of the oligonucleotide. Linkers of various lengths are introduced between the conjugation moiety and the oligonucleotide to facilitate enzyme access to the oligonucleotide sequence (e.g., access to the polymerase when a fluorescently labeled polynucleotide strand complementary to a sample nucleic acid strand is synthesized by DNA or RNA polymerase using a template immobilized on a carrier particle). Examples of linkers are thymidine monophosphate xn (n=1-40), PEG3, PEG4, PEG5 and (PEG6-P(O)(OH)O-) xn (n=1-12), where PEG means polyethylene glycol and PEGN, with N=3, 4, 5 and 6, means a polymer of N ethylene glycol units.

Substrate preparation. A 300 μm thick, 100 mm double-sided polished silicon wafer (e.g., from Virginia Semiconductor, Fredericksburg, VA, or Rogue Valley, VA) is prepared with a 30 nm SiN layer deposited by low pressure chemical vapor deposition (LPCVD) on each side of the wafer. A negative e-beam resist is spun on each side, and then the resist on one side of the wafer (the "front" side) is exposed in an electron beam lithography (EBL) machine to pattern reaction wells on the SiN layer on that side. The resist is then developed and the unexposed resist is removed. A 5 nm adhesion layer of chromium or titanium is deposited on the front side of the wafer by e-beam evaporation, followed by e-beam deposition of a selected thickness of an opaque metal layer (e.g., 200 nm Au or Al) onto the front side of the wafer. The wafer is then placed in a solution that removes the remaining exposed resist from the front side, known as "lift-off," leaving reaction wells in the metal membrane layer with diameters of 40-120 nm and well depths of 100-250 nm , or other dimensions depending on the user's preference.

Blockage of the proximal through-holes by the carrier particles was monitored by measuring the cis-trans current from an onset (opening) current of about 1000 nAmp to a current of less than about 200 nAmp when the rate of change of the current reached a steady state. Blockage of all 16 wells was complete in about 30 seconds. The diameter of the carrier particles was larger than the diameter of the through-holes , preventing the carrier particles from passing through the through-holes from the cis side to the trans side of the substrate. In addition, the carrier particles were sparsely loaded with fluorescently labeled polynucleotide strands, so that each reaction well was loaded with no more than one fluorescently labeled polynucleotide strand.