Systems and methods for writing, reading, and controlling data stored in a polymer

• US 10,640,822 B2
• Filed: 05/02/2018
• Issued: 05/05/2020
• Est. Priority Date: 02/29/2016
• Status: Active Grant

• Current Assignee: Iridia, Inc.
• Original Assignee: Iridia, Inc.
• Inventors: Predki, Paul F., Cassidy, Maja
• Primary Examiner(s): Bashar, Mohammed A
• Application Number:
  US15/969,745
• Publication Number:
  US 20190136307A1
• Time in Patent Office: 734 Days
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 33/48721 Investigating individual macromolecules, e.g. by translocation through nanopores Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68
• CPC Class Codes: H01P 7/00 Resonators of the waveguide type
• CPC Class Codes: H03H 7/06 including resistors H03H7/075, H03H7/09, H03H7/12, H03H7/13 take precedence

Abstract

The disclosure provides a novel system of storing information using a charged polymer, e.g., DNA, the monomers of which correspond to a machine-readable code, e.g., a binary code, and which can be synthesized and/or read using a novel nanochip device comprising nanopores; novel methods and devices for synthesizing oligonucleotides in a nanochip format; novel methods for synthesizing DNA in the 3′ to 5′ direction using topoisomerase; novel methods and devices for reading the sequence of a charged polymer, e.g., DNA, by measuring capacitive or impedance variance, e.g., via a change in a resonant frequency response, as the polymer passes through the nanopore; and further provides compounds, compositions, methods and devices useful therein.

Claims

1. A method for synthesizing a charged polymer comprising at least two distinct monomers or oligomers in a nanopore-based device, the nanopore-based device comprising

one or more addition chambers or channels containing buffer solution and reagents for addition of one or more monomers or oligomers to the charged polymer in blocked form, such that only a single monomer or oligomer can be added in one reaction cycle;

and

one or more deblocking chambers or channels containing buffer solution and reagents for removing the blocker group from the charged polymer,

wherein the addition chambers or channels are separated from the deblocking chambers or channels by one or more membranes comprising one or more nanopores, and

wherein the charged polymer can pass through a nanopore and at least one of the reagents for addition of one or more monomers or oligomers cannot,

the method comprising

a) moving the first end of a charged polymer having a first end and a second end, by electrical attraction, into an addition chamber or channel, whereby monomers or oligomers are added to said first end in blocked form,

b) moving the first end of the charged polymer with the added monomer or oligomer in blocked form into a deblocking chamber or channel, whereby the blocking group is removed from the added monomer or oligomer, and

c) repeating steps a) and b), wherein the monomers or oligomers added in step a) are the same or different, until the desired polymer sequence is obtained, and

d) detecting the sequence of the polymer as it passes through the nanopore to confirm that the desired sequence has been synthesized.

2. The method of claim 1, wherein the charged polymer is DNA.

3. The method of claim 1, wherein the second end of the polymer is bound to a surface.

4. The method of claim 1, wherein the device comprises one or more first addition chambers or channels containing reagents suitable for adding a first type of monomer or oligomer and one or more second addition chambers containing reagents suitable for adding a second type of monomer or oligomer, and wherein in step a), the first end of the charged polymer is moved into either the first addition chamber or the second addition chamber, depending on whether it is desired to add a first type of monomer or oligomer or a second type of monomer or oligomer.

5. The method of claim 2, wherein the reagents for addition of one or more monomers or oligomers to the charged polymer comprise reagents selected from a topoisomerase, a DNA polymerase, or combinations thereof.

6. The method of claim 2 wherein the step of detecting the sequence of the polymer as it passes through a nanopore to confirm that the desired sequence has been synthesized is performed by measuring the capacitive variance in a resonant RF circuit as the DNA is drawn through the nanopore.

7. The method of claim 1, wherein step of detecting the sequence of the polymer as it passes through the nanopore to confirm that the desired sequence has been synthesized comprises:

i) providing an LC resonator having an effective impedance;

ii) providing a cell, the cell having at least the addition chamber, the deblocking chamber, and the nanopore disposed therebetween, the effective impedance being affected by the polymer passing through the nanopore, the resonator having an AC output voltage resonant frequency response at a probe frequency, which is based on the effective impedance, in response to an AC input voltage at the probe frequency;

iii) providing the AC input voltage having at least the probe frequency; and

iv) monitoring the AC output voltage at least at the probe frequency, the AC output voltage at the probe frequency being indicative of the data stored in the polymer at the time of monitoring.

8. The method of claim 7 wherein the polymer comprises at least two types of monomers or oligomers having different properties causing different resonant frequency responses.

9. The method of claim 8 wherein the at least two types of monomers or oligomers comprises at least a first monomer or oligomer having a first property that causes a first resonant frequency response when the first monomer or oligomer is in the nanopore, and a second monomer or oligomer having a second property that causes a second resonant frequency response when the second monomer or oligomer is in the nanopore.

10. The method of claim 9 wherein a characteristic of the first frequency response at the probe frequency is different from the same characteristic of the second frequency response at the probe frequency.

11. The method of claim 10 wherein the characteristic of the first and second frequency responses comprises at least one of magnitude and phase response.

12. The method of claim 9 wherein the first property and the second property of the monomers comprises a dielectric property.

13. The method of claim 7 wherein the cell comprises at least a top electrode and a bottom electrode, the nanopore being disposed between the electrodes, and the cell having the buffer solution and reagents therein, and wherein the electrodes, the nanopore, and the buffer solution and reagents have an effective cell capacitance that changes when the polymer passes through the nanopore.

14. The method of claim 13 wherein the effective impedance comprises an inductor connected in series with the effective capacitance to create the resonator, a combination of the inductor and effective capacitance being related to the resonant frequency response.

15. The method of claim 13 wherein the polymer is moved through the nanopore via a DC steering voltage applied to the electrodes.

16. The method of claim 13 wherein the cell has at least three chambers, comprising two of the addition chambers and the deblocking chamber, at least two nanopores, and at least three electrodes for moving the polymer through the nanopores between the addition chambers and the deblocking chamber.

17. The method of claim 8 wherein at least part of the sequence of the at least two types of monomers or oligomers of the polymer stores data in the form of a computer-readable code.

18. The method of claim 7 wherein the polymer comprises DNA, and wherein the DNA comprises at least two types of nucleotides, each type of nucleotide providing a unique frequency response at the probe frequency.

19. The method of claim 7 wherein the probe frequency is about 1 MHz to 100 GHz.

20. The method of claim 8 wherein the at least two types of monomers or oligomers have a dielectric property that affects the frequency response of the resonator to produce at least two different frequency responses at the probe frequency.

21. The method of claim 7 wherein the resonator comprises at least one of a longitudinal resonator and a transverse resonator.

22. The method of claim 21 wherein the transverse resonator comprises a split-ring resonator having the nanopore disposed in a gap of the split-ring resonator.

23. The method of claim 7 wherein the nanopore comprises a nano-channel having dimensions that allow for substantially linear flow of the polymer without folding on itself.

24. The method of claim 7 further comprising a plurality of the resonators in the cell, each resonator driven by a common AC input voltage and monitored from a common AC output voltage.

25. The method of claim 24 wherein the plurality of resonators in the cell comprises at least one longitudinal resonator and at least one transverse resonator.

26. The method of claim 25 wherein the frequency response of the longitudinal resonator and of the transverse resonator are monitored simultaneously.

27. The method of claim 7 further comprising a plurality of cells, each cell having the resonator tuned to a different resonant frequency band, the cells being driven by a common AC input voltage and measured by a common AC output voltage.