Method and system of nanopore-based information encoding
This invention provides methods and systems of DNA synthesis including providing an encoding unit comprising an enzyme, a single-stranded DNA (ssDNA) and a nanopore, providing a lipid bilayer having on opposite sides a cis and a trans reservoir each having a different buffer composition, wherein the nanopore is within the lipid bilayer and the enzyme and the ssDNA are in the cis reservoir, providing an electrode over the lipid bilayer wherein the electrode can modulate voltage across the lipid bilayer, wherein the enzyme catalyzes DNA synthesis in response to the voltage.
1. A method of encoding information through synthesis of a DNA strand comprising
providing an encoding unit comprising a DNA polymerase, a single-stranded DNA (ssDNA) and a nanopore, wherein the DNA polymerase is conjugated to the nanopore,
providing a lipid bilayer having on opposite sides a cis and a trans reservoir each having a different buffer composition, wherein the nanopore is within the lipid bilayer and the DNA polymerase and the ssDNA are in the cis reservoir,
introducing into the cis reservoir a specific nucleotide corresponding to an encoding scheme for information storage in synthesized DNA, and
applying a voltage across the lipid bilayer via an electrode wherein the electrode can modulate the voltage across the lipid bilayer,
wherein catalytic ions Mg 2+ or Co 2+ are injected to the cis side from the trans side, thereby activating the DNA polymerase to catalyze addition of the specific nucleotide to the single stranded DNA during DNA synthesis, and
repeating catalytic addition to create synthesized DNA wherein the information is encoded and stored in the synthesized DNA.
2. The method of claim 1 , wherein the DNA polymerase is a template dependent or independent DNA polymerase.
3. The method of claim 2 , wherein the DNA polymerase is a ϕ29 DNA polymerase or a terminal deoxynucleotidyl transferase.
4. The method of claim 1 , wherein the ssDNA is immobilized to the nanopore.
5. The method of claim 1 , wherein the ssDNA is a template or an initiator for DNA synthesis.
6. The method of claim 1 , wherein the nanopore is a protein membrane channel.
7. The method of claim 6 , wherein the protein membrane channel comprises αHL, MspA, or ϕ29 connector.
8. The method of claim 1 , wherein the cis reservoir contains non-catalytic buffer comprising non-catalytic ion Ca 2+ that prevents base addition during DNA synthesis, and wherein the trans reservoir contains catalytic buffer comprising catalytic ion Mg 2+ or Co 2+ that promotes base addition during DNA synthesis.
9. The method of claim 1 , further comprising providing nucleotide bases to the cis reservoir and turning on the voltage such that catalytic ions Mg 2+ or Co 2+ are injected to the cis side from the trans side, thereby activating the enzyme that catalyzes the addition of the nucleotide bases during DNA synthesis.
10. The method of claim 1 , wherein the nucleotide base can be added as a single base or a homopolymer run during DNA synthesis.
11. The method of claim 1 , further comprising turning off the voltage, flushing the cis reservoir with fresh non-catalytic buffer and removing any nucleotide bases and catalytic ions.
12. The method of claim 1 , wherein the method is capable of single molecule measurement.
13. The method of claim 1 , wherein the method records voltage polarity applied across the lipid bilayer over time by encoding this information into a complement strand of DNA during DNA synthesis.
14. The method of claim 13 , wherein the information is stored in the complement strand in the form of regions of bases either containing or lacking cytosine bases at the sites which complement the inosine bases of the template strand, which translates to the “1” or “0” state of a single bit of information.
15. The method of claim 1 , wherein the ssDNA is immobilized to the nanopore within the cis reservoir.
16. The method of claim 1 , wherein the 5′ end of the ssDNA is immobilized to the nanopore.
17. The method of claim 1 , wherein the 5′ end of the ssDNA is immobilized to the nanopore within the cis reservoir.
18. A method of DNA synthesis comprising applying a voltage across a lipid bilayer having one or more encoding units associated therewith, wherein each encoding unit comprises a DNA polymerase an enzyme, a single-stranded DNA (ssDNA) and a nanopore, wherein the lipid bilayer has on opposite sides a cis and a trans reservoir each having a different buffer composition, wherein the trans reservoir comprises catalytic ions, wherein the nanopore is within the lipid bilayer and the DNA polymerase enzyme and the ssDNA are conjugated to the nanopore and are in the cis reservoir, and wherein the catalytic ions move from the trans reservoir to the cis reservoir in response to the voltage and the DNA polymerase enzyme is activated and catalyzes DNA synthesis in the presence of a nucleotide response to the voltage.
19. The method of claim 18 , wherein the cis reservoir further comprises nucleotide bases.
20. The method of claim 18 , wherein the voltage is applied via an electrode that modulates the voltage across the lipid bilayer.
21. The method of claim 18 , wherein the ssDNA is linear.
22. The method of claim 18 wherein the ssDNA is a circular single-stranded DNA.