Controlling fluorescent signal composition in optically-based nanopore sequencing
The invention is directed to optically based methods for nanopore sequencing of polynucleotides which comprise steps of (i) translocating a polynucleotide through a bore of a nanopore at a translocation speed, wherein nucleotides of the polynucleotide are labeled with fluorescent labels such that in free solution fluorescent labels of nucleotides are substantially quenched and wherein fluorescent labels within the bore are constrained such that substantially no detectable fluorescent signal is generated therein; (ii) exciting the fluorescent label of each nucleotide upon exiting the nanopore and prior to quenching with a preceding mutually quenching fluorescent label or a quenching agent; (iii) measuring a fluorescent signal generated by fluorescent labels exiting the nanopore, wherein the translocation speed is selected so that the measured fluorescent signal comprises fluorescence from substantially a single fluorescent label; and (iv) determining a nucleotide sequence of the polynucleotide from a sequence of measured fluorescent signals.
1. A method for determining a nucleotide sequence of a polynucleotide comprising the steps of:
translocating a polynucleotide through a bore of a nanopore at a translocation speed, wherein nucleotides of the polynucleotide are labeled with fluorescent labels such that, when exposed to fluorescence excitation light, fluorescent labels of nucleotides that are in free solution are substantially quenched, and fluorescent labels of nucleotides within the bore are constrained such that substantially no detectable fluorescent signal is generated by the fluorescent labels therein;
exciting the fluorescent label of each nucleotide upon exiting the nanopore and prior to being quenched by a preceding mutually quenching fluorescent label of the polynucleotide or by a quenching agent in the solution;
measuring a fluorescent signal generated by fluorescent labels of the nucleotides exiting the nanopore, wherein the translocation speed is selected so that a fluorescent label of an exiting nucleotide is quenched before it travels more than an inter-nucleotide distance from the nanopore; and
determining a nucleotide sequence of the polynucleotide from a sequence of measured fluorescent signals.
2. The method of claim 1 wherein said step of measuring includes selecting said translocation speed based on the relative values of fluorescent intensities distributed among a plurality of optical detection channels.
3. The method of claim 2 wherein said plurality of optical detection channels includes at least one channel for an emission maximum of each fluorescent label.
4. A method of determining a nucleotide sequences of polynucleotides, the method comprising the steps of:
(a) translocating single stranded polynucleotides through nanopores at a translocation speed, wherein nucleotides of the single stranded polynucleotides are labeled with fluorescent labels such that, when exposed to fluorescence excitation light, fluorescent labels of adjacent nucleotides are in a mutually quenched state in free solution and wherein the nanopores force the fluorescent labels within the nanopore into a constrained state wherein substantially no detectable signal is generated;
(b) exciting the fluorescent labels of nucleotides upon exiting the nanopores and prior to being quenched by the label of an adjacent nucleotide;
(c) measuring fluorescent signals generated by the fluorescent labels exiting the nanopores and determining from the measured fluorescent signals whether a single fluorescent label contributes to the measured fluorescent signal or a plurality of fluorescent labels contributes to the measured fluorescent signal at each nanopore;
(d) reducing the translocation speed by a predetermined value whenever a measured fluorescent signal at a nanopore is generated by a plurality of fluorescent labels;
(e) repeating steps (a) through (d) until the translocation speed corresponds to substantially every measured fluorescent signal at each nanopore comprising fluorescence from substantially a single fluorescent label; and
(f) identifying nucleotides of the translocating polynucleotides from measured fluorescent signals.
5. The method of claim 4 wherein said step of reducing comprises lowering a voltage potential across said nanopores.
6. The method of claim 4 wherein nucleotides of said polynucleotides are labeled with second members of a FRET pair, each second member producing a FRET signal indicative of the nucleotide to which it is attached, and wherein nucleotides of said polynucleotides pass in sequence by a first member of the FRET pair positioned adjacent to said nanopore so that each second member upon exiting said nanopore passes within a FRET distance of the first member of the FRET pair; and
wherein said step of exciting includes exposing the first member to excitation energy of a first wavelength so that FRET occurs between the first and second members of the FRET pair within the FRET distance to generate a FRET signal of a second wavelength indicative of the nucleotide exiting said nanopore.
7. The method of claim 6 wherein said nanopore is disposed in a solid phase membrane and wherein said first member of said FRET pair is attached to the solid phase membrane adjacent to said nanopore.
8. The method of claim 6 wherein said nanopore is a protein nanopore and wherein said first member of said FRET pair is attached to the protein nanopore.
9. The method of claim 6 wherein said first member of said FRET pair is a donor and said second member of said FRET pair is an acceptor.
10. The method of claim 9 wherein said donor is a quantum dot.
11. The method of claim 9 wherein said acceptor is a fluorescent organic dye.