U.S. patent application number 16/978027 was filed with the patent office on 2022-03-17 for high throughput nucleic acid profiling of single cells.
This patent application is currently assigned to WISCONSIN ALUMNI RESEARCH FOUNDATION. The applicant listed for this patent is WISCONSIN ALUMNI RESEARCH FOUNDATION. Invention is credited to Leland Bradford Hyman, Philip Anthony Romero.
Application Number | 20220081711 16/978027 |
Document ID | / |
Family ID | |
Filed Date | 2022-03-17 |
United States Patent
Application |
20220081711 |
Kind Code |
A9 |
Romero; Philip Anthony ; et
al. |
March 17, 2022 |
HIGH THROUGHPUT NUCLEIC ACID PROFILING OF SINGLE CELLS
Abstract
Methods of profiling the nucleic acid composition of single
cells and tools for same. The methods can include isolating a
single cell in a liquid droplet, lysing the single cell in the
liquid droplet to release template nucleic acid from the cell,
amplifying the template nucleic acid in the liquid droplet to
generate amplified nucleic acid, and detecting the amplified
nucleic acid in the liquid droplet. The methods can be useful for
profiling expression patterns and/or detecting genetic
characteristics such as single nucleotide polymorphisms. The tools
include nucleic acid logic gates, including polymerase-dependent
logic gates. The logic gates can perform logical operations such as
YES, NOT, AND, OR, AND-NOT, NOT-AND, NOT-OR, EXCLUSIVE-OR,
EXCLUSIVE-NOR, and IMPLY. The tools also include microfluidic
systems for performing the methods.
Inventors: |
Romero; Philip Anthony;
(Madison, WI) ; Hyman; Leland Bradford; (Madison,
WI) |
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Applicant: |
Name |
City |
State |
Country |
Type |
WISCONSIN ALUMNI RESEARCH FOUNDATION |
Madison |
WI |
US |
|
|
Assignee: |
WISCONSIN ALUMNI RESEARCH
FOUNDATION
Madison
WI
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20210363573 A1 |
November 25, 2021 |
|
|
Appl. No.: |
16/978027 |
Filed: |
March 6, 2019 |
PCT Filed: |
March 6, 2019 |
PCT NO: |
PCT/US2019/020926 PCKC 00 |
371 Date: |
September 3, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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62639822 |
Mar 7, 2018 |
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International
Class: |
C12Q 1/6844 20060101
C12Q001/6844 |
Claims
1. A method of profiling a nucleic acid composition of a single
cell comprising: isolating the single cell in a liquid droplet;
lysing the single cell in the liquid droplet to release template
nucleic acid from the cell; amplifying the template nucleic acid in
the liquid droplet to generate amplified nucleic acid; and
detecting the amplified nucleic acid in the liquid droplet.
2. The method of claim 1, wherein the isolating comprises isolating
the cell in an aqueous liquid droplet suspended in a
water-immiscible medium.
3. The method of claim 1, wherein the isolating comprises isolating
the cell in the liquid droplet with a lysis reagent, a DNA
polymerase, amplification primers, deoxynucleotide triphosphates,
an RNAse inhibitor, a nucleic acid logic gate, and a reporter.
4-20. (canceled)
21. The method of claim 1, wherein the detecting comprises
profiling the amplified nucleic acid by performing a molecular
computation with a polymerase-dependent nucleic acid logic
gate.
22. The method of claim 21, wherein the polymerase-dependent logic
gate comprises an output strand annealed to a gate strand, the
logic gate being configured to release the output strand from the
gate strand in the presence of an input strand and a DNA
polymerase, wherein: the output strand anneals to an output-strand
annealing portion of the gate strand and does not anneal to an
output-strand non-annealing portion of the gate strand, wherein the
output-strand annealing portion is closer to a 5' end of the gate
strand than the output-strand non-annealing portion; the input
strand anneals to an input-strand annealing portion of the gate
strand and does not anneal to an input-strand non-annealing portion
of the gate strand, wherein the input-strand annealing portion is
closer to a 3' end of the gate strand than the input-strand
non-annealing portion; and annealing of the input strand to the
gate strand and polymerase-mediated extension of the input strand
along the gate strand to form an extended input strand are together
necessary and sufficient for release of the output strand from the
gate strand.
23. The method of claim 22, wherein the output-strand annealing
portion and the input-strand non-annealing portion at least
partially overlap, and wherein the input-strand annealing portion
and the output-strand non-annealing portion at least partially
overlap.
24-31. (canceled)
32. The method of claim 22, wherein: the output strand comprises a
reporter-gate annealing portion that anneals to either a reporter
strand or a quencher strand of a reporter gate.
33. The method of claim 32, wherein: the output strand further
comprises a second-input-strand annealing portion that anneals to a
second input strand, wherein the second-input-strand annealing
portion is closer to a 3' end of the output strand than the
reporter-gate annealing portion.
34. The method of claim 22, wherein the logic gate further
comprises a threshold strand configured to anneal to the output
strand.
35-36. (canceled)
37. The method of claim 22, wherein the input strand is comprised
by the amplified nucleic acid.
38. The method of claim 22, wherein the input strand is an output
strand from a second logic gate configured to detect a second input
strand.
39. The method of claim 22, wherein the output strand is an input
strand for a second logic gate.
40. The method of claim 22, wherein the output strand is an input
strand for a reporter gate.
41-42. (canceled)
43. The method of claim 21, wherein a substantially constant
temperature is maintained throughout and between each of the
lysing, the amplifying, and the profiling the amplified nucleic
acid.
44-45. (canceled)
46. The method of claim 1, wherein the detecting comprises
detecting a single nucleotide polymorphism in the nucleic acid
composition of the single cell.
47. The method of claim 1, wherein the isolating, the lysing, the
amplifying, and the detecting all occur in a single, continuous
network of channels.
48. The method of claim 1, wherein the isolating occurs prior to
the lysing, the lysing occurs prior to the amplifying, and the
amplifying occurs prior to the detecting.
49. (canceled)
50. The method of claim 1, wherein the lysing, the amplifying, and
the detecting all occur without diluting the liquid droplet, adding
additional reagents to the liquid droplet, and/or removing reagents
or liquid from the liquid droplet after the isolating.
51. A polymerase-dependent logic gate comprising an output strand
annealed to a gate strand, the logic gate being configured to
release the output strand from the gate strand in the presence of
an input strand and a DNA polymerase, wherein: the output strand
anneals to an output-strand annealing portion of the gate strand
and does not anneal to an output-strand non-annealing portion of
the gate strand, wherein the output-strand annealing portion is
closer to a 5' end of the gate strand than the output-strand
non-annealing portion; the input strand anneals to an input-strand
annealing portion of the gate strand and does not anneal to an
input-strand non-annealing portion of the gate strand, wherein the
input-strand annealing portion is closer to a 3' end of the gate
strand than the input-strand non-annealing portion; and annealing
of the input strand to the gate strand and polymerase-mediated
extension of the input strand along the gate strand to form an
extended input strand are together necessary and sufficient for
release of the output strand from the gate strand.
52. A system for performing the method of claim 1.
Description
FIELD OF THE INVENTION
[0001] The invention is directed to high-throughput methods,
systems, and devices for profiling the nucleic acid composition of
single cells within a heterogeneous population of cells.
BACKGROUND
[0002] Cellular heterogeneity and its impact on biological function
and disease are becoming increasingly important for questions in
human immunology, stem cell biology, and cancer research. By
transcriptionally analyzing individual cells with
reverse-transcriptase polymerase chain reaction (RT-PCR), for
example, it is possible to identify rare cells or transient cell
states that are unobservable when studying the entire population in
bulk (Bendall et al. 2012, Kalisky and Blainey et al. 2011, Kalisky
and Quake 2011, Levsky et al. 2003). However, obtaining meaningful
information on these cells necessitates tools capable of
high-throughput analysis. Current methods for manipulating,
isolating, and transcriptionally profiling single cells with RT-PCR
are cumbersome and limited in throughput, enabling the examination
of just hundreds of individual cells.
[0003] The ultrahigh-throughput capability of droplet-based
microfluidics is ideal for single-cell analysis applications (Guo
et al. 2012, Novak et al. 2011, Vyawahare et al. 2010). These
microfluidic techniques rely on microdroplets, tiny spheres of
aqueous liquid ranging from 1 to 100 .mu.m in diameter, to
encapsulate biological components in an oil-based emulsion (The et
al. 2008). The drops serve, essentially, as very tiny "test tubes,"
compartmentalizing millions of reactions. A major advantage of this
approach is that a minimal amount of reagent is used, greatly
reducing the cost for a given experiment. In addition, with
microfluidic techniques, the drops can be formed, split, injected
with reagent, and sorted at kilohertz rates, holding potential for
performing millions of single-cell reactions at unprecedented
throughput. However, an obstacle to realizing the potential of this
approach is that, at concentrations of a single cell in a
microdroplet, cell lysate is a potent inhibitor of RT-PCR (Arezi et
al. 2010, Hedman et al. 2013, White et al. 2011).
[0004] To avoid cell lysate inhibition of RT-PCR, previous
drop-based methods have utilized large droplets (2 nL) in which the
lysate concentration is no longer inhibitory, or agarose droplets
that can be solidified, rinsed, and stained with DNA dyes (Mary et
al. 2011, Zhang et al. 2012). Methods using large droplets have
only been able to analyze .about.100 cells in total, while the
agarose method is unable to use TaqMan probes or cell staining,
precluding correlation of specific cell types with associated
transcriptional targets. An alternative strategy for performing
single-cell RT-PCR on cells is to isolate the cells in microwells
fabricated into an elastomeric device. This approach allows robust
and specific single-cell transcriptional profiling. However,
because each microwell and its control valves must be fabricated
and individually controlled, throughput is also limited to just a
few hundred cells in total (Kalisky and Blainey et al. 2011,
Kalisky and Quake 2011, White et al. 2013). Yet other strategies
for performing single-cell RT-PCR on single cells is lyse cells in
microdroplets and then dilute the microdroplets prior to RT-PCR
(Eastburn et al. 2013). While this dilution approach is effective,
it is a highly cumbersome process that limits processivity.
[0005] To enable expression analysis of large numbers of cells in a
heterogeneous population, new methods, systems, and devices are
needed that combine the throughput of droplet-based microfluidic
techniques with the specificity of microwell reactions. Aspects of
the invention provided herein address these needs.
SUMMARY OF THE INVENTION
[0006] One aspect of the invention is directed to methods of
profiling a nucleic acid composition of a single cell. The methods
comprise isolating the single cell in a liquid droplet, lysing the
single cell in the liquid droplet to release template nucleic acid
from the cell, amplifying the template nucleic acid in the liquid
droplet to generate amplified nucleic acid, and detecting the
amplified nucleic acid in the liquid droplet.
[0007] Another aspect of the invention is directed to
polymerase-dependent logic gates. The polymerase-dependent logic
gates can be used in the profiling methods of the invention.
[0008] Another aspect of the invention is directed to microfluidic
systems for performing the profiling methods of the invention.
[0009] The objects and advantages of the invention will appear more
fully from the following detailed description of the preferred
embodiment of the invention made in conjunction with the
accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 shows a schematic of an exemplary version of a
process of the invention.
[0011] FIG. 2 shows a schematic of an exemplary branch
migration-mediated strand displacement reporter. The half
arrowheads indicate 3' ends of depicted nucleic acid strands. In
FIGS. 2-4, nucleic acid portions designated with a given number
(e.g., "1") are substantially identical to nucleic acid portions
designated with the same number and are substantially complementary
to nucleic acid portions designated with the same number and an
asterisk (e.g., "1*").
[0012] FIG. 3 shows an exemplary branch migration-mediated strand
displacement logic gate employing a YES logical operation. The half
arrowheads indicate 3' ends of depicted nucleic acid strands.
[0013] FIG. 4 shows a schematic of an exemplary
polymerase-dependent logic gate employing a YES logical operation.
The half arrowheads indicate 3' ends of depicted nucleic acid
strands.
[0014] FIG. 5 shows a schematic of an exemplary
polymerase-dependent logic gate employing an OR logical operation.
The half arrowheads indicate 3' ends of depicted nucleic acid
strands.
[0015] FIG. 6 shows a schematic of an exemplary
polymerase-dependent logic gate employing an AND logical operation.
The half arrowheads indicate 3' ends of depicted nucleic acid
strands.
[0016] FIGS. 7A-7D show a schematic of an alternative exemplary
polymerase-dependent logic gate employing an AND logical operation.
FIG. 7A shows components of the logic gate. FIG. 7B show operation
of the logic gate in the presence of both of two targeted inputs.
FIGS. 7C and 7D show operation of the logic gate in the presence of
only one of the two targeted inputs. The half arrowheads indicate
3' ends of depicted nucleic acid strands. The logic gate shown in
FIG. 7A can constitute an inclusive-OR logical operation if the
threshold strand is absent.
[0017] FIG. 8 shows a schematic of an exemplary
polymerase-dependent logic gate employing an AND-NOT logical
operation. The half arrowheads indicate 3' ends of depicted nucleic
acid strands.
[0018] FIGS. 9A-9D show a schematic of an alternative exemplary
polymerase-dependent logic gate employing an AND-NOT logical
operation. The half arrowheads indicate 3' ends of depicted nucleic
acid strands. FIG. 9A shows components of the logic gate. FIG. 9B
shows operation of the logic gate in the presence of both of two
targeted inputs, wherein the presence of input 2 negates the signal
generated from the presence of input 1. FIGS. 9C and 9D show
operation of the logic gate in the presence of only one of the two
targeted inputs. The half arrowheads indicate 3' ends of depicted
nucleic acid strands.
[0019] FIG. 10 shows a schematic of an exemplary
polymerase-dependent logic gate employing a NOT logical operation.
The half arrowheads indicate 3' ends of depicted nucleic acid
strands.
[0020] FIG. 11 shows a schematic of an exemplary
polymerase-dependent logic gate employing a YES logical operation
that uses the 3' end of a LAMP dumbbell product as an input. The
half arrowheads indicate 3' ends of depicted nucleic acid
strands.
[0021] FIG. 12 shows an exemplary device suitable for performing
the methods described herein.
[0022] FIG. 13 shows an exemplary device for sorting droplets, as
performed in the methods described herein.
[0023] FIGS. 14A and 14B show effects of lysate on catalytic
hairpin assembly (CHA) amplification. FIG. 14A shows high
background using CHA in the presence of lysate. FIG. 14B shows
concentration-dependent lysate inhibition. In FIG. 14A, error bars
denote +/-1 standard deviation of the mean.
[0024] FIGS. 15A and 15B show RT-LAMP amplification of mRNA for the
epithelial marker CK19 (KRT19) in CK19+ human breast cancer cells
(SK-BR-3) versus CK19-leukocytes (MOLT-4). FIG. 15A shows
amplification at a lysate concentration of 10.sup.6 cells/mL, which
is equivalent to the lysate concentration of a single cell in a
microdroplet.
[0025] FIG. 15B shows amplification above and below a lysate
concentration of 10.sup.6 cells/mL. Error bars denote +/-1 standard
deviation of the mean.
[0026] FIG. 16 shows fluorescent detection patterns of LAMP
products of the epithelial phenotype marker KRT19 and/or the
mesenchymal phenotype marker VIM using polymerase-dependent logic
gates employing a YES logical operation, a NOT logical operation,
an OR logical operation, an AND logical operation, or an AND-NOT
logical operation. The YES logic gate was structured as shown in
FIG. 4. The NOT logic gate was structured as shown in FIG. 10. The
OR logic gate was structured as shown in FIGS. 7A-7D, with the
threshold strand omitted. The AND logic gate was structured as
shown in FIGS. 7A-7D. The AND-NOT logic gate was structured as
shown in FIGS. 9A-9D.
[0027] FIGS. 17A and 17B show fluorescent detection of KRT19 RNA
(FIG. 17A) or VIM RNA (FIG. 17B) using RT-LAMP with a multiplex
primer format and YES nucleic acid logic gates structured as shown
in FIG. 4. Error bars denote +/-1 standard deviation of the
mean.
[0028] FIGS. 18A and 18B show fluorescent detection of KRT19 and/or
VIM RNA using RT-LAMP and an OR logic gate (FIG. 18A) or an AND
logic gate (FIG. 18B). The OR and logic gates were both structured
as shown in FIGS. 7A-7D except that OR logic gate lacked a
threshold strand. Error bars denote +/-1 standard deviation of the
mean.
[0029] FIGS. 19A and 19B show fluorescent detection of KRT19 and/or
VIM RNA using RT-LAMP and a NOT logic gate (FIG. 19A) or an AND-NOT
logic gate (FIG. 19B). The NOT gate was structured as shown in FIG.
10, and the AND-NOT gate was structured as shown in FIGS. 9A-9D.
Error bars denote +/-1 standard deviation of the mean.
[0030] FIGS. 20A and 20B show RT-LAMP amplification of mRNA for the
epithelial marker CK19 (KRT19) in CK19+ human breast cancer cells
(SK-BR-3) (FIG. 20A) versus CK19- leukocytes (MOLT-4) (FIG. 20B) in
aqueous droplets suspended in fluorinated oil, using a
dsDNA-specific dye to indicate amplification.
[0031] FIG. 21 shows a histogram of RT-LAMP amplification of the
Estrogen Receptor (ER) mRNA transcript ESR1 in ER+ human breast
cancer cells (MCF7) versus ER- breast cancer cells (SK-BR-3) in
aqueous droplets suspended in fluorinated oil, using a
dsDNA-specific dye to indicate amplification. Droplets were
collected and heated in batch prior to detection.
[0032] FIG. 22 shows a histogram of RT-LAMP amplification of a
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA transcript in
human leukocyte cell line MOLT-4 in aqueous droplets suspended in
fluorinated oil, using a dsDNA-dependent dye to indicate
amplification. Droplets were generated, incubated, and analyzed in
an integrated microfluidic device implementing the entire
workflow.
[0033] FIGS. 23A and 23B show histograms of RT-LAMP amplification
of the epithelial marker CK19 (KRT19) and the mesenchymal marker
VIM (VIM) using multiplexed and orthogonal YES logic gates and
reporter complexes, where KRT19 amplification activates the Alexa
Fluor 647 reporter, and VIM amplification activates the HEX
reporter. FIG. 23A shows histograms for Alexa Fluor 647 signal with
a human CK19- osteosarcoma cell line (U-2 OS) and a human CK19+
breast cancer cell line (SK-BR-3), after a 60-minute or 5-minute
incubation. FIG. 23B shows histograms for HEX signal with a human
VIM- breast cancer cell line (SK-BR-3) and a human VIM+
osteosarcoma cell line (U-2 OS), after a 60-minute or 5-minute
incubation. Each YES gate was structured as shown in FIG. 4, and
each reporter complex was structured as shown in FIG. 2.
[0034] FIGS. 24A and 24B show droplets collected from the waste
outlet (FIG. 24A) or sorted outlet (FIG. 24B) of a droplet sorting
device discriminating between human ER+ breast cancer line MCF7 and
ER- breast cancer line SK-BR-3 based on ESR1 (ER) LAMP
amplification.
[0035] FIG. 25 shows LAMP-based SNP detection in total RNA samples
from two cell lines: SK-BR-3, with only WT ACTB transcripts (SEQ ID
NO:1), and MOLT-4, which contains an A->C SNP at position 960 of
an ACTB transcript (SEQ ID NO:2). Error bars denote +/-1 standard
deviation of the mean.
DETAILED DESCRIPTION OF THE INVENTION
[0036] One aspect of the invention is directed to a method of
profiling the nucleic acid composition of a single cell. The method
may comprise isolating the single cell in a liquid droplet, lysing
the single cell in the liquid droplet to release template nucleic
acid from the cell, amplifying the template nucleic acid in the
liquid droplet to generate amplified nucleic acid, and detecting
the amplified nucleic acid in the liquid droplet.
[0037] A schematic of an exemplary version of this process is shown
in FIG. 1. The exemplary process is for profiling nucleated blood
cells for the presence of circulating tumor cells (CTCs). The
isolating in FIG. 1 is depicted as "encapsulation of single cells,"
the lysing is depicted as "cell lysis," the amplifying is depicted
as "amplification," and the detecting is depicted as "DNA-based
logical computation" and "High-throughput fluorescence detection."
As shown in FIG. 1, the isolating, the lysing, the amplifying, and
the detecting can all occur in a single network of channels in a
continuous, high-throughput manner. This is contrasted with
processes that conduct certain steps in batch. The channels can be
microfluidic channels generated using any suitable method. An
exemplary method is provided in the following examples. As used
herein, "channels" refers to passages embedded in a solid device as
well as exposed tubing.
[0038] The isolating may comprise isolating the cell in an aqueous
liquid droplet suspended in a water-immiscible medium. This can be
performed by feeding the cells through microfluidic channels in a
continuous aqueous solution stream past inlets for the
water-immiscible medium. The water-immiscible medium disrupts the
continuous aqueous solution stream and "pinches off" distinct
liquid droplets such that the liquid droplets become suspended in
the water-immiscible medium. The cells can be present in the
continuous aqueous solution stream at a low enough concentration
such that one or fewer cells become isolated in per aqueous liquid
droplet formed. The water-immiscible medium may comprise an oil. An
exemplary oil is fluorinated oil, such as QX200.TM. Droplet
Generation Oil for EvaGreen #1864005 (BioRad, Hercules, Calif.).
Various surfactants present in the water-immiscible medium and or
the aqueous phase may help to stabilize the liquid droplets within
the water-immiscible medium. The liquid droplets preferably have a
volume of from about 1 pL to about 100 nL, such as a volume from
about 10 pL to about 10 nL or from about 100 pL to about 1 nL.
Amounts above and below these amounts are acceptable.
[0039] For downstream steps, such as the lysing, amplification, and
detecting, each cell is preferably isolated in the liquid droplet
with one or more reagents such as a lysis reagent, a DNA
polymerase, amplification primers, deoxynucleotide triphosphates,
RNase inhibitor, one or more nucleic acid logic gates, and a
reporter. This can be performed by merging one or more solutions
containing these reagents with a cell-containing solution upstream
of where the droplets are formed. The solutions may form adjacent
laminar flows prior to droplet formation. The solutions become
co-encapsulated by the water-immiscible medium to form the droplet.
Formation of the droplets thereby isolates individual cells with
the reagents.
[0040] The lysis reagent isolated with the cell in the droplet may
comprise a detergent. The detergent preferably comprises a
non-denaturing detergent. The non-denaturing detergent preferably
comprises a non-ionic detergent. Exemplary non-denaturing,
non-ionic detergents include Tween 20 (Millipore Sigma, Burlington,
Mass.), TRITON X-100 (Dow Chemical Company, Midland, Mich.)
(4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol,
t-octylphenoxypolyethoxyethanol, polyethylene glycol
tert-octylphenyl ether), NP-40 (nonyl phenoxypolyethoxylethanol),
NONIDET P-40 (Shell Chemical Co., The Hague, The Netherlands)
(octyl phenoxypolyethoxylethanol), and others. The detergent is
preferably present in the liquid droplet at a concentration of from
about 0.5% v/v to about 5% v/v, such as a concentration of from
about 1% v/v to about 5% v/v or a concentration of about 2.5% v/v.
The lysis reagent may alternatively or additionally comprise a
lytic enzyme. Exemplary lytic enzymes include lysozyme and phage
lysins. Suitable lysozyme concentrations of lysozyme include
concentrations between about 1 kU/ml and 60 kU/ml, such as about 30
kU/ml. Concentrations above and below these amounts are also
acceptable. Optimal concentrations of other particular lytic
enzymes can be easily determined. A detergent should be sufficient
to lyse most cells, particularly when heated. For cells with cell
walls, such as certain types of bacteria, the lysis reagent
preferably includes a detergent in addition to a lytic enzyme. In
some versions of the invention, a lysis reagent is absent and cell
lysis occurs by virtue of heating alone.
[0041] The polymerase isolated with the cell in the droplet can be
any polymerase suitable for downstream amplification of the target
nucleic acid. For amplification of DNA target nucleic acid, the
polymerase is preferably a DNA-dependent DNA polymerase.
DNA-dependent DNA polymerases are enzymes that catalyze the
replication of DNA from a DNA template. An exemplary DNA-dependent
DNA polymerase is Taq polymerase. For the amplification of RNA
target nucleic acid, the polymerase is preferably a DNA- and
RNA-dependent DNA polymerase or a combination of a DNA-dependent
DNA polymerase with a separate RNA-dependent DNA polymerase.
RNA-dependent DNA polymerases are enzymes that catalyze the
production of DNA from a RNA template. RNA-dependent DNA
polymerases are sometimes known as "reverse transcriptases." DNA-
and RNA-dependent DNA polymerases are enzymes that have both
DNA-dependent DNA polymerase activity and RNA-dependent DNA
polymerase (reverse transcriptase) activity. "Amplification" is
used herein as is typically used in the art except that it is
understood herein also to encompass the reverse transcription of an
RNA target nucleic acid to DNA.
[0042] For the certain types of amplification, such as isothermal
amplification, the polymerase additionally has strand displacement
activity and lacks 5'.fwdarw.3'' exonuclease activity. The
polymerase may also contain or lack and 3'.fwdarw.5' exonuclease
activity. The polymerase is preferably thermostable. An exemplary
polymerase is the Bst 2.0 DNA Polymerase (New England BioLabs,
Inc., Ipswich, Mass.). This enzyme has RNA-dependent DNA polymerase
(reverse transcriptase) activity, DNA-dependent DNA polymerase
activity, and strand displacement activity and lacks 5'.fwdarw.3''
exonuclease activity. Other polymerases with these characteristics
are well-known in the art.
[0043] The amplification primers isolated with the cell in the
droplet include any primers suitable for amplification of the
nucleic acid template. A number of amplification methods and the
design of suitable primers therefor are known in the art. The dNTPs
serve as the building blocks for the amplified nucleic acids in the
amplification.
[0044] The nucleic acid logic gate isolated with the cell in the
droplet may comprise any one or more nucleic acid gates suitable
for profiling nucleic acids. Exemplary nucleic acid logic gates are
known in the art and are described elsewhere herein.
[0045] The reporter isolated with the cell in the droplet may
comprise any reagent suitable for indicating the presence of
nucleic acids generally or particular nucleic acids specifically.
Exemplary reporters are known in the art and are described
elsewhere herein.
[0046] The lysing step may comprise heating the cell either in the
presence or absence of a lysis reagent within the droplet. This
step may be performed by heating the droplet containing the cell or
the cell and lysis reagent. Heating the droplet may be performed by
flowing the droplet containing the cell or the cell and lysis
reagent through a heated zone. The heated zone may comprise an
entire channel-containing device or subsections thereof. In some
versions, a heating step is not required, as mixing the lysis
reagent with the cell may be sufficient on its own to lyse the
cell.
[0047] The amplifying may comprise amplifying a DNA template or
reverse transcribing template RNA into DNA and amplifying the
reverse transcribed DNA. The type of amplification used depends on
the type of nucleic acid targeted for profiling. DNA templates, for
example, will typically require only amplification of the DNA
itself. RNA templates, by contrast, will typically require reverse
transcription and DNA amplification.
[0048] The amplification method may comprise any method suitable
for amplifying nucleic acids. Exemplary methods comprise PCR and
isothermal amplification methods. Reverse transcription may be
combined with PCR or the isothermal amplification method. Exemplary
isothermal amplification methods include enzyme-free isothermal
amplification methods and enzyme-dependent isothermal amplification
methods. Exemplary enzyme-free isothermal amplification methods
include hybridization chain reaction (HCR), catalytic hairpin
assembly (CHA), and others. Exemplary enzyme-dependent isothermal
amplification methods include loop-mediated isothermal
amplification (LAMP), rolling circle amplification (RCA), multiple
displacement amplification (MDA), recombinase polymerase
amplification (RPA), nucleic acid sequence-based amplification
(NASBA), among others.
[0049] The presence of lysis reagent and/or undiluted cell lysate
during amplification can inhibit certain amplification methods such
as PCR and enzyme-free isothermal amplification methods such as HCR
and CHA. As shown in the following examples, LAMP was capable of
producing a strong amplification signal in the presence of the
lysis reagent and undiluted cell lysate. Thus, preferred
amplification methods for performing in the presence of lysis
reagent and/or undiluted cell lysate include enzyme-mediated
isothermal amplification methods such as LAMP.
[0050] After nucleic acid amplification, the amplified nucleic acid
can be detected by a number of methods. Preferred methods include
fluorescent methods. The methods can include non-specific nucleic
acid detection and specific nucleic acid detection.
[0051] Non-specific nucleic acid detection detects nucleic acid
regardless of the particular sequence using a non-specific nucleic
acid reporter, such as a non-specific fluorescent DNA reporter.
Exemplary non-specific nucleic acid reporters include ethidium
bromide, propidium iodide, crystal violet, dUTP-conjugated probes,
DAPI (4',6-diamidino-2-phenylindole), 7-AAD (7-aminoactinomycin D),
Hoechst 33258, Hoechst 33342, Hoechst 34580, PICOGREEN (Molecular
Probes, Inc., Eugene, Oreg.), Helixyte (AAT Bioquest, Sunnyvale,
Calif.), YOYO-1, DiYO-1, TOTO-1, DiTO-1, and SYBR dyes (Molecular
Probes, Inc., Eugene, Oreg.), such as SYBR Green I, SYBR Green II,
SYBR Gold, etc. Many of these non-specific nucleic acid reporters
are DNA intercalating agents.
[0052] Specific nucleic acid detection detects species of nucleic
acid having a particular sequence with a sequence-specific nucleic
acid reporter, such as a sequence-specific fluorescent nucleic acid
reporter. A number of sequence-specific nucleic acid fluorescent
reporters are known in the art. Exemplary sequence-specific
fluorescent nucleic acid reporters include quenched reporters that
release the quencher upon binding to a particular sequence. An
example of a sequence-specific fluorescent nucleic acid reporter is
shown in FIG. 2. This reporter is configured to operate through
branch migration-mediated strand displacement. The reporter
comprises a quencher strand 22 (black) annealed to a substantially
complementary reporter strand 20 (red). The quencher strand 22
comprises a quencher 23 (Q). The reporter strand 20 comprises a
fluorophore 21 (F). Annealing of the quencher strand 22 to the
reporter strand 20 places the quencher 23 in close proximity to the
fluorophore 21, thereby quenching any fluorescence emitted from the
fluorophore 21. The reporter strand 20 anneals only to a first
portion (1*) of the quencher strand 22, leaving a second portion
(2*) of the quencher strand 22, referred to as a toehold, exposed.
The quencher strand 22 is designed to be substantially
complementary to an input strand 24 (blue). The input strand 24 is
a nucleic acid intended to be detected. The input strand 24
comprises a portion (2) substantially complementary to the toehold
(1*). When present, the input strand 24 binds to the toehold via
the substantially complementary portion and displaces the reporter
strand 20 from the quencher strand 22 upon annealing to the
remainder of the quencher strand 22. Displacement of the reporter
strand 20 removes the fluorophore 21 from the proximity of the
quencher 23, thereby resulting in an activated reporter (shown as
"output" in FIG. 2). The activated reporter can then fluoresce upon
excitation with light. In alternative versions, the reporter strand
is substantially complementary to the input strand and comprises a
toehold such that binding of the input strand to the toehold on the
reporter strand followed by branch migration displaces the quencher
strand from the reporter strand. The resulting input
strand/reporter strand duplex is then capable of fluorescing.
[0053] The detection may comprise profiling the composition of
amplified nucleic acids by performing a molecular logical
computation using a molecular logic circuit. The molecular logic
circuit inputs one or more particular species of nucleic acid,
performs a logical computation, and outputs one or more different
species of nucleic acid. The molecular logic circuit comprises one
or more nucleic acid logic gates used alone or in various
combinations. As used herein, "molecular logical computation" or
"molecularly computing" refers to the production of one or more
output nucleic acids (e.g., output strands) from one or more
nucleic acid logic gates in response to one or more input nucleic
acids (e.g., input strands). "Production" in this context refers to
the displacement of the output nucleic acid from a nucleic acid
logic gate such that the output nucleic acid can be detected or
used as an input for one or more downstream nucleic acid logic
gates, as described in further detail below. The molecular logic
circuit may be a DNA-based molecular logic circuit containing one
or more DNA logic gates, an RNA-based molecular logic circuit
containing one or more logic gates, or a combination thereof.
[0054] Each logic gate is configured to perform a specific logical
operation. Exemplary logical operations include YES, NOT, AND, OR,
AND-NOT, NOT-AND, NOT-OR, EXCLUSIVE-OR, EXCLUSIVE-NOR, and
IMPLY.
[0055] YES and NOT gates are each configured for profiling a single
input.
[0056] A YES gate produces an output (1) if and only if the input
is in high abundance. This means that the gate fails to produce an
output (0) if the input is not in high abundance. A YES gate may
also be referred to as a "transducer." A logical operation
performed by a YES gate for input A is shown in the following truth
table:
TABLE-US-00001 YES gate A A 1 1 0 0
[0057] The NOT gate is a circuit that produces an inverted version
of the input at its output. It is also known as an inverter. If the
input variable is A, the inverted output is NOT A. This is also
shown as A', or A with a bar over the top. A logical operation
performed by a NOT gate for input A is shown in the following truth
table:
TABLE-US-00002 NOT gate A 0 1 1 0
[0058] The AND, OR, AND-NOT, NOT-AND, NOT-OR, EXCLUSIVE-OR, and
EXCLUSIVE-NOR operations are each configured for profiling multiple
inputs.
[0059] The AND gate is configured to give a high output (1) only if
all its inputs are high and otherwise fails to produce a high
output (0). A dot (.) is used to show the AND operation: A.B. The
dot is sometimes omitted: AB. A logical operation performed by an
AND gate for inputs A and B is shown in the following truth
table:
TABLE-US-00003 2 Input AND gate A B A.B 0 0 0 0 1 0 1 0 0 1 1 1
[0060] An OR gate (otherwise known as an INCLUSIVE-OR gate) is
configured to give a high output (1) if one or more of its inputs
are high. A plus (+) is used to show the OR operation. A logical
operation performed by an OR gate for inputs A and B is shown in
the following truth table:
TABLE-US-00004 2 Input OR gate A B A + B 0 0 0 0 1 1 1 0 1 1 1
1
[0061] A NOT-AND (NAND) gate is equal to an AND gate followed by a
NOT gate. The outputs of all NAND gates are high (1) if any of the
inputs are low (0). A logical operation performed by a NAND gate
for inputs A and B is shown in the following truth table:
TABLE-US-00005 2 Input NAND gate A B A.B 0 0 1 0 1 1 1 0 1 1 1
0
[0062] An AND-NOT gate detects the presence of only one of two
possible inputs. The outputs of AND-NOT gates are high (1) if one
and only one of two possible inputs are high (1) and low (0) if any
other conditions obtain. A truth table for an AND-NOT gate in which
input B is negated (A AND-NOT B) is as follows:
TABLE-US-00006 2 Input AND-NOT gate A B Output 0 0 0 0 1 0 1 0 1 1
1 0
[0063] A NOT-OR (NOR) gate is equal to an OR gate followed by a NOT
gate. The outputs of all NOR gates are low (0) if any of the inputs
are high (1). A logical operation performed by a NOR gate for
inputs A and B is shown in the following truth table:
TABLE-US-00007 2 Input NOR gate A B A + B 0 0 1 0 1 0 1 0 0 1 1
0
[0064] An EXCLUSIVE-OR (EXOR) gate is configured to give a high
output if either, but not both, of its two inputs are high (1). An
encircled plus sign is used to show the EXOR operation. A logical
operation performed by an EXCLUSIVE-OR gate for inputs A and B is
shown in the following truth table:
TABLE-US-00008 2 Input EXOR gate A B A .sym. B 0 0 0 0 1 1 1 0 1 1
1 0
[0065] An EXCLUSIVE-NOR (EXNOR) gate does the opposite of the EXOR
gate. The EXCLUSIVE-NOR gate is configured to give a high output if
both of two inputs are high (1) or if both of two inputs are low
(0). It gives a low output if either, but not both, of its two
inputs are high. A logical operation performed by an EXCLUSIVE-NOR
gate for inputs A and B is shown in the following truth table:
TABLE-US-00009 2 Input EXNOR gate A B A .sym. B 0 0 1 0 1 0 1 0 0 1
1 1
[0066] An IMPLY gate employs a CONDITIONAL logical operation, by
conveying "if-then" logic.
[0067] Nucleic acid logic gates configured to perform logical
operations are known in the art. See, e.g., Baccouche et al. 2014,
Chen et al. 2015, Deng et al. 2014, Li et al. 2011, Li et al. 2013,
Li et al. 2016, Massey et al. 2017, Okamoto et al. 2004, Qian and
Winfree 2011, Qian and Winfree et al. 2011, Ravan et al. 2017,
Thubagere et al. 2017, Wei et al. 2016, Xu et al. 2014, Yang et al.
2013, Yang et al. 2016, Yang et al. 2014, Yao et al. 2015, Zhang et
al. 2010, Zhu et al. 2013, Zou et al. 2017, US 2007/0072215, and WO
2017/141068.
[0068] Exemplary logic gates operate through branch
migration-mediated strand displacement, also known as
toehold-mediated branch migration or random-walk branch migration.
A schematic of an exemplary branch migration-mediated strand
displacement logic gate employing a YES logical operation is shown
in FIG. 3. The logic gate comprises an output strand 33 (brown
strand) annealed to a substantially complementary gate strand 32
(blue). An input strand 31 (green) that is substantially
complementary to the gate strand 32 binds to an exposed toehold
(3*) portion of the gate strand 32 and subsequently displaces the
output strand 33 upon further annealing to the gate strand 32. The
branch migration-mediated strand displacement mechanism can be
configured to generate logic gates employing other operations, such
as NOT, AND, OR, NOT-AND, NOT-OR, EXCLUSIVE-OR, EXCLUSIVE-NOR
operations, among others. See, e.g., the references provided
above.
[0069] Other exemplary logic gates include polymerase-dependent
logic gates. Polymerase-dependent logic gates require
polymerase-mediated extension of an input strand along a gate
strand to displace an output strand from the gate strand. These
logic gates are designed to operate with polymerases that have
strand displacement activity and lack 5'.fwdarw.3' exonuclease
activity. A schematic of an exemplary polymerase-dependent logic
gate employing a YES logical operation is shown in FIG. 4. The
polymerase-dependent logic gate comprises an output strand 43
(brown) annealed to a gate strand 42 (blue). The output strand 43
anneals to an output-strand annealing portion (2*) of the gate
strand 42. The output strand 43 does not anneal to an output-strand
non-annealing portion (1*) of the gate strand 42, leaving an
exposed portion on the gate strand 42. The output-strand annealing
portion is closer to the 5' end of the gate strand 42 (brown) than
the output-strand non-annealing portion. An input strand 41 (green)
anneals to an input-strand annealing portion (1*) of the gate
strand 42. In the example in FIG. 4, the input-strand annealing
portion of the gate strand 42 is the same as the output-strand
non-annealing portion, but it does not have to be so. The input
strand 41 does not anneal to an input-strand non-annealing portion
(2*) of the gate strand 42. In the example in FIG. 4, the
input-strand non-annealing portion of the gate strand 42 is the
same as the output-strand annealing portion, but it does not have
to be so. Mere binding of the input strand 41 to the input-strand
annealing portion of the gate strand 42 is on its own insufficient
to displace the output strand 43 from the gate strand 42. Extension
of the input strand 41 along the gate strand 42 must occur to
displace the output strand 43 from the gate strand 42. The output
strand 43 can then be directly detected with a reporter gate or
serve as input for one or more additional logic gates.
[0070] The input-strand non-annealing portion of the gate strand
does not have to be the same as the output-strand annealing
portion. In order for there to be an exposed portion near the 3'
end of the input strand to facilitate binding of the input strand,
however, the input-strand annealing portion and the output-strand
non-annealing portion at least partially overlap. In some versions,
the input-strand annealing portion is a sub-portion of the
output-strand non-annealing portion. For binding of the input
strand not to be sufficient displace the output strand (i.e., for
extension of the input strand along the gate strand to be necessary
for displacement of the output strand), the output-strand annealing
portion and the input-strand non-annealing portion at least
partially overlap. In some versions, the output-strand annealing
portion and the input-strand annealing portion at least partially
overlap. Too much overlap, however, will induce displacement of the
output strand merely by the input strand binding to the exposed
portion on the gate strand without requiring polymerase-mediated
extension of the input strand. Thus, any overlap between the
output-strand annealing portion and the input-strand annealing
portion is an amount less than that sufficient for the input strand
to displace the output strand without polymerase-mediated
extension. In preferred versions of the invention, the
output-strand annealing portion and the input-strand annealing
portion do not overlap. This is thought to permit faster binding
kinetics between the input strand and the gate strand.
[0071] In some versions, the input-strand annealing portion the
gate strand can serve as a primer for an amplification reaction,
wherein the input-strand annealing portion binds to a sequence on a
template nucleic acid. In some versions, the amplification reaction
is LAMP. The gate strand can serve as a forward internal primer
(FIP) or a backward internal primer (BIP) in the LAMP reaction.
Amplification from the forward outer primer (FP) and/or backward
outer primer (BP), respectively, can displace an output strand
initially bound to the gate strand.
[0072] The polymerase-dependent logic gates can be configured to
perform any of the logical operations described herein, including
the YES, NOT, AND, OR, AND-NOT, NOT-AND, NOT-OR, EXCLUSIVE-OR,
EXCLUSIVE-NOR, and IMPLY operations, among others.
[0073] An exemplary polymerase-dependent OR logic gate is shown in
FIG. 5. The input-strand annealing portion of the gate strand 53
(green) has a first input-strand annealing portion (01*) and a
second input-strand annealing portion (02*). The first input-strand
annealing portion is substantially complementary to and anneals to
a first input strand 51 (red). The second input-strand annealing
portion (02*) is substantially complementary to and anneals to a
second input strand 52 (teal). Annealing of either or both of the
first 51 and second 52 input strands induces polymerase-mediated
extension thereof and displaces the output strand 54 (blue) from
the gate strand.
[0074] An exemplary polymerase-dependent AND logic gate is shown in
FIG. 6. This logic gate has a gate strand 63 (green) without a
pre-annealed output strand and instead employs an output strand 64
(red/blue) that is generated in situ. As with the OR logic gate,
the input-strand annealing portion (02*01*) of the gate strand 63
has a first input-strand annealing portion (01*) and a second
input-strand annealing portion (02*). The first input-strand
annealing portion (01*) is substantially complementary to and
anneals to a first input strand 61 (red). The second input-strand
annealing portion (02*) is substantially complementary to and
anneals to a second input strand 62 (teal). The output strand 64 is
generated in situ through binding of the first input strand 61 to
the first input-strand annealing portion and polymerase-mediated
extension of the first input strand 61 along the gate strand. The
generated output strand 64 is then displaced upon binding of the
second input strand 62 to the second input-strand annealing portion
and extension of the second input strand to generate an extended
input strand 65 (teal/red/blue).
[0075] An alternative exemplary polymerase-dependent AND logic gate
is shown in FIGS. 7A-7D. This logic gate includes at least two
distinct gate strands 73,74 (green, fuschia), output strands 75
(blue), and a threshold strand 76. "Distinct" used with reference
to nucleic acid means that the nucleic acids have different
nucleotide sequences. The output strands 75 are initially bound to
the gate strands 73,74 but are substantially complementary to and
are capable of annealing to the threshold strand 76. The threshold
strand 76 each of the at least two distinct gate strands 73,74 with
the output strands 75 bound thereto are present in a substantially
equimolar concentration. "Substantially equimolar" in this context
refers to equimolar amounts or a molar excess of one strand over
each other relevant strand less than about 40%, less than about
35%, less than about 30%, less than about 25%, less than about 20%,
less than about 15%, less than about 10%, less than about 5%, less
than about 4%, less than about 3%, less than about 2%, or less than
about 1%. A slight molar excess of the threshold strand over each
of the at least two distinct gate strands 73,74 with the output
strands 75 bound thereto is acceptable. The slight excess may be a
molar excess less than about 40%, less than about 35%, less than
about 30%, less than about 25%, less than about 20%, less than
about 15%, less than about 10%, less than about 5%, less than about
4%, less than about 3%, less than about 2%, or less than about 1%.
In the present example, the output strands 75 for each of the two
gate strands 73,74 are identical. However, they need not be
identical, so long as they each have a portion that is capable of
binding to the threshold strand 76 (and any downstream logic gate
or reporter gate), and are therefore substantially identical. The
input-strand annealing portion of a first 73 (green) of the at
least two distinct gate strands 73,74 anneals to a first 71
(red/Input 1) of at least two distinct input strands 71,72 but not
to a second 72 (teal/Input 2) of the at least two distinct input
strands 71,72. The input-strand annealing portion of a second 74
(fuschia) of the at least two distinct gate strands 73,74 anneals
to the second 72 (teal/Input 2) of the at least two distinct input
strands 71,72 but not to the first 71 (red/Input 1) of the at least
two distinct input strands 71,72. As shown in FIG. 7C and FIG. 7D,
the presence of only one of the two input strands 71,72 displaces
an amount of output strand 75 (1.times.) that is less than or equal
to the amount of threshold strand 76. The threshold strand 76 thus
binds to all of the displaced output strand 75 and quarantines it
from detection or further signaling through downstream logic gates.
As shown in FIG. 7B, however, the presence of both of the two input
strands 71,72, displaces an amount of output strand 75 (2.times.)
that is greater than the amount of threshold strand 76. The
threshold strand 76 is therefore capable of binding only to subset
of the displaced output strand 75, leaving a second subset free for
detection or further signaling through downstream logic gates. If
the threshold strand 76 is omitted, then activation of either
transducer 73,75 or 74,75 is sufficient to produce free output
strand 75 (blue), and this forms an alternative, exemplary OR
gate.
[0076] An exemplary polymerase-dependent AND-NOT logic gate is
shown in FIG. 8.
[0077] This logic gate comprises at least two distinct gate strands
83,84 (green, fuchsia). Each gate strand 83,84 is bound to a
distinct output strand 85,86 (blue, red, respectively). The output
strands 85,86 are substantially complementary to each other and are
capable of annealing to each other once displaced from the gate
strands 83,84. The input-strand annealing portion of a first 83
(green) of the at least two distinct gate strands 83,84 anneals to
a first 81 (red/Input 1) of at least two distinct input strands
81,82 but not to a second 82 (teal/Input 2) of the at least two
distinct input strands 81,82. The input-strand annealing portion of
a second 84 (fuschia/Input 2) of the at least two distinct gate
strands 83,84 anneals to the second 82 (fuchsia/Input 2) of the at
least two distinct input strands 81,82 but not to the first 81
(teal/Input 2) of the at least two distinct input strands 81,82.
When only one 81 (teal/Input 2 or fuschia/Input 2) of the two
distinct input strands 81,82 is present, the output strand 85 or 86
(blue or red, respectively) is displaced and can be detected or
used for signaling in downstream logic gates. When both of the
input strands 81,82 (teal/Input 2 and fuschia/Input 2) are present,
the displaced output strands 85,86 anneal to each other when
released from the gate strands 83,84 and quarantine each other from
detection or signaling in downstream logic gates.
[0078] An alternative exemplary polymerase-dependent AND-NOT logic
gate is shown in FIGS. 9A-9D. As shown in FIG. 9A, this logic gate
comprises a gate strand 116 (green) comprising a site (Input 1*)
capable of binding to a first input strand 114 (red/Input 1). The
gate strand 116 is initially bound to an output strand 115 (blue).
The output strand 115 (blue) is substantially complementary to a
reporter strand 118 (black), and additionally includes a site
(Input 2*) capable of binding to a second input strand 119
(fuschia/Input 2). The input-strand annealing portion of the gate
strand 116 anneals to the first input strand 114 (red/Input 1) but
not to the second input strand 119 (fuschia/Input 2). When only the
second input strand 119 (fuschia/Input 2) is present, as shown in
FIG. 9D, no reaction occurs. However, when only the first input
strand 114 (red/Input 1) is present, as shown in FIG. 9C, the
output strand 115 (blue) is displaced upon polymerization of the
first input strand 114 (red/Input 1) and can be detected or used
for signaling in downstream logic gates. In this example, the
output strand 115 binds to a toehold region of reporter strand 118
(black), displacing quencher strand 117 (black), and producing a
fluorescent signal. (The output strand 115 could alternatively bind
to the quencher strand 117 (black) or to either a reporter strand
or a quencher strand of a polymerase-dependent reporter gate,
depending on the desired format.) When the second input strand 119
(fuschia/Input 2) is also present, as in FIG. 9B, the output strand
115 anneals to the second input strand 119 (fuschia/Input 2), and
reporter strand 118 (black) is displaced from output strand 115
upon polymerization of the second input strand 119 (fuschia/Input
2). Displaced reporter strand 118 then binds to displaced quencher
strand 117, removing the fluorescent signal.
[0079] Another exemplary polymerase-dependent NOT logic gate is
shown in FIG. 10. This logic gate comprises an activated output
complex comprising a reporter strand 118 (black) serving as an
output strand and a gate strand 115 (blue). The gate strand 115
(blue) is substantially complementary to input strand 119 (fuschia)
at portion Input*. In its initial state, reporter strand 118 is not
bound to a quenching moiety and therefore yields a fluorescent
signal. When input strand 119 binds output strand 115, it displaces
reporter strand 118 upon polymerization. Displaced reporter strand
118 can then bind to quencher strand 117 (black), which can be
referred to as a "NOT strand," to quench the fluorescent signal. In
this example, strands 118 and 117 comprise a fluorophore-quencher
reporter complex, but in principle this NOT operation can act on
any reversible logic gate or output strand, wherein strands 118 and
117 could be devoid of a fluorophore or quencher, strand 117 could
constitute an output strand from another gate or any other strand
substantially complementary to strand 118, and binding of strand
118 with strand 117 would effectively quarantine strand 118 and
prevent downstream signaling. Similar to the embodiment above, the
output strand 115 could alternatively bind to the quencher strand
117 (black) or to either a reporter strand or a quencher strand of
a polymerase-dependent reporter gate, thereby making the initially
non-bound member of the reporter gate the NOT strand.
[0080] Exemplary lengths of the gate strand in the
polymerase-dependent logic gates include lengths of from about 4,
about 10, about 15, about 20, about 25, about 30, about 35, about
40, about 45, about 50 or more nucleotide bases to about 10, about
15, about 20, about 25, about 30, about 35, about 40, about 45,
about 50, about 60, about 70, about 80, about 90, about 100, about
150, about 200, about 250, about 300 or more nucleotide bases.
[0081] Exemplary lengths of the output-strand annealing portion of
the gate strand in the polymerase-dependent logic gates include
lengths of from about 2, about 4, about 10, about 15, about 20,
about 25, about 30, about 35, about 40, about 45, about 50 or more
nucleotide bases to about 5, about 10, about 15, about 20, about
25, about 30, about 35, about 40, about 45, about 50, about 60,
about 70, about 80, about 90, about 100, about 150, about 200,
about 250, about 300 or more nucleotide bases.
[0082] Exemplary lengths of the output-strand non-annealing portion
of the gate strand in the polymerase-dependent logic gates include
lengths of from about 1, about 2, about 4, about 10, about 15,
about 20, about 25, about 30, about 35, about 40, about 45, about
50 or more nucleotide bases to about 5, about 10, about 15, about
20, about 25, about 30, about 35, about 40, about 45, about 50,
about 60, about 70, about 80, about 90, about 100, about 150, about
200, about 250, about 300 or more nucleotide bases.
[0083] Exemplary lengths of the input-strand annealing portion of
the gate strand in the polymerase-dependent logic gates include
lengths of from about 1, about 2, about 4, about 10, about 15,
about 20, about 25, about 30, about 35, about 40, about 45, about
50 or more nucleotide bases to about 5, about 10, about 15, about
20, about 25, about 30, about 35, about 40, about 45, about 50,
about 60, about 70, about 80, about 90, about 100, about 150, about
200, about 250, about 300 or more nucleotide bases.
[0084] Exemplary lengths of the output strand in the
polymerase-dependent logic gates include lengths of from about 2,
about 4, about 10, about 15, about 20, about 25, about 30, about
35, about 40, about 45, about 50 or more nucleotide bases to about
5, about 10, about 15, about 20, about 25, about 30, about 35,
about 40, about 45, about 50, about 60, about 70, about 80, about
90, about 100, about 150, about 200, about 250, about 300 or more
nucleotide bases.
[0085] The input strand in any polymerase-dependent logic gate can
comprise a species of the amplified nucleic acid or can comprise an
output strand from an upstream logic gate in the circuit. When
employing LAMP, for example, the input strand can comprise a LAMP
amplification product, such as a portion of a LAMP dumbbell
product. A 3' portion of the LAMP dumbbell product, for example,
can bind to the input-strand annealing portion of a gate strand. A
schematic of such a mechanism is shown in FIG. 11, wherein a LAMP
dumbbell product 91 serves as an input strand. A 3' portion of the
LAMP dumbbell product 91 binds to and extends along a gate strand
92 to generate an extended input strand 93, thereby displacing an
output strand 94 from the gate strand 92.
[0086] The output strand in any polymerase-dependent logic gate can
comprise an input strand for a downstream logic gate or an input
strand for a reporter gate.
[0087] A reporter gate configured to detect an output strand can
comprise a branch migration-mediated strand displacement reporter
as shown in FIG. 2 or a polymerase-dependent reporter gate. A
polymerase-dependent reporter gate can be generated by placing a
fluorophore on the gate strand 42 (such as on the 5' end) and a
quencher on the output strand 43 (such as on the 3' end) of a
polymerase-dependent logic gate as shown in FIG. 4, thereby
generating a reporter strand and a quencher strand. Alternatively,
a polymerase-dependent reporter gate can be generated by placing a
fluorophore on the output strand 43 (such as on the 5' end) and a
quencher on the gate strand 42 (such as on the 3' end) of a
polymerase-dependent logic gate as shown in FIG. 4, thereby
generating a reporter strand and a quencher strand. In either case,
binding and polymerase-mediated extension of an input strand will
displace the quencher strand from the reporter strand and permit
fluorescence to be detected from the fluorophore.
[0088] As used herein, "identical" refers to nucleic acid sequences
that are the same. "Substantially identical" refers to nucleic
acids sequences that are identical or sufficiently identical as to
bind to a same third nucleic acid sequence under conditions
suitable for enzyme-dependent isothermal amplification, such as
loop-mediated isothermal amplification (LAMP). Substantial identity
therefore encompasses identity, and any embodiment described herein
as having substantial identity can therefore have identity.
"Complementary" refers to nucleic acid sequences that have perfect
base pairing. "Substantially complementary" refers to nucleic acid
sequences that have perfect base pairing (complementarity) or
sufficient base pairing as to bind to each other under conditions
suitable for enzyme-dependent isothermal amplification, such as
LAMP. Substantial complementarity therefore encompasses
complementarity, and any embodiment described herein as having
substantial complementarity can therefore have complementarity
(perfect base pairing). In any embodiment described herein, the
identity, substantial identity, complementarity or substantial
complementarity can occur over a length of 2-200 bases or more,
such as at least 2, at least 3, at least 4, at least 5, at least 6,
at least 7, at least 8, at least 9, at least 10, at least 11, at
least 12, at least 13, at least 14, at least 15, at least 16, at
least 17, at least 18, at least 19, at least 20, at least 21, at
least 22, at least 23, at least 24, at least 25, at least 26, at
least 27, at least 28, at least 29, at least 30, at least 31, at
least 32, at least 33, at least 34, at least 35, at least 36, at
least 37, at least 38, at least 39, at least 40, at least 41, at
least 42, at least 43, at least 44, at least 45, at least 46, at
least 47, at least 48, at least 49, at least 50, at least 60, at
least 70, at least 80, at least 90, at least 100, at least 110, at
least 120, at least 130, at least 140, at least 150, at least 160,
at least 170, at least 180, or at least 190 to at least 3, at least
4, at least 5, at least 6, at least 7, at least 8, at least 9, at
least 10, at least 11, at least 12, at least 13, at least 14, at
least 15, at least 16, at least 17, at least 18, at least 19, at
least 20, at least 21, at least 22, at least 23, at least 24, at
least 25, at least 26, at least 27, at least 28, at least 29, at
least 30, at least 31, at least 32, at least 33, at least 34, at
least 35, at least 36, at least 37, at least 38, at least 39, at
least 40, at least 41, at least 42, at least 43, at least 44, at
least 45, at least 46, at least 47, at least 48, at least 49, at
least 50, at least 60, at least 70, at least 80, at least 90, at
least 100, at least 110, at least 120, at least 130, at least 140,
at least 150, at least 160, at least 170, at least 180, at least
190, at least 200 or more bases.
[0089] In the methods described herein, the isolating can occur
prior to the lysing, the lysing can occur prior to the amplifying,
and/or the amplifying can occur prior to the detecting. In
preferred versions, the liquid droplet remains at a substantially
constant volume throughout and between each of the lysing, the
amplifying, and the detecting. In preferred versions, the lysing,
the amplifying, and the detecting all occur without diluting the
liquid droplet, adding additional reagents to the liquid droplet,
and/or removing reagents or liquid from the liquid droplet after
the isolating.
[0090] The lysing, the amplifying, and/or the molecular logical
computation in some versions are performed at substantially the
same temperature. "Substantially same temperature" refers to a
temperature range spanning about 50.degree. C., about 45.degree.
C., about 40.degree. C., about 35.degree. C., about 30.degree. C.,
about 25.degree. C., about 20.degree. C., about 20.degree. C.,
about 15.degree. C., about 10.degree. C., about 5.degree. C. or
less.
[0091] The lysing, the amplifying, and/or the molecular logical
computation in some versions are performed at a substantially
constant temperature. "Substantially constant temperature" refers
to temperature maintained over time within a range of about
50.degree. C., about 45.degree. C., about 40.degree. C., about
35.degree. C., about 30.degree. C., about 25.degree. C., about
20.degree. C., about 20.degree. C., about 15.degree. C., about
10.degree. C., about 5.degree. C. or less.
[0092] In some versions, a substantially constant temperature is
maintained throughout and between each of the lysing, the
amplifying, and/or performing the molecular logical
computation.
[0093] The lysing, the amplifying, and the molecular logical
computation in some versions are performed at a temperature of from
about 45.degree. C., about 50.degree. C., about 55.degree. C., or
about 60.degree. C. to about 70.degree. C., about 75.degree. C.,
about 80.degree. C., about 85.degree. C., or about 90.degree.
C.
[0094] The methods described herein can be used to profile a number
of aspects of the nucleic acid composition of a single cell using
the cell's RNA or DNA as a nucleic acid template. The methods, for
example, can be used in expression profiling to characterize the
expression pattern of the cell's mRNA or miRNA. Certain cell types,
such as circulating tumor cells (CTCs) or cells having particular
lineages, have distinguishable expression patterns. See, e.g.,
Sieuwerts et al. 2011. The methods of the invention can be used to
distinguish CTCs from non-CTCs, or cells of one lineage from cells
of another lineage. The methods of the invention can also be used
to detect certain genetic mutations in a cell, such as single
nucleotide polymorphisms (SNPs) or other types of mutations. See,
e.g., Yongkiettrakul et al. 2016 and Badolo et al. 2012 for
appropriate LAMP primer design for detecting SNPs.
[0095] An exemplary device 100 suitable for performing the methods
described herein is shown in FIG. 12. The device 100 comprises a
single, continuous network of microfluidic channels 101 and tubing
107, in combination with a number of additional elements. The
additional elements include a cell-solution inlet 102,
reagent-solution inlets 103, an oil inlet 104, a droplet former
105, an oil extractor 106, an incubation section comprising tubing
107 running through a heating element 109, an oil injector 110, a
laser 111, and a fluorescence detector 112. Cell solution and
reagent solutions are introduced in the channels via the
cell-solution inlet 102 and the reagent-solution inlets 103,
respectively. The cell solution can include any collection of cells
desired to be profiled. The reagent solution can include any
reagent described herein such as a lysis reagent, a DNA polymerase,
amplification primers, deoxynucleotide triphosphates, RNAse
inhibitors, one or more nucleic acid logic gates, and one or more
reporters, or any other reagent. The device can optionally include
more than one reagent-solution inlets 103, such as two, three, or
more, for introducing the reagents separately (see the examples).
The cell solution and reagent solution(s) merge upstream of the
droplet former 105 to form a cell-and-reagent solution. The
cell-and-reagent solution may comprise laminar flows of each of the
cell solution and reagent solution(s). The cell-and-reagent
solution flows to the droplet former 105. The droplet former 105
includes oil channels connected to the oil inlet 104 that inject
oil perpendicularly into the aqueous flow. The injected oil
separates the cell-and-reagent solution into aqueous droplets
suspended in the oil. The droplets are then compacted in the oil by
flowing past the oil extractor 106, which removes some of the oil
surrounding the droplets using suction. The compacted droplets flow
into the incubation section. The length of tubing section 107
determines the time each droplet spends in the incubation section
to allow controlled cell lysis, nucleic acid template
amplification, and logical computation with the nucleic acid logic
gates. Heating of the tubing 107 by the heating element 109
facilitates the cell lysis and amplification. The heating element
109 may heat the entire device 100 or any sub-portion thereof, such
as the incubation section. The droplets are then decompacted within
the channel by virtue of the oil injector 110 injecting oil into
the channel. This permits analysis of each individual liquid
droplet without interference from other droplets and provides data
acquisition software time to collect and process collected data.
Activated reporters generated during the logical computation are
then illuminated with the laser 111, and fluorescence is detected
with the fluorescence detector 112. Droplet sorting can occur with
a sorting device depending on the emitted fluorescence. The devices
of the invention may include some or all of the exemplary elements
described herein and may include others.
[0096] In some versions, the device 100 can be configured to split
the droplets at any point after formation to increase
throughput.
[0097] An exemplary device 113 capable of sorting droplets is shown
in FIG. 13. This device comprises a continuous network of
microfluidic channels 129 suitable for processing droplets, as well
as a set of liquid electrodes 123,124 capable of conducting
electricity. Droplets first flow into injection port 120 and are
spaced out by reinjecting oil via an additional port 121. Droplets
pass through sorting junction 122 after having their fluorescence
measured via laser(s) 130 and detector(s) 131. Based on their
fluorescence, droplets are directed into sorted outlet 126 or waste
outlet 127 through application of an electric field via liquid
electrodes 123 and 124. Intermittent application of this field
creates a dielectric force on the droplet, deflecting it into the
sorted outlet 126. Sorting is tuned by the amplitude and frequency
of DC current application between reference electrode 123 and
sorting electrode 124. Bias oil, applied through inlet 125, also
tunes sorting. Pressure relief shunts 128 equilibrate the oil
pressure between the sorting outlet 126 and waste outlet 127,
thereby preventing pressure fluctuations which can disrupt
sorting.
[0098] Systems of the invention include any combination of elements
described herein for performing the method steps recited
herein.
[0099] The elements and method steps described herein can be used
in any combination whether explicitly described or not.
[0100] All combinations of method steps as used herein can be
performed in any order, unless otherwise specified or clearly
implied to the contrary by the context in which the referenced
combination is made.
[0101] As used herein, the singular forms "a," "an," and "the"
include plural referents unless the content clearly dictates
otherwise.
[0102] Numerical ranges as used herein are intended to include
every number and subset of numbers contained within that range,
whether specifically disclosed or not. Further, these numerical
ranges should be construed as providing support for a claim
directed to any number or subset of numbers in that range. For
example, a disclosure of from 1 to 10 should be construed as
supporting a range of from 2 to 8, from 3 to 7, from 5 to 6, from 1
to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.
[0103] All patents, patent publications, and peer-reviewed
publications (i.e., "references") cited herein are expressly
incorporated by reference to the same extent as if each individual
reference were specifically and individually indicated as being
incorporated by reference. In case of conflict between the present
disclosure and the incorporated references, the present disclosure
controls.
[0104] It is understood that the invention is not confined to the
particular construction and arrangement of parts herein illustrated
and described, but embraces such modified forms thereof as come
within the scope of the claims.
EXAMPLES
Background
[0105] The following examples show aspects of the invention for
profiling the nucleic acid composition of each individual cell
within a large population of hematopoietic cells for detecting
circulating tumor cells (CTCs) therein.
[0106] During cancer progression, cells detach from the primary
tumor and disseminate throughout the blood stream. These CTCs serve
as valuable biomarkers for real-time monitoring of carcinogenesis,
and the methods described herein for detecting them provide a
critical tool in cancer detection and treatment.
[0107] Detecting CTCs remains technically challenging due to their
ultra-low abundance among normal hematopoietic cells and their
highly heterogeneous phenotypes. The present invention provides a
high-throughput platform for single-cell analysis that affords the
ability to detect CTCs with high sensitivity and specificity.
[0108] The CTC analysis platform combines the specificity of
molecular computation with the massive throughput of droplet-based
microfluidics. Single cells are encapsulated in microdroplets
containing a DNA-based logic circuit that inputs cellular
transcripts, performs a logical computation, and outputs a
fluorescence signal based on the cell's phenotypic state (FIG. 1).
The microdroplets are generated, incubated, and analyzed on a
microfluidic device that can process millions of cells per hour.
This approach is highly scalable and can be applied to tens of RNA
inputs, providing a detailed profile of a cell's phenotypic
state.
Materials and Methods
Device Fabrication
[0109] Microfluidic devices were fabricated from
polydimethylsiloxane (PDMS) via a soft photolithography process.
SU-8 3025 or 3010 Photoresist (Microchem) was deposited onto a
silicon wafer and spun to achieve the desired layer height. A
photomask was used to create patterns of polymerized photoresist
during UV exposure. The patterned wafer was then placed in a petri
dish to form a mold, and Sylgard.RTM. 184 PDMS (Dow Corning, Cat.
No. 4019862) in an 11:1 polymer:activator ratio was added. After
polymerization, patterned PDMS was excised and bonded to a glass
microscope slide via plasma treatment. Finally, Aquapel (Pittsburgh
Glass Works) was applied to the channels to provide a hydrophobic
coating.
DNA Complexes and Primer Mixes
[0110] All DNA oligos were ordered from Integrated DNA Technologies
(Coralville, Iowa) using standard desalting, with the exception of
reporter complex strands, which were HPLC purified. All LAMP
primers were diluted in DNase/RNase-free water (Invitrogen Cat. No.
10977023) prior to storage at -20.degree. C. Logic gate and
reporter strands were diluted in DEPC-treated 10 mM
Phosphate-Buffered Saline (pH 7.4) prior to storage at -20.degree.
C. 100.times. stocks of each DNA complex were prepared in diethyl
pyrocarbonate (DEPC)-treated phosphate buffered phosphate buffered
saline (PBS) and stored at -20.degree. C. On the day of experiment,
each DNA complex was separately annealed via heating to 97.degree.
C. for 5 minutes and cooling at a rate of -2.degree. C./min to
23.degree. C. DNA complexes were stored on ice until the time of
experiment. 20.times.LAMP primer mixes were prepared in
DNase/RNase-free water and stored at -20.degree. C. Sequences of
primers and logic gates used in LAMP experiments are shown in Table
1.
TABLE-US-00010 TABLE 1 Sequences of primers and logic gates used in
LAMP experiments. SEQ Strand Sequence ID NO KRT19 LAMP Primers
KRT19 LAMP-3 F3 AGTGACATGCGAAGCCAAT 3 KRT19 LAMP-3 B3
GCTTTCATGCTCAGCTGTGA 4 KRT19 LAMP-3 FIP AGCGACCTCCCGGTTCAATTCTC 5
GAGCAGAACCGGAAGGAT KRT19 LAMP-3 BIP CACACGGAGCAGCTCCAGATGTG 6
CAGCTCAATCTCAAGACC KRT19 LAMP-3 LF TGGTGAACCAGGCTTCAGC 7 KRT19
LAMP-3 LB AGGTCCGAGGTTACTGACCTGC 8 VIM LAMP Primers VIM LAMP-2 F3
CCGCACCAACGAGAAGG 9 VIM LAMP-2 B3 TGGTTAGCTGGTCCACCT 10 VIM LAMP-2
FIP TCCAGGAAGCGCACCTTGTCGGA 11 GCTGCAGGAGCTGAA VIM LAMP-2 BIP
AAGATCCTGCTGGCCGAGCTCCC 12 GCATCTCCTCCTCGTAG VIM LAMP-2 LF
AGTTGGCGAAGCGGTCA 13 VIM LAMP-2 LB CAGCTCAAGGGCCAAGGCAA 14 ESR1
LAMP Primers ESR1 LAMP-1 F3 AGAGCTGCCAACCTTTGG 15 ESR1 LAMP-1 B3
TGAACCAGCTCCCTGTCTG 16 ESR1 LAMP-1 FIP GGCACTGACCATCTGGTCGGAAG 17
CCCGCTCATGATCAAAC ESR1 LAMP-1 BIP TTGTTGGATGCTGAGCCCCCCCC 18
ATCATCGAAGCTTCACT ESR1 LAMP-1 LF GCCAGGCTGTTCTTCTTAGAGC 19 ESR1
LAMP-1 LB ACTCTATTCCGAGTATGATCCTA 20 CC GAPDH LAMP Primers GAPDH
LAMP-2 F3 GCTGCCAAGGCTGTGG 21 GAPDH LAMP-2 B3 CCCAGGATGCCCTTGAGG 22
GAPDH LAMP-2 FIP GTTGGCAGTGGGGACACGGAAC 23 AAGGTCATCCCTGAGCTGA
GAPDH LAMP-2 BIP TGTCAGTGGTGGACCTGACCTGT 24 CCGACGCCTGCTTCA GAPDH
LAMP-2 LF GGCCATGCCAGTGAGCTT 25 GAPDH LAMP-2 LB
CGTCTAGAAAAACCTGCCAAATA 26 TG ACTB SNP Detection LAMP Primers ACTB
4 B3-SNP GGCTGGAAGAGTGCCGC 27 ACTB 4 F3 GCGGCTACAGCTTCACCA 28 ACTB
4 FIP CGTGGCCATCTCTTGCTCGAAGG 29 GGAAATCGTGCGTGACATT ACTB 4 BIP
GCTTCCAGCTCCTCCCTGGACCG 30 CTCATTGCCAATGGT ACTB 4 LF
ACGTAGCACAGCTTCTCCTT 31 ACTB 4 LB GAAGAGCTACGAGCTGCCT 32 ACTB 4
B3-Sink GCGGCACTCTTCCAGCC 33 Reporter Complexes RepF 6-FAM- 34
CGAGTGCTGCGTATGACAAGGGC TAGCGTT RepF-HEX HEX- 35
CGAGTGCTGCGTATGACAAGGGC TAGCGTT RepQ CCCTTGTCATACGCAGCACTCG- 36
IowaBlackFQ RepF2-AF647 AlexaFluor647- 37 CGCCGCGTCCTGATCTAACTGAC
TGACTGC RepQ2 TCAGTTAGATCAGGACGCGGCG- 38 IowaBlackRQ Transducer
Orthogonality Experiments KRT19 -> Rep Transducer Gate
CGAGTGCTGCGTATGACAAGGGC 39 TAGCGTTATGCTACGAGCGACCT CCCGGTTCAATTCT
KRT19 -> Rep Transducer AACGCTAGCCCTTGTCATACGCA 40 Output
GCACTCG VIM -> Rep Transducer Gate CGAGTGCTGCGTATGACAAGGGC 41
TAGCGTTATGCTACGTCCAGGAA GCGCACCTTGTC VIM -> Rep Transducer
Output AACGCTAGCCCTTGTCATACGCA 42 GCACTCG Exemplary Logic Gates
Corresponding to Gates Shown in FIGS. 5 and 6 KRT19 AND VIM Strand
1 CGAGTGCTGCGTATGACAAGGGC 43 TAGCGTTATGCTACGTCCAGGAA
GCGCACCTTGTCATGCTACGAGC GACCTCCCGGTTCAATTCT KRT19 AND VIM Strand 2
CGAGTGCTGCGTATGACAAGGGC 44 TAGCGTTATGCTACGAGCGACCT
CCCGGTTCAATTCTATGCTACGT CCAGGAAGCGCACCTTGTC KRT19 OR VIM Gate
CGAGTGCTGCGTATGACAAGGGC 45 TAGCGTTATGCTACGTCCAGGAA
GCGCACCTTGTCATGCTACGAGC GACCTCCCGGTTCAATTCT KRT19 OR VIM Output
AACGCTAGCCCTTGTCATACGCA 46 GCACTCG VIM F1 GACAAGGTGCGCTTCCTGGA 47
KRT19 F1 AGAATTGAACCGGGAGGTCGCT 48 AND Gate with LAMP Inputs KRT19
Transducer 2 Gate CTGCTCTCACGGAGGCGCACCGG 49
TAAGGGTCATCGATGAGCGACCT CCCGGTTCAATTCT VIM Transducer 2 Gate
CTGCTCTCACGGAGGCGCACCGG 50 TAAGGGTCATCGATGTCCAGGAA GCGCACCTTGTC
KRT19/VIM Transducer 2 CGATGACCCTTACCGGTGCGCCT 51 Output
CCGTGAGAGCAG KRT19 AND VIM Gate CGAGTGCTGCGTATGACAAGGGC 52
TAGCGTTATGCTGCTCTCACGG KRT19 AND VIM Out AACGCTAGCCCTTGTCATACGCA 53
GCACTCG KRT19 AND VIM Threshold CCGCTGGTGATCACTCTGCTCTC 54
ACGGAGGCGCACCGGTAAGGGT CATCG OR Gate with LAMP Inputs KRT19
Transducer 3 Gate CGCGATCCGAGTGCTGCGTATGA 55
CAAGGGCTAGCGTTTGCCGGAAG CGACCTCCCGGTTC VIM Transducer 3 Gate
CGCGATCCGAGTGCTGCGTATGA 56 CAAGGGCTAGCGTTTGCCGGATC CAGGAAGCGCACCT
KRT19/VIM Transducer 3 TCCGGCAAACGCTAGCCCTTGTC 57 Output
ATACGCAGCACTCGGATCGCG NOT/AND-NOT Gates with LAMP Inputs VIM
Transducer 4 Gate CCATCGCGGAGACACGGACATCG 58
TTAAGGCAGCCTGTAGGCAGCCT CCAGGAAGCGCACC KRT19 Transducer 4 Gate
GTGTCTCCGCGATGGCGAGTGCT 59 GCGTATGACAAGGGCTAGCGTTA GCGACCTCCCGGTT
KRT19 AND-NOT VIM GGCTGCCTACAGGCTGCCTTAAC 60 Inhibitor
GATGTCCGTGTCTCCGCGATGG KRT19 AND-NOT VIM Output
AACGCTAGCCCTTGTCATACGCA 61 GCACTCGCCATCGCGGAGACAC Transducer
Orthogonality Experiments in Droplets KRT19 -> Rep2 Transducer
Gate GTGTCTCCGCGATGGCGCCGCGT 62 CCTGATCTAACTGACTGACTGCA
GCGACCTCCCGGTT KRT19 -> Rep2 Transducer GCAGTCAGTCAGTTAGATCAGGA
63 Output CGCGGCGCCATCGCGGAGACAC VIM Transducer 3 Gate
CGCGATCCGAGTGCTGCGTATGA 56 CAAGGGCTAGCGTTTGCCGGATC CAGGAAGCGCACCT
KRT19/VIM Transducer 3 TCCGGCAAACGCTAGCCCTTGTC 57 Output
ATACGCAGCACTCGGATCGCG
TABLE-US-00011 TABLE 2 Sequences of oligos used in CHA experiments.
SEQ ID Strand Sequence NO CK19 GAGTTACCAGCCTGGAGTTCTCAATGGTGGCCT 64
Sensor GGTAACTCACTGACCGAGCTAA H1 CGACATCTAACCTAGCTCACTGACCGAGCTAAG
65 CTGTTCTCGATTAGCTCGGTCAGTGAGTTACCA G H2
GCTGTTCTCGATCACTGACCGAGCTAATCGAGA 66 ACAGCTTAGCTCG H3
GTCAGTGAGCTAGGTTAGATGTCGCCATGTGTA 67
GACGACATCTAACCTAGCCCTTGTCATAGAGCA C H4
AGATGTCGTCTACACATGGCGACATCTAACCTA 68 GCCCATGTGTAGA RepF-CHA
6-FAM-CGAGTGCTCTATGACAAGGGCTAGGTT 69 RepQ-CHA
CCCTTGTCATAGAGCACTCG-IowaBlackFQ 70 CK19 Input
GCCACCATTGAGAACTCCAGG 71
In Vitro Transcription
[0111] DNA templates for KRT19 and VIM transcripts were synthesized
as "gBlocks" (SEQ ID NO:72 and SEQ ID NO:73, respectively) by
Integrated DNA Technologies (Coralville, Iowa) and cloned into a
pET-22b(+) vector (available from Novagen, Cat. No. 69744-3) under
a T7 promoter. In vitro transcription was performed with a
HiScribe.TM. T7 High Yield RNA Synthesis Kit (New England Biolabs,
Cat. No. E2040S), and resulting RNA was purified using a GeneJET
RNA Purification Kit (Thermo Scientific, Cat. No. K0731). RNA
concentration was quantified on a NanoDrop.TM. Spectrophotometer
(Thermo Scientific) and stocks were stored at -80.degree. C. in
DEPC-treated PBS.
Cell Culture and Staining
[0112] MOLT-4 cells (American Type Culture Collection) were
subcultured in a 1:8 ratio every two days and grown in RPMI-1640
Medium (Gibco, Cat. No. 11875093) supplemented with 10% FBS (Gibco,
Cat. No. 10082147) and 1.times. Antibiotic-Antimycotic (Gibco, Cat.
No. 15240062). SK-BR-3, U-2 OS, and MCF7 cells (American Type
Culture Collection) were subcultured in a 1:4 ratio every two days
and grown in DMEM, high glucose (Gibco, Cat. No. 11965-092)
supplemented with 10% FBS and 1.times. Antibiotic-Antimycotic. On
the day of experiment, each cell type was collected and washed
twice with Dulbecco's Phosphate-Buffered Saline (DPBS) (Gibco, Cat.
No. 14190144). Cells were then stained on ice using either 1 .mu.M
CellTrace.TM. Calcein Red-Orange AM (Invitrogen, Cat. No. C34851),
1 .mu.M CellTrace.TM. Calcein Violet AM for 405 nm Excitation
(Invitrogen, Cat. No. C34858), or 0.6 .mu.M Calcein AM (BD
Biosciences, Cat. No. 564061) in DPBS for 30 minutes. Cells were
subsequently washed twice with DPBS and resuspended in 18.75% v/v
Optiprep.TM. Density Gradient Medium (Sigma Aldrich, Cat. No.
D1556) in DPBS for microfluidic assays.
Batch RT-LAMP Experiments
[0113] Batch RT-LAMP assays were performed in triplicate on a
BioRad CFX Connect at 65.degree. C. Reactions were prepared at a
total volume of 10 with 1.6 .mu.M each FIP/BIP primer, 0.2 .mu.M
each F3/B3 Primer, 0.4 .mu.M each LoopF/B Primer, 1.times.
WarmStart LAMP Master Mix (New England Biolabs, Cat. No. E1700S),
0.5 U/.mu.L SUPERase.cndot.In.TM. RNase Inhibitor (Invitrogen, Cat.
No. AM2696), and 0.5.times. Phosphate Buffered Saline. DNA
complexes were added at varying concentrations. LAMP primer and
logic gate sequences are shown in Table 1. In reactions without any
DNA complexes, LAMP Fluorescent Dye (New England Biolabs) was added
as a general LAMP indicator. In lysate experiments, 1% Triton X-100
(Sigma Aldrich, Cat. No. T8787) was included in the reaction
mixture. Intact, unstained cells were added to each well
immediately before the start of each experiment. For all
experiments, "standard concentrations" of LAMP primers are defined
as 1.6 .mu.M of each FIP/BIP primer, 0.2 .mu.M of each F3/B3
Primer, and 0.4 .mu.M of each LoopF/B Primer for a given LAMP
primer set.
Transducer Orthogonality Experiments
[0114] Experiments were performed in triplicate as described above
in "Batch RT-LAMP Experiments," with slight modifications. Each
reaction included two LAMP primer sets: one specific to a KRT19
transcript, and another specific to a VIM transcript. Only one
transducer was added to each reaction, recognizing either VIM or
KRT19 amplification products. In vitro transcribed KRT19 or VIM
RNAs were added to each reaction at a concentration of 10 nM. PBS
was also added in non-template control reactions for each
transducer. Each reaction included 400 nM transducer gate strand
pre-annealed to 200 nM transducer output strand, and 200 nM
reporter quenching strand pre-annealed to 100 nM reporter
fluorophore strand. Logic gate and LAMP primer sequences are shown
in Table 1.
AND Logic Gate Experiment with LAMP Inputs
[0115] Experiments were performed in triplicate, similarly to
"Batch RT-LAMP Experiments." Each reaction included both KRT19 and
VIM LAMP primer sets at standard concentrations (as defined in
"Batch RT-LAMP Experiments"). In vitro transcribed KRT19 and/or VIM
RNAs were added at 10 nM concentrations. Each reaction also
contained 50 nM RepF pre-annealed to 100 nM RepQ. Every reaction
included both KRT19 and VIM transducers: 60 nM KRT19 Transducer 2
pre-annealed to 120 nM KRT19/VIM Transducer 2 Output, and 60 nM VIM
Transducer 2 pre-annealed to 120 nM KRT19 I VIM Transducer 2
Output. Each reaction contained 55 nM KRT19 AND VIM Output strand
pre-annealed to 60 nM KRT19 AND VIM Gate strand. In KRT19 AND VIM
logic experiments, the KRT19 AND VIM Threshold strand was included
at 65 nM. All LAMP primer and logic strand sequences are given in
Table 1.
OR Logic Gate Experiment with LAMP Inputs
[0116] Each reaction included both KRT19 and VIM LAMP primer sets
at standard concentrations (as defined in "Batch RT-LAMP
Experiments"). In vitro transcribed KRT19 and/or VIM RNAs were
added at 1 nM concentrations, or an equivalent volume of water as a
non-template control. Each reaction contained 100 nM RepF
pre-annealed to 200 nM RepQ, 220 nM KRT19 Transducer 3 pre-annealed
to 200 nM KRT19/VIM Transducer 3 Output, and 220 nM VIM Transducer
3 pre-annealed to 200 nM KRT19/VIM Transducer 3 Output. Experiments
were performed in triplicate, as described in "Batch RT-LAMP
Experiments." All LAMP primer and logic strand sequences are given
in Table 1.
YES Logic Gate Experiment with LAMP Inputs
[0117] Each reaction included the KRT19 primer set at standard
concentrations (as defined in "Batch RT-LAMP Experiments"). In
vitro transcribed KRT19 RNA was added at 1 nM concentrations, or an
equivalent volume of water as a non-template control. Each reaction
contained 100 nM RepF pre-annealed to 200 nM RepQ, and 220 nM KRT19
Transducer 3 pre-annealed to 200 nM KRT19/VIM Transducer 3 Output.
Experiments were performed in triplicate, as described in "Batch
RT-LAMP Experiments." All LAMP primer and logic strand sequences
are given in Table 1.
NOT Logic Gate Experiment with LAMP Inputs
[0118] Each reaction included 200 nM KRT19 AND-NOT VIM Output
pre-annealed to 100 nM RepF, 240 nM VIM Transducer 4 Gate
pre-annealed to 220 nM KRT19 AND-NOT VIM Inhibitor, and 200 nM
unbound RepQ. Each reaction also included the VIM LAMP primer set
at standard concentrations (as defined in "Batch RT-LAMP
Experiments"). 1 nM VIM in vitro transcribed RNA was added, or
water as a negative control. Experiments were performed in
triplicate as described in "Batch RT-LAMP Experiments."
AND-NOT Logic Gate Experiment with LAMP Inputs
[0119] Each reaction included 220 nM KRT19 Transducer 4 Gate
pre-annealed to 200 nM KRT19 AND-NOT VIM Output, 240 nM VIM
Transducer 4 Gate pre-annealed to 220 nM KRT19 AND-NOT VIM
Inhibitor, and 200 nM RepQ pre-annealed to 100 nM RepF. Each
reaction also included the KRT19 and VIM LAMP primer set at
standard concentrations (as defined in "Batch RT-LAMP
Experiments"). 1 nM KRT19 and/or VIM in vitro transcribed RNA was
added, or water as a negative control. Experiments were performed
in triplicate as described in "Batch RT-LAMP Experiments."
Droplet RT-LAMP Experiments with dsDNA-Specific Reporter
[0120] Experiments were performed as described above in "Droplet
RT-LAMP Experiments with Fluorogenic Logic Gate Reporter," with
some modifications. Prior to the experiment, MOLT-4 and SK-BR-3
cells were stained separately with Calcein Red-Orange AM
(Invitrogen), as described above in "Cell Culture and Staining." A
coflow microfluidic dropmaker with 100 .mu.M square channels was
used to generate droplets approximately 1 nL in volume. The
dropmaker combined three inlets containing: [0121] 1.) A 4.times.
mixture of cells (900 cells/.mu.L), suspended in an 18.75% v/v
mixture of Optiprep.TM. Density Medium (Sigma Aldrich, Cat. No.
D1556) and Phosphate Buffered Saline, [0122] 2.) 2.times. WarmStart
LAMP Master Mix (New England Biolabs, Cat. No. E1700S), [0123] 3.)
A 4.times. mixture containing LAMP primers, SUPERase.cndot.In.TM.
RNase Inhibitor (Invitrogen, Cat. No. AM2696), 4.times. WarmStart
LAMP Fluorescent Dye (New England Biolabs), and 4% v/v Triton X-100
(Sigma Aldrich Cat. No. T8787).
[0124] These aqueous flows were combined into droplets suspended in
fluorinated oil (QX200.TM. Droplet Generation Oil for EvaGreen (Bio
Rad, Cat. No. 1864005)). Flow rates were 50 .mu.L/hour for inlets 1
and 3, 100 .mu.L/hour for inlet 2, and 800 .mu.L/hour for the oil
inlet. The cell concentration (3,600 cells/.mu.l) was chosen such
that approximately one in every ten droplets contained a single
cell. For microscopy experiments, droplets were collected into a
microcentrifuge tube and incubated at 65.degree. C. for 20 minutes
prior to fluorescence measurements. Droplets were placed into a
PDMS imaging chamber and imaged on a Nikon Eclipse Ti
Epifluorescence Microscope.
[0125] Final RT-LAMP conditions included 1.6 .mu.M each FIP/BIP
primer, 0.2 .mu.M each F3/B3 Primer, 0.4 .mu.M each LoopF/B Primer,
1.times. WarmStart LAMP Master Mix (New England Biolabs), 0.5
U/.mu.L SUPERase.cndot.In.TM. RNase Inhibitor (Invitrogen), 1%
Triton X-100 (Sigma Aldrich), 4.7% v/v Optiprep.TM. Density
Gradient Medium (Sigma Aldrich), 1.times. LAMP Fluorescent Dye (New
England Biolabs), and 0.5.times. Phosphate Buffer.
Droplet RT-LAMP Experiments with Multiplexed Transducers
[0126] A coflow microfluidic dropmaker was used to encapsulate
cells with LAMP components, logic gates, and lysis reagents. Prior
to the experiment, U-2 OS and SK-BR-3 cells were stained separately
with Calcein AM (BD Biosciences, Cat. No. 564061), as described
above in "Cell Culture and Staining." 60 .mu.m was chosen as the
microfluidic channel width and height, generating droplets
approximately 250 pL in volume. This dropmaker combined three
aqueous inlets containing: [0127] 1.) A 10.times. mixture of cells
(5,000 cells/.mu.L), suspended in Phosphate Buffered Saline, [0128]
2.) 2.times. WarmStart LAMP Master Mix (New England Biolabs, Cat.
No. E1700S), [0129] 3.) A 2.5.times. mixture containing LAMP
primers, DNA complexes, SUPERase.cndot.In.TM. RNase Inhibitor
(Invitrogen, Cat. No. AM2696), and 6.25% v/v Tween-20 (Sigma
Aldrich Cat. No. P9416).
[0130] These aqueous flows were combined into droplets suspended in
fluorinated oil (QX200.TM. Droplet Generation Oil for EvaGreen (Bio
Rad, Cat. No. 1864005)). Flow rates were 80 .mu.L/hour for inlet 1,
400 .mu.L/hour for inlet 2, 320 .mu.L/hour for inlet 3, and 1200
.mu.L/hour for the oil inlet. The cell concentration (5,000
cells/.mu.L) was chosen such that approximately one in every ten
droplets contained a single cell. Droplets were collected into a
microcentrifuge tube on ice and incubated at 65.degree. C. for 60
minutes or 5 minutes (for negative controls) prior to fluorescence
measurements. Droplets were then re-injected into a second
microfluidic device for fluorescence measurements, with a droplet
injection rate of 400 .mu.L/hour and an oil reinjection rate of
1,200 .mu.L/hour. Each droplet passed a set of lasers (Changchun
New Industries Optoelectronics Tech. Co., Changchun, China) with
488 nm, 530 nm, and 637 nm excitation wavelengths, and fluorescence
was measured via one photomultiplier tube (ThorLabs, Newton, N.J.)
per channel. Fluorescence data was acquired via an FPGA card
(National Instruments) and analyzed with LabView software (National
Instruments).
[0131] Each condition included KRT19 and VIM primer sets, as listed
in Table 1. Final RT-LAMP conditions included 1.6 .mu.M each
FIP/BIP primer, 0.2 .mu.M each F3/B3 Primer, 0.4 .mu.M each LoopF/B
Primer, 100 nM RepF-HEX strand pre-annealed to 200 nM RepQ strand,
100 nM RepF2-AF647 strand pre-annealed to 200 nM RepQ2 strand, 110
nM KRT19->Rep Transducer Gate strand pre-annealed to 100 nM
KRT19->Rep Transducer Output, 110 nM VIM->Rep Transducer Gate
pre-annealed to 100 nM VIM->Rep Transducer Output, 1.times.
WarmStart LAMP Master Mix (New England Biolabs), 0.5 U/.mu.L
SUPERase.cndot.In.TM. RNase Inhibitor (Invitrogen), and 2.5% v/v
Tween-20 (Sigma Aldrich Cat. No. P9416).
Droplet ESR1 RT-LAMP
[0132] Experiments were carried out as in "Droplet RT-LAMP
Experiments with Multiplexed Transducers," with some alterations.
MCF7 and SK-BR-3 cells were stained separately with Calcein
Red-Orange AM (Invitrogen), as described above in "Cell Culture and
Staining." Cells were loaded into the device at a concentration of
9,000 cells/.mu.L in Phosphate Buffered Saline with 125 pg/.mu.L
RNase A (Thermo Scientific, Cat. No. EN0531). Each condition
included the ESR1 LAMP primer set at standard concentrations (as
defined in "Batch RT-LAMP Experiments"). ESR1 LAMP primer sequences
are listed in Table 1. Warmstart LAMP Dye (New England Biolabs) was
added into inlet 3 as a general LAMP indicator. Final RT-LAMP
conditions included 1.times. WarmStart LAMP Master Mix (New England
Biolabs) with 1.times. WarmStart LAMP Dye, 0.5 U/.mu.L
SUPERase.cndot.In.TM. RNase Inhibitor (Invitrogen), and 2.5% v/v
Tween-20 (Sigma Aldrich Cat. No. P9416). Droplets were collected on
ice, incubated at 65.degree. C. for 50 minutes, and fluorescence
was measured using 473 nm and 532 nm lasers (Changchun New
Industries Optoelectronics Tech. Co.) for excitation, similarly to
the above experiment. Flow rates for droplet generation were 40
.mu.L/hour for inlet 1, 100 .mu.L/hour for inlet 2, 80 .mu.L/hour
for inlet 3, and 800 .mu.L/hour for the oil inlet.
Droplet RT-LAMP with Integrated Device
[0133] Experiments were carried out as in "Droplet ESR1 RT-LAMP,"
with some alterations. MOLT-4 cells were stained with Calcein
Red-Orange AM (Invitrogen), as described above in "Cell Culture and
Staining." Cells were loaded into the device at a concentration of
9,000 cells/.mu.L in Phosphate Buffered Saline. Each condition
included the GAPDH LAMP primer set at standard concentrations (as
defined in "Batch RT-LAMP Experiments"). Primer sequences are
listed in Table 1. Final RT-LAMP conditions included 1.times.
WarmStart LAMP Master Mix (New England Biolabs) with 1.times.
WarmStart LAMP Dye, 0.5 U/.mu.L SUPERase.cndot. In.TM. RNase
Inhibitor (Invitrogen), and 2.5% v/v Tween-20 (Sigma Aldrich Cat.
No. P9416). Droplets were generated, incubated, and analyzed in a
single integrated device as shown in FIG. 12. Droplets travelled
out of the droplet generator into a length of PE tubing (Scientific
Commodities, Inc., Lake Havasu City, Ariz.), after having excess
oil extracted for tight droplet packing. The PE tubing length was
such that droplets spent 45 minutes within a 65.degree. C.
incubator before being reinjected into a microfluidic device for
fluorescence measurement. Droplets were excited with 473 nm and 532
nm lasers, and data was acquired and analyzed similarly to the
above experiment. Flow rates for droplet generation were 20
.mu.L/hour for inlet 1, 50 .mu.L/hour for inlet 2, 40 .mu.L/hour
for inlet 3, and 400 .mu.L/hour for the oil inlet. Oil was
subsequently extracted at 390 .mu.L/hour and reinjected at 200
.mu.L/hour prior to droplet measurement.
Catalyzed Hairpin Assembly Experiments
[0134] Catalyzed Hairpin Assembly (CHA) was performed in 10 .mu.L
reactions at 37.degree. C. on a Tecan Spark plate reader.
Fluorescence was measured with an excitation wavelength of 490 nm
and an emission wavelength of 535. The reaction buffer consisted of
TENaK (20 mM Tris, 1 mM EDTA, 140 mM NaCl, and 5 mM KCl, pH 8.0)
plus 0.5% SDS. Each reaction contained DNA oligos including 50 nM
H1, 400 nM H2, 50 nM H3, 400 nM H4, 10 nM CK19 Sensor, and 50 nM
RepF-CHA pre-annealed to 100 nM RepQ-CHA. Oligos were separately
annealed as described in "DNA Complexes and Primer Mixes," except
for RepF-CHA and RepQ-CHA, which were annealed together. Sequences
of each oligo are given in Table 2. For CHA background experiments,
reactions were performed in triplicate with or without 100 pM of
the DNA oligo "CK19 Input" present to initiate the reaction.
Reactions proceeded for 12 hours.
[0135] For lysate inhibition experiments, MOLT-4 cells were
titrated into the LAMP reactions with final concentrations ranging
from 3.9*10.sup.5 cells/.mu.L to 1.0*10.sup.8 cells/.mu.L. In
"CK19+" reactions, 10 nM of the DNA oligo "CK19 Input" was added to
initiate the reaction, otherwise no initiator was added. Reactions
proceeded for 8 hours.
Droplet Sorting Experiments
[0136] Droplets were generated similarly to the methods used in
"Droplet ESR1 RT-LAMP" above, with some alterations. MCF7 cells
were stained with Calcein Red-Orange AM (Invitrogen) and SK-BR-3
cells were stained separately with Calcein Violet AM (Invitrogen),
as described above in "Cell Culture and Staining." Cells were mixed
in a 1:1 ratio and loaded into the device at a concentration of
9,000 cells/.mu.L total in Phosphate Buffered Saline. Each
condition included the ESR1 LAMP primer set at standard
concentrations (as defined in "Batch RT-LAMP Experiments"). Primer
sequences are listed in Table 1. Warmstart LAMP Dye (New England
Biolabs) was added into inlet 3 as a general LAMP indicator. Final
RT-LAMP conditions included 1.times. WarmStart LAMP Master Mix (RNA
& DNA) (New England Biolabs) with 1.times. WarmStart LAMP Dye,
0.5 U/.mu.L SUPERase.cndot.In.TM. RNase Inhibitor (Invitrogen), and
2.5% v/v Tween-20 (Sigma Aldrich Cat. No. P9416). Flow rates for
droplet generation were 40 .mu.L/hour for inlet 1, 100 .mu.L/hour
for inlet 2, 80 .mu.L/hour for inlet 3, and 800 .mu.L/hour for the
oil inlet. Droplets were collected on ice, incubated at 65.degree.
C. for 1 hour, and loaded into a sorting device as shown in FIG.
13. See also (Sciambi and Abate 2015). Sorting flow rates were 100
.mu.L/hour for droplets, 400 .mu.L/hour for reinjection oil, and
1,000 .mu.L/hour for bias oil. Fluorescence was measured using 405
nm, 473 nm, and 532 nm lasers (Changchun New Industries
Optoelectronics Tech. Co.) for excitation, similarly to the above
experiment. Droplets were sorted based on high fluorescence in the
LAMP dye channel (473 nm excitation). Each sorting pulse was
applied through the sorting (positive) and reference (negative)
electrodes using a 10 kHz square wave, with 800V amplitude and 250
burst cycles, generated by a Trek model 2210 high voltage amplifier
(Trek Inc., Lockport, N.Y.). Sorting and reference electrodes were
filled with 1M NaCl dissolved in de-ionized water. After sorting,
droplets were placed into a PDMS imaging chamber and imaged on a
Nikon Eclipse Ti Epifluorescence Microscope.
ACTB SNP-LAMP Detection Experiments
[0137] Experiments were performed in duplicate similarly to the
methods used in "Batch RT-LAMP Experiments" above, with some
modifications. Total RNA was extracted from MOLT-4 or SK-BR-3 cells
using a GeneJET RNA Purification Kit (Thermo Scientific, Cat. No.
K0731) and was added at a final concentration of 1 ng/.mu.L. ACTB
LAMP primers were added at standard concentrations (as defined in
"Batch RT-LAMP Experiments"). Additionally, 1 .mu.M "ACTB 4
B3-Sink" strand was added. All oligo sequences can be found in
Table 1. Warmstart LAMP Dye (New England Biolabs) was added as a
general LAMP indicator.
Results
DNA-Based CTC Detection Circuit
[0138] The following examples show a DNA-based circuit that inputs
multiple cellular transcripts, performs a logical computation, and
outputs a binary CTC classification. Early experiments indicated
that DNA strand displacement cascades were insufficient to detect
transcript levels from single cells and therefore required an input
amplification step. We therefore sought to develop amplification
mechanisms that are sensitive, selective, and resistant to high
lysate concentrations.
[0139] Signal amplification: We initially explored a catalytic
hairpin assembly (CHA) amplification mechanism. CHA uses an input
nucleic acid as a catalyst to assemble metastable hairpins,
resulting in linear signal amplification. CHA amplification
suffered from high signal background and strong cell lysate
inhibition (FIGS. 14A and 14B). After thorough characterization, we
concluded that CHA was insufficient to amplify transcripts from
single cells in microfluidic droplets. We also tested hybridization
chain reaction (HCR) as an amplification mechanism and found
results similar to CHA. In FIG. 14A, error bars denote +/-1
standard deviation of the mean. These experiments were performed as
described in "Catalyzed Hairpin Assembly Experiments."
[0140] We identified reverse transcriptase loop-mediated isothermal
amplification (RT-LAMP) as a robust technique for amplifying
transcripts from single cells. LAMP uses six primers and a
strand-displacing DNA polymerase to exponentially amplify nucleic
acid targets. With RT-LAMP, we've achieved sub-nanomolar detection
sensitivity and lysate resistance beyond the target working
concentration of 10.sup.6 cells/ml. We tested RT-LAMP's ability to
distinguish between human breast cancer cells (SK-BR-3) and
leukocytes (MOLT-4) using primers specific to the epithelial marker
Cytokeratin 19 (CK19, KRT19). These experiments revealed a broad
(50 minute) detection window with a signal-to-background
ratio>150 (FIGS. 15A and 15B). This outstanding signal
amplification is more than sufficient to feed into downstream
molecular logic gates and detect on microfluidic chips.
Furthermore, we determined that CK19 LAMP reliably distinguishes
SK-BR-3 and MOLT-4 lysates even at 10.sup.7 cells/mL, which is
equivalent to one cell per 100 pL droplet (FIG. 15B). These
experiments show that RT-LAMP on the epithelial marker KRT19 can
distinguish between human breast cancer cells (SK-BR-3) and
leukocytes (MOLT-4) under conditions equivalent to microdroplet
reactions. Error bars denote +/-1 standard deviation of the mean in
all experiments. These experiments were performed as described in
"Batch RT-LAMP Experiments."
[0141] Molecular logic: A CTC detection circuit must integrate the
signals from multiple transcripts to perform a CTC classification.
DNA logic gates can perform complex molecular logic and computation
on nucleic acid inputs. We have designed new logic gates that
harness the strand-displacement activity of the LAMP polymerase to
drive strand displacement. This new mechanism provides superior
kinetics and less signal leakage than traditional random-walk
branch migration cascades.
[0142] We have designed all fundamental one- and two-input logic
gates using the polymerase-driven mechanism (FIGS. 4-11). In
theory, any arbitrarily complex logical function could be computed
by cascading these fundamental gates. As an initial demonstration,
we built a YES gate that detects an epithelial marker (KRT19) (FIG.
4), a NOT gate that detects a mesenchymal marker (VIM) (FIG. 10),
an OR gate that recognizes epithelial (KRT19) or mesenchymal (VIM)
markers (FIGS. 7A-D), an AND gate that recognizes epithelial
(KRT19) and mesenchymal (VIM) markers (FIGS. 7A-7D), and an AND-NOT
gate that recognizes epithelial (KRT19) and mesenchymal (VIM)
markers (FIGS. 9A-D). These gates are important for identifying
CTCs that occupy states along the epithelial-mesenchymal or
mesenchymal-epithelial transitions. The experiments on these gates
demonstrated a strong and uniform fluorescence signal in the
presence of KRT19 and/or VIM consistent with the designed logical
operations of the logic gates (FIG. 16). FIG. 16 includes endpoint
results from the time traces shown for the NOT gate (FIG. 19A), OR
gate (FIG. 18A), AND gate (FIG. 18B), and AND-NOT gate (FIG. 19B).
These experiments were performed as described under "AND Logic Gate
Experiment with LAMP Inputs," "OR Logic Gate Experiment with LAMP
Inputs," "YES Logic Gate Experiment with LAMP Inputs," "NOT Logic
Gate Experiment with LAMP Inputs," and "AND-NOT Logic Gate
Experiment with LAMP Inputs." Error bars denote +/-1 standard
deviation of the mean in all plots.
[0143] We additionally demonstrated that YES gates can operate
orthogonally to detect specific amplification events in a
multiplexed LAMP reaction. Experiments were conducted using
orthogonal, multiplexed LAMP and YES logic gates recognizing either
VIM (FIG. 17B) or KRT19 (FIG. 17A), as described above under
"Transducer Orthogonality Experiments." Error bars denote +/-1
standard deviation of the mean.
Ultra-High-Throughput Microfluidic CTC Screening Device
[0144] The following examples provide an integrated microfluidic
device that monitors the output of the DNA-based logic circuit
developed in the experiments outlined above. The experiments
demonstrate the ability to miniaturize RT-LAMP reactions in
microfluidic droplets and apply this to distinguish phenotypic
states of single cells.
[0145] Single-cell analysis in microfluidic droplets: After
optimizing LAMP conditions in bulk assays, we scaled them down to a
microemulsion format. Single cells were loaded onto a microfluidic
device and encapsulated in .about.1 nL droplets containing lysis
reagents, RT-LAMP reagents, and a dsDNA-specific LAMP indicator. A
cell stain (Calcein Red-Orange AM, Invitrogen) was used to verify
co-localization of cells and LAMP signal. The droplets were
incubated for 20 minutes and imaged using fluorescence microcopy.
Leukocytes (MOLT-4) displayed no KRT19 signal above background
(FIG. 20B), whereas breast cancer cells (SK-BR-3) displayed strong
KRT19 amplification (FIG. 20A). This experiment demonstrates the
ability to distinguish cell types using LAMP in microemulsion
drops. The experiment was performed as described in "Droplet
RT-LAMP Experiments with dsDNA-Specific Reporter."
[0146] We further demonstrated single-cell analysis with higher
throughput using a fully microfluidic workflow with serial
detection. We successfully applied this technique to analyze
Estrogen Receptor (ER, ESR1) expression for thousands of MCF7 (ER+)
and SK-BR-3 (ER-) cells, as shown in FIG. 21. As expected, a
significantly larger portion of the MCF7 cells showed high
fluorescence for the LAMP indicator than did the SK-BR-3 cells.
This demonstrates that our method can successfully distinguish
cells based on their mRNA expression in a high-throughput manner.
This experiment was performed as described in "Droplet ESR1
RT-LAMP."
[0147] We additionally showed that multiple transcripts can be
assayed simultaneously in these droplet assays, as shown in FIGS.
23A and 23B. We performed a multiplexed transducer experiment
wherein KRT19 amplification activates an Alexa Fluor 647 reporter,
and VIM amplification activates a HEX reporter. We encapsulated
SK-BR-3 (CK19+/VIM-) and U-2 OS (CK19-/VIM+) cells into droplets
with these transducers and reporters and saw that each cell type
only activated its expected reporter. This experiment was performed
as described in "Droplet RT-LAMP Experiments with Multiplexed
Transducers."
[0148] Integrated microfluidic device: We integrated all three
microfluidic steps into a continuous workflow that requires no user
intervention. This device comprises three modules. The modules: (1)
Encapsulate single cells in droplets containing the DNA circuit and
amplification reagents; (2) Incubate the reaction for a specified
time and temperature; and (3) Detect the circuit output using
fluorescence spectroscopy. An exemplary system is shown in FIG. 12.
As shown in FIG. 22, we were able to amplify a GAPDH transcript in
thousands of MOLT-4 cells using this device. Based on these
experiments, a fully automated microfluidic device for analyzing
millions of cells per hour can be made. This experiment was
performed as described in "Droplet RT-LAMP with Integrated
Device."
[0149] Microfluidic droplet sorting: We employed a
dielectrophoretic sorting device to separate two cell types based
on expression of an Estrogen Receptor (ER) transcript (ESR1). MCF7
(ER+) and SK-BR-3 (ER-) cells were separately stained, mixed
together, then sorted based on RT-LAMP amplification. As shown in
FIGS. 24A and 24B, this device successfully enriched green
fluorescent droplets in the sorted outlet, which indicates
successful ESR1 amplification. This experiment demonstrates our
ability to physically separate cell populations based on their
transcriptional status. These sorted and unsorted pools could be
further analyzed by RNA sequencing, or other assays. This sorting
device is depicted in FIG. 13. See also Sciambi and Abate 2015.
[0150] Additional logic circuits: The experiments outlined above
demonstrate the ability to profile single cells in microfluidic
droplets using strand displacement cascades. One of the advantages
of the molecular logic circuits is the ability to swap in/out new
elements to tailor assays to cancer types or even patients. Logic
circuits can be built to profile multiple transcriptional inputs,
such as informative molecular marker panels for specific cancer
types. The microfluidic device can be used to detect and/or
classify low-abundance CTCs in human blood samples.
[0151] Additional samples and profiling characteristics: The assays
and devices described herein can be used on samples other than
blood samples. Cells from tissue biopsies, for example, can be
dispersed and subjected to nucleic acid profiling as described
herein. The profiling can be used to profile any of a number of
nucleic acid characteristics and to classify cells in any of a
number of formats. Profiling expression patterns of mRNAs and/or
miRNAs and detecting or profiling SNPs provide only a few,
non-limiting examples. We have demonstrated that LAMP-based SNP
detection could be feasible in droplets, by discriminating between
MOLT-4 and SK-BR-3 total RNA. MOLT-4 RNA, which contains a SNP in
an ACTB transcript, amplified before SK-BR-3 RNA. These results are
shown in FIG. 25. Error bars denote +/-1 standard deviation of the
mean. This SNP experiment was performed as described in "ACTB
SNP-LAMP Detection Experiments."
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Sequence CWU 1
1
7311921DNAHomo sapiens 1gagtgagcgg cgcggggcca atcagcgtgc gccgttccga
aagttgcctt ttatggctcg 60agcggccgcg gcggcgccct ataaaaccca gcggcgcgac
gcgccaccac cgccgagacc 120gcgtccgccc cgcgagcaca gagcctcgcc
tttgccgatc cgccgcccgt ccacacccgc 180cgccagctca ccatggatga
tgatatcgcc gcgctcgtcg tcgacaacgg ctccggcatg 240tgcaaggccg
gcttcgcggg cgacgatgcc ccccgggccg tcttcccctc catcgtgggg
300cgccccaggc accagggcgt gatggtgggc atgggtcaga aggattccta
tgtgggcgac 360gaggcccaga gcaagagagg catcctcacc ctgaagtacc
ccatcgagca cggcatcgtc 420accaactggg acgacatgga gaaaatctgg
caccacacct tctacaatga gctgcgtgtg 480gctcccgagg agcaccccgt
gctgctgacc gaggcccccc tgaaccccaa ggccaaccgc 540gagaagatga
cccagatcat gtttgagacc ttcaacaccc cagccatgta cgttgctatc
600caggctgtgc tatccctgta cgcctctggc cgtaccactg gcatcgtgat
ggactccggt 660gacggggtca cccacactgt gcccatctac gaggggtatg
ccctccccca tgccatcctg 720cgtctggacc tggctggccg ggacctgact
gactacctca tgaagatcct caccgagcgc 780ggctacagct tcaccaccac
ggccgagcgg gaaatcgtgc gtgacattaa ggagaagctg 840tgctacgtcg
ccctggactt cgagcaagag atggccacgg ctgcttccag ctcctccctg
900gagaagagct acgagctgcc tgacggccag gtcatcacca ttggcaatga
gcggttccgc 960tgccctgagg cactcttcca gccttccttc ctgggcatgg
agtcctgtgg catccacgaa 1020actaccttca actccatcat gaagtgtgac
gtggacatcc gcaaagacct gtacgccaac 1080acagtgctgt ctggcggcac
caccatgtac cctggcattg ccgacaggat gcagaaggag 1140atcactgccc
tggcacccag cacaatgaag atcaagatca ttgctcctcc tgagcgcaag
1200tactccgtgt ggatcggcgg ctccatcctg gcctcgctgt ccaccttcca
gcagatgtgg 1260atcagcaagc aggagtatga cgagtccggc ccctccatcg
tccaccgcaa atgcttctag 1320gcggactatg acttagttgc gttacaccct
ttcttgacaa aacctaactt gcgcagaaaa 1380caagatgaga ttggcatggc
tttatttgtt ttttttgttt tgttttggtt tttttttttt 1440ttttggcttg
actcaggatt taaaaactgg aacggtgaag gtgacagcag tcggttggag
1500cgagcatccc ccaaagttca caatgtggcc gaggactttg attgcacatt
gttgtttttt 1560taatagtcat tccaaatatg agatgcgttg ttacaggaag
tcccttgcca tcctaaaagc 1620caccccactt ctctctaagg agaatggccc
agtcctctcc caagtccaca caggggaggt 1680gatagcattg ctttcgtgta
aattatgtaa tgcaaaattt ttttaatctt cgccttaata 1740cttttttatt
ttgttttatt ttgaatgatg agccttcgtg cccccccttc cccctttttt
1800gtcccccaac ttgagatgta tgaaggcttt tggtctccct gggagtgggt
ggaggcagcc 1860agggcttacc tgtacactga cttgagacca gttgaataaa
agtgcacacc ttaaaaatga 1920g 192121921DNAHomo sapiens 2gagtgagcgg
cgcggggcca atcagcgtgc gccgttccga aagttgcctt ttatggctcg 60agcggccgcg
gcggcgccct ataaaaccca gcggcgcgac gcgccaccac cgccgagacc
120gcgtccgccc cgcgagcaca gagcctcgcc tttgccgatc cgccgcccgt
ccacacccgc 180cgccagctca ccatggatga tgatatcgcc gcgctcgtcg
tcgacaacgg ctccggcatg 240tgcaaggccg gcttcgcggg cgacgatgcc
ccccgggccg tcttcccctc catcgtgggg 300cgccccaggc accagggcgt
gatggtgggc atgggtcaga aggattccta tgtgggcgac 360gaggcccaga
gcaagagagg catcctcacc ctgaagtacc ccatcgagca cggcatcgtc
420accaactggg acgacatgga gaaaatctgg caccacacct tctacaatga
gctgcgtgtg 480gctcccgagg agcaccccgt gctgctgacc gaggcccccc
tgaaccccaa ggccaaccgc 540gagaagatga cccagatcat gtttgagacc
ttcaacaccc cagccatgta cgttgctatc 600caggctgtgc tatccctgta
cgcctctggc cgtaccactg gcatcgtgat ggactccggt 660gacggggtca
cccacactgt gcccatctac gaggggtatg ccctccccca tgccatcctg
720cgtctggacc tggctggccg ggacctgact gactacctca tgaagatcct
caccgagcgc 780ggctacagct tcaccaccac ggccgagcgg gaaatcgtgc
gtgacattaa ggagaagctg 840tgctacgtcg ccctggactt cgagcaagag
atggccacgg ctgcttccag ctcctccctg 900gagaagagct acgagctgcc
tgacggccag gtcatcacca ttggcaatga gcggttccgc 960tgccctgcgg
cactcttcca gccttccttc ctgggcatgg agtcctgtgg catccacgaa
1020actaccttca actccatcat gaagtgtgac gtggacatcc gcaaagacct
gtacgccaac 1080acagtgctgt ctggcggcac caccatgtac cctggcattg
ccgacaggat gcagaaggag 1140atcactgccc tggcacccag cacaatgaag
atcaagatca ttgctcctcc tgagcgcaag 1200tactccgtgt ggatcggcgg
ctccatcctg gcctcgctgt ccaccttcca gcagatgtgg 1260atcagcaagc
aggagtatga cgagtccggc ccctccatcg tccaccgcaa atgcttctag
1320gcggactatg acttagttgc gttacaccct ttcttgacaa aacctaactt
gcgcagaaaa 1380caagatgaga ttggcatggc tttatttgtt ttttttgttt
tgttttggtt tttttttttt 1440ttttggcttg actcaggatt taaaaactgg
aacggtgaag gtgacagcag tcggttggag 1500cgagcatccc ccaaagttca
caatgtggcc gaggactttg attgcacatt gttgtttttt 1560taatagtcat
tccaaatatg agatgcgttg ttacaggaag tcccttgcca tcctaaaagc
1620caccccactt ctctctaagg agaatggccc agtcctctcc caagtccaca
caggggaggt 1680gatagcattg ctttcgtgta aattatgtaa tgcaaaattt
ttttaatctt cgccttaata 1740cttttttatt ttgttttatt ttgaatgatg
agccttcgtg cccccccttc cccctttttt 1800gtcccccaac ttgagatgta
tgaaggcttt tggtctccct gggagtgggt ggaggcagcc 1860agggcttacc
tgtacactga cttgagacca gttgaataaa agtgcacacc ttaaaaatga 1920g
1921319DNAArtificial SequencePrimer KRT19 LAMP-3 F3 3agtgacatgc
gaagccaat 19420DNAArtificial SequencePrimer KRT19 LAMP-3 B3
4gctttcatgc tcagctgtga 20541DNAArtificial SequencePrimer KRT19
LAMP-3 FIP 5agcgacctcc cggttcaatt ctcgagcaga accggaagga t
41641DNAArtificial SequenceKRT19 LAMP-3 BIP 6cacacggagc agctccagat
gtgcagctca atctcaagac c 41719DNAArtificial SequencePrimer KRT19
LAMP-3 LF 7tggtgaacca ggcttcagc 19822DNAArtificial SequencePrimer
KRT19 LAMP-3 LB 8aggtccgagg ttactgacct gc 22917DNAArtificial
SequencePrimer VIM LAMP-2 F3 9ccgcaccaac gagaagg
171018DNAArtificial SequencePrimer VIM LAMP-2 B3 10tggttagctg
gtccacct 181138DNAArtificial SequencePrimer VIM LAMP-2 FIP
11tccaggaagc gcaccttgtc ggagctgcag gagctgaa 381240DNAArtificial
SequencePrimer VIM LAMP-2 BIP 12aagatcctgc tggccgagct cccgcatctc
ctcctcgtag 401317DNAArtificial SequencePrimer VIM LAMP-2 LF
13agttggcgaa gcggtca 171420DNAArtificial SequencePrimer VIM LAMP-2
LB 14cagctcaagg gccaaggcaa 201518DNAArtificial SequencePrimer ESR1
LAMP-1 F3 15agagctgcca acctttgg 181619DNAArtificial SequencePrimer
ESR1 LAMP-1 B3 16tgaaccagct ccctgtctg 191740DNAArtificial
SequencePrimer ESR1 LAMP-1 FIP 17ggcactgacc atctggtcgg aagcccgctc
atgatcaaac 401840DNAArtificial SequencePrimer ESR1 LAMP-1 BIP
18ttgttggatg ctgagccccc cccatcatcg aagcttcact 401922DNAArtificial
SequencePrimer ESR1 LAMP-1 LF 19gccaggctgt tcttcttaga gc
222025DNAArtificial SequencePrimer ESR1 LAMP-1 LB 20actctattcc
gagtatgatc ctacc 252116DNAArtificial SequencePrimer GAPDH LAMP-2 F3
21gctgccaagg ctgtgg 162218DNAArtificial SequencePrimer GAPDH LAMP-2
B3 22cccaggatgc ccttgagg 182341DNAArtificial SequencePrimer GAPDH
LAMP-2 FIP 23gttggcagtg gggacacgga acaaggtcat ccctgagctg a
412438DNAArtificial SequencePrimer GAPDH LAMP-2 BIP 24tgtcagtggt
ggacctgacc tgtccgacgc ctgcttca 382518DNAArtificial SequencePrimer
GAPDH LAMP-2 LF 25ggccatgcca gtgagctt 182625DNAArtificial
SequencePrimer GAPDH LAMP-2 LB 26cgtctagaaa aacctgccaa atatg
252717DNAArtificial SequencePrimer ACTB 4 B3-SNP 27ggctggaaga
gtgccgc 172818DNAArtificial SequencePrimer ACTB 4 F3 28gcggctacag
cttcacca 182942DNAArtificial SequencePrimer ACTB 4 FIP 29cgtggccatc
tcttgctcga aggggaaatc gtgcgtgaca tt 423038DNAArtificial
SequencePrimer ACTB 4 BIP 30gcttccagct cctccctgga ccgctcattg
ccaatggt 383120DNAArtificial SequencePrimer ACTB 4 LF 31acgtagcaca
gcttctcctt 203219DNAArtificial SequencePrimer ACTB 4 LB
32gaagagctac gagctgcct 193317DNAArtificial SequencePrimer ACTB 4
B3-Sink 33gcggcactct tccagcc 173430DNAArtificial SequenceReporter
RepF 34cgagtgctgc gtatgacaag ggctagcgtt 303530DNAArtificial
SequenceReporter RepF-HEX 35cgagtgctgc gtatgacaag ggctagcgtt
303622DNAArtificial SequenceReporter RepQ 36cccttgtcat acgcagcact
cg 223730DNAArtificial SequenceReporter RepF2-AF647 37cgccgcgtcc
tgatctaact gactgactgc 303822DNAArtificial SequenceReporter RepQ2
38tcagttagat caggacgcgg cg 223960DNAArtificial SequenceKRT19 ->
Rep Transducer Gate 39cgagtgctgc gtatgacaag ggctagcgtt atgctacgag
cgacctcccg gttcaattct 604030DNAArtificial SequenceKRT19 -> Rep
Transducer Output 40aacgctagcc cttgtcatac gcagcactcg
304158DNAArtificial SequenceVIM -> Rep Transducer Gate
41cgagtgctgc gtatgacaag ggctagcgtt atgctacgtc caggaagcgc accttgtc
584230DNAArtificial SequenceVIM -> Rep Transducer Output
42aacgctagcc cttgtcatac gcagcactcg 304388DNAArtificial
SequenceKRT19 AND VIM Strand 1 43cgagtgctgc gtatgacaag ggctagcgtt
atgctacgtc caggaagcgc accttgtcat 60gctacgagcg acctcccggt tcaattct
884488DNAArtificial SequenceKRT19 AND VIM Strand 2 44cgagtgctgc
gtatgacaag ggctagcgtt atgctacgag cgacctcccg gttcaattct 60atgctacgtc
caggaagcgc accttgtc 884588DNAArtificial SequenceKRT19 OR VIM Gate
45cgagtgctgc gtatgacaag ggctagcgtt atgctacgtc caggaagcgc accttgtcat
60gctacgagcg acctcccggt tcaattct 884630DNAArtificial SequenceKRT19
OR VIM Output 46aacgctagcc cttgtcatac gcagcactcg
304720DNAArtificial SequenceVIM F1 47gacaaggtgc gcttcctgga
204822DNAArtificial SequenceKRT19 F1 48agaattgaac cgggaggtcg ct
224960DNAArtificial SequenceKRT19 Transducer 2 Gate 49ctgctctcac
ggaggcgcac cggtaagggt catcgatgag cgacctcccg gttcaattct
605058DNAArtificial SequenceVIM Transducer 2 Gate 50ctgctctcac
ggaggcgcac cggtaagggt catcgatgtc caggaagcgc accttgtc
585135DNAArtificial SequenceKRT19/VIM Transducer 2 Output
51cgatgaccct taccggtgcg cctccgtgag agcag 355245DNAArtificial
SequenceKRT19 AND VIM Gate 52cgagtgctgc gtatgacaag ggctagcgtt
atgctgctct cacgg 455330DNAArtificial SequenceKRT19 AND VIM Out
53aacgctagcc cttgtcatac gcagcactcg 305450DNAArtificial
SequenceKRT19 AND VIM Threshold 54ccgctggtga tcactctgct ctcacggagg
cgcaccggta agggtcatcg 505560DNAArtificial SequenceKRT19 Transducer
3 Gate 55cgcgatccga gtgctgcgta tgacaagggc tagcgtttgc cggaagcgac
ctcccggttc 605660DNAArtificial SequenceVIM Transducer 3 Gate
56cgcgatccga gtgctgcgta tgacaagggc tagcgtttgc cggatccagg aagcgcacct
605744DNAArtificial SequenceKRT19/VIM Transducer 3 Output
57tccggcaaac gctagccctt gtcatacgca gcactcggat cgcg
445860DNAArtificial SequenceVIM Transducer 4 Gate 58ccatcgcgga
gacacggaca tcgttaaggc agcctgtagg cagcctccag gaagcgcacc
605960DNAArtificial SequenceKRT19 Transducer 4 Gate 59gtgtctccgc
gatggcgagt gctgcgtatg acaagggcta gcgttagcga cctcccggtt
606045DNAArtificial SequenceKRT19 AND-NOT VIM Inhibitor
60ggctgcctac aggctgcctt aacgatgtcc gtgtctccgc gatgg
456145DNAArtificial SequenceKRT19 AND-NOT VIM Output 61aacgctagcc
cttgtcatac gcagcactcg ccatcgcgga gacac 456260DNAArtificial
SequenceKRT19 -> Rep2 Transducer Gate 62gtgtctccgc gatggcgccg
cgtcctgatc taactgactg actgcagcga cctcccggtt 606345DNAArtificial
SequenceKRT19 -> Rep2 Transducer Output 63gcagtcagtc agttagatca
ggacgcggcg ccatcgcgga gacac 456455DNAArtificial SequenceCK19 Sensor
64gagttaccag cctggagttc tcaatggtgg cctggtaact cactgaccga gctaa
556567DNAArtificial SequenceOligo H1 65cgacatctaa cctagctcac
tgaccgagct aagctgttct cgattagctc ggtcagtgag 60ttaccag
676646DNAArtificial SequenceOligo H2 66gctgttctcg atcactgacc
gagctaatcg agaacagctt agctcg 466767DNAArtificial SequenceOligo H3
67gtcagtgagc taggttagat gtcgccatgt gtagacgaca tctaacctag cccttgtcat
60agagcac 676846DNAArtificial SequenceOligo H4 68agatgtcgtc
tacacatggc gacatctaac ctagcccatg tgtaga 466927DNAArtificial
SequenceOligo RepF-CHA 69cgagtgctct atgacaaggg ctaggtt
277020DNAArtificial SequenceOligo RepQ-CHA 70cccttgtcat agagcactcg
207121DNAArtificial SequenceOligo CK19 Input 71gccaccattg
agaactccag g 21721496DNAArtificial SequenceKRT19 gBlock
72caggtctcgt atgagatatc cgcccctgac accattcctc ccttcccccc tccaccggcc
60gcgggcataa aaggcgccag gtgagggcct cgccgctcct cccgcgaatc gcagcttctg
120agaccagggt tgctccgtcc gtgctccgcc tcgccatgac ttcctacagc
tatcgccagt 180cgtcggccac gtcgtccttc ggaggcctgg gcggcggctc
cgtgcgtttt gggccggggg 240tcgcctttcg cgcgcccagc attcacgggg
gctccggcgg ccgcggcgta tccgtgtcct 300ccgcccgctt tgtgtcctcg
tcctcctcgg gggcctacgg cggcggctac ggcggcgtcc 360tgaccgcgtc
cgacgggctg ctggcgggca acgagaagct aaccatgcag aacctcaacg
420accgcctggc ctcctacctg gacaaggtgc gcgccctgga ggcggccaac
ggcgagctag 480aggtgaagat ccgcgactgg taccagaagc aggggcctgg
gccctcccgc gactacagcc 540actactacac gaccatccag gacctgcggg
acaagattct tggtgccacc attgagaact 600ccaggattgt cctgcagatc
gacaatgccc gtctggctgc agatgacttc cgaaccaagt 660ttgagacgga
acaggctctg cgcatgagcg tggaggccga catcaacggc ctgcgcaggg
720tgctggatga gctgaccctg gccaggaccg acctggagat gcagatcgaa
ggcctgaagg 780aagagctggc ctacctgaag aagaaccatg aggaggaaat
cagtacgctg aggggccaag 840tgggaggcca ggtcagtgtg gaggtggatt
ccgctccggg caccgatctc gccaagatcc 900tgagtgacat gcgaagccaa
tatgaggtca tggccgagca gaaccggaag gatgctgaag 960cctggttcac
cagccggact gaagaattga accgggaggt cgctggccac acggagcagc
1020tccagatgag caggtccgag gttactgacc tgcggcgcac ccttcagggt
cttgagattg 1080agctgcagtc acagctgagc atgaaagctg ccttggaaga
cacactggca gaaacggagg 1140cgcgctttgg agcccagctg gcgcatatcc
aggcgctgat cagcggtatt gaagcccagc 1200tgggcgatgt gcgagctgat
agtgagcggc agaatcagga gtaccagcgg ctcatggaca 1260tcaagtcgcg
gctggagcag gagattgcca cctaccgcag cctgctcgag ggacaggaag
1320atcactacaa caatttgtct gcctccaagg tcctctgagg cagcaggctc
tggggcttct 1380gctgtccttt ggagggtgtc ttctgggtag agggatggga
aggaagggac ccttaccccc 1440ggctcttctc ctgacctgcc aataaaaatt
tatggtccaa gggtgagcga gaccac 149673706DNAArtificial SequenceVIM
gBlock 73caggtctcgt atgccaccca cacccaccgc gccctcgttc gcctcttctc
cgggagccag 60tccgcgccac cgccgccgcc caggccatcg ccaccctccg cagccatgtc
caccaggtcc 120gtgtcctcgt cctcctaccg caggatgttc ggcggcccgg
gcaccgcgag ccggccgagc 180tccagccgga gctacgtgac tacgtccacc
cgcacctaca gcctgggcag cgcgctgcgc 240cccagcacca gccgcagcct
ctacgcctcg tccccgggcg gcgtgtatgc cacgcgctcc 300tctgccgtgc
gcctgcggag cagcgtgccc ggggtgcggc tcctgcagga ctcggtggac
360ttctcgctgg ccgacgccat caacaccgag ttcaagaaca cccgcaccaa
cgagaaggtg 420gagctgcagg agctgaatga ccgcttcgcc aactacatcg
acaaggtgcg cttcctggag 480cagcagaata agatcctgct ggccgagctc
gagcagctca agggccaagg caagtcgcgc 540ctgggggacc tctacgagga
ggagatgcgg gagctgcgcc ggcaggtgga ccagctaacc 600aacgacaaag
cccgcgtcga ggtggagcgc gacaacctgg ccgaggacat catgcgcctc
660cgggagaaat tgcaggagga gatgcttcag agatgagcga gaccac 706
* * * * *