US11866775

Extracted document text. Layout, formulas and symbols may not survive extraction; consult the preserved PDF for the original presentation.

Text extraction covers 17 pages, including 17 transcribed by optical character recognition. Some transcribed passages remain difficult to read; use the preserved page images to check them.

Page 1

a2) United States Patent
Gang et al.

US 11,866,775 B2
Jan. 9, 2024

US011866775B2

(10) Patent No.:
(45) Date of Patent:

(54) METHODS FOR ISOTHERMAL
MOLECULAR AMPLIFICATION WITH
NANOPARTICLE-BASED REACTIONS

(71) Applicant: Brookhaven Science Associates, LLC,
Upton, NY (US)

(72) Inventors: Oleg Gang, Setauket, NY (US);
Suchetan Pal, New York, NY (US)

(73) Assignee: Brookhaven Science Associates, LLC,
Upton, NY (US)

(*) Notice: Subject to any disclaimer, the term of this
patent is extended or adjusted under 35
U.S.C. 154(b) by 491 days.

(21) Appl. No.: 17/065,255

(22) Filed: Oct. 7, 2020

(65) Prior Publication Data

US 2021/0024990 Al Jan. 28, 2021

Related U.S. Application Data

Division of application No. 15/983,804, filed on May
18, 2018, now abandoned.

Provisional application No. 62/508,682, filed on May
19, 2017.

(62)

(60)

(51) Int. Cl.
C12Q 1/6862
B82Y 5/00
C12@ 1/6853
C12Q 1/6844
US. Cl.
CPC veces . €12Q 1/6862 (2013.01), B82 5/00
(2013.01); C12Q 1/6844 (2013.01); C12@
1/6853 (2013.01); C12Q 2545/101 (2013.01)
Field of Classification Search
CPC .. C12Q 1/6862; C12Q 1/6844; C12Q 1/6853;
C12Q 1/682; C12Q 2545/101; C12Q
2563/149; C12Q 2563/155; B82Y 5/00
See application file for complete search history.

(2018.01)
(2011.01)
(2018.01)
(2018.01)
(52)

(58)

(56) References Cited

U.S. PATENT DOCUMENTS

11,513,076 B2* 11/2022 Grabmayr .. . GOLN 21/6428

OTHER PUBLICATIONS

Zhang et al. A DNA-origami chip platform for label-free SNP
genotyping using tochold-mediate strand displacement. Small (2010)
vol. 6, No. 17, pp. 1854-1858. (Year: 2010).*

Zhu et al. Toehold-mediated strand displacement reaction triggered.
isothermal DNA amplification for highly sensitive and selective
fluorescent detection of single-base mutation. Biosensors and
Bioelectronics (2014) 59:276-281. (Year: 2014).*

Dirks et al. Triggered amplification by hybridization chain reaction.
Proc. Natl. Acad. Sci. (2004) vol. 101, No. 43, pp. 15275-15278.
(Year: 2004).*

* cited by examiner

Primary Examiner — David C Thomas
(74) Attorney, Agent, or Firm — Dorene Price

(57) ABSTRACT

The present method of detection involves increasing an
amount of analyte molecules by an isothermal molecular
amplification approach. In the present approach a starting
molecule of interest may be amplified through a reaction it
induces with specifically engineered and functionalized par-
ticles, namely protected particles A and storage particles B.
This reaction may result in a set of output DNA molecules
that is larger in number than the input DNA molecules. Thus
the reaction between nanoparticles for amplification of a
certain DNA sequence (input DNA molecules) may occur
when there is a match with a targeted molecule (stored
molecules on storage particles B) and if the DNA sequence
of the input DNA molecules does not match (partially or
completely) the targeted molecule the reaction may not
occur. Without a certain molecular input of the input DNA
molecule the reaction may not occur.

18 Claims, 7 Drawing Sheets
(7 of 7 Drawing Sheet(s) Filed in Color)

Specification includes a Sequence Listing.

Page 2

US 11,866,775 B2

Sheet 1 of 7

Jan. 9, 2024

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Page 9

US 11,866,775 B2

1
METHODS FOR ISOTHERMAL
MOLECULAR AMPLIFICATION WITH
NANOPARTICLE-BASED REACTIONS

CROSS-RELATED APPLICATION

The present application is a divisional application of U.S.
application Ser. No. 15/983,804, filed on May 18, 2018,
which claims priority from U.S. Provisional Application No.
62/508.682, filed May 19, 2017, each of which is incorpo-
rated herein by reference in their entirety.

STATEMENT OF GOVERNMENT RIGHTS

The present application was made with government sup-
port under contract number DE-SC0012704 awarded by the
U.S. Department of Energy. The United States government
has certain rights in the invention(s).

FIELD OF THE INVENTION

This application relates to isothermal molecular amplifi-
cation with nanoparticle-based reactions

BACKGROUND OF THE INVENTION

Bio-sensing, counterfeit detection and chemical safety
require sensitive detection of minute amounts of target
molecules. That is, such detection requires complex and
costly methods to reveal infinitesimal amounts of materials.
In some cases, single molecule level detection may be
achieved through optical methods. However challenges may
remain for use of such optical methods for practical real-life
applications. Other methods of detection may use physical
detection such as, for example, fluorescent detection, Raman
signal, plasmonic shift or the like.

State of the art physical detection methods focus on the
detection side of molecular sensing, i.e. they are based on
enhancing the sensitivity of the detection apparatus or
applying methods that permit more sensitive detection
modes/principles. However, there is still a need to address
the other side of the detection process by increasing the
incoming molecular signal.

SUMMARY OF THE INVENTION

The present isothermal molecular amplification approach
relates to a method of detection by increasing an amount of
analyte molecules. In the present approach a starting mol-

ecule of interest (e.g., DNA with specific input sequence as 5

the analyte molecule or referred to also as an input DNA
molecule (yellow)) may be amplified through a reaction it
induces with specifically engineered and functionalized par-
ticles, namely protected particles A and storage particles B.
This reaction may result in a set of output DNA molecules
(yellow) that is larger in number than the input DNA
molecules (yellow).

The reaction between nanoparticles for amplification of a
certain DNA sequence (input DNA molecules (yellow)) may
occur when there is a match with a targeted molecule (stored
molecules (yellow)) on storage particles B, and if the DNA
sequence of the input DNA molecules (yellow) does not
match the targeted molecule the reaction may not occur.
Without a certain molecular input of the input DNA mol-
ecule (yellow) the reaction may not occur.

The amplification by this reaction may be by a factor of
about 100-500, and larger factors may be achieved through

2

optimization and system design that one skilled in the art
may carry out. The present isothermal molecular amplifica-
tion system may be utilized with known detection methods,
thus, it may enhance the sensitivity of other methods.

A sensor apparatus for detecting targeted molecules com-
prising one or more protected particles A, storage particles
B and input DNA molecules wherein protected particles A
comprise first sequence (red) strands and second sequence
(green) strands functionalized thereon, storage particles B

10 comprise third sequence (purple) strands grafted thereon and

a

40

60

6

stored targeted DNA molecule (yellow) partially hybridized
thereon, protected particles A, deprotected by input DNA
molecules, react with storage particles B by a duplex
between third sequence (purple) strands and first sequence
(red strands), and wherein input DNA molecules have a
matching DNA sequence with stored targeted DNA (yellow)
molecules and output DNA molecules (yellow).

The foregoing summary is illustrative only and is not
intended to be in any way limiting. In addition to the
illustrative aspects, embodiments, and features described
above, further aspects, embodiments, and features will
become apparent by reference to the drawings and the
following detailed description.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the
Office upon request and payment of the necessary fee.

The foregoing and other features of this disclosure will
become more fully apparent from the following description
and appended claims, taken in conjunction with the accom-
panying drawings. Understanding that these drawings depict
only several embodiments in accordance with the disclosure
and are, therefore, not to be considered limiting of its scope,
the disclosure will be described with additional specificity
and detail through use of the accompanying drawings, in
which:

FIG. 1(a) shows an energy landscape of present isother-
mal DNA amplification and illustration of an example of an
overall scheme of an isothermal molecular amplification
reaction system;

FIG. 1(4) shows DNA replacement reaction induced

5 aggregation and isothermal amplification of yellow DNA

molecule (about 50 strands per 10 nm particle);

FIG. 2(a) shows schematic representation of origami
protected particle A;

FIG. 2(6) AFM topography of the construct respectively.
The scale bar is 200 nm;

FIG. 2(c) shows a histogram of the number of origami
attached to the 20 nm AuNP A (bar) and a Gaussian function
fitting (line curve) to the number distribution, on average
there are approximately 3-4 origami attached to each DNA
coated AUNP A;

FIG, 3(a) is a schematic representation of origami pro-
tected particle A;

FIG, 3(5) is a schematic representation of origami pro-
tected particle A and storage particle B;

FIG. 3(c) is a schematic representation of origami pro-
tected particle A, storage particle B and input DNA molecule
(yellow strand);

FIG. 3(d@) DLS hydrodynamic diameter of construct of
system shown in FIGS. 3(a)-(c): origami protected particles
A (FIG. 3(@)) shown by curve labeled (a); origami protected
particles A and storage particles B (FIG. 3(6)) mixed but not
activated by input DNA molecule (yellow strand) shown by

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US 11,866,775 B2

3

curve labeled (b); origami protected particles A, storage
particles B with added input DNA molecule (yellow strand)
(FIG. 3(c)) added in different concentrations—10 nM shown
by curve labeled (cl), 100 nM shown by curve labeled (c2),
and 1000 nM shown by curve labeled (c3) respectively;

FIG. 4(a) shows a schematic representation and a change
of fluorescence of storage particle B (2.1 nM) with the
addition of excess of first sequence (red) DNA strand;

FIG. 4(4) shows a schematic representation and a change
of fluorescence with a 1:1 ratio of protected particles A and
storage particles B (2.1 nM each), and 0.5 nM of input DNA
molecules (yellow strands) combined together;

FIG. 4(c) relative change of fluorescence intensity with
increasing amount of first sequence (red) DNA strand added
shown as black squares. The x-axis intercept line is a straight
black dotted line labeled as the number of yellow DNA
grafied/particle and corresponds to the number of stored
input molecules (yellow DNA strands) on storage particles

B. The change in the intensity for the fluorescence enhance- 2

ment assay experiments for origami protected particles A
and storage particles B-Y-Cy5 is approximately 1.2 (portion
of dotted line parallel to x axis and labeled “Number of
yellow DNA released/particle”) corresponds to 40-50 output
DNA molecules (yellow strands) released per particle (por-
tion of dotted line parallel toy axis and labeled “Number of
yellow DNA released/particle”);

FIG. 4(d) shows the kinetics of the present amplification
reaction, monitored by in situ fluorescence and DLS mea-
surements; the reaction is completed within 1 hour of
initiation;

FIG. 5 shows the effect of DNA mutation on the aggre-
gation. A+B represents a 1:1 ratio of origami protected
particles A and storage particles B-Y-Cy5. A+B+Y repre-
sents when 10 nM of input DNA molecules (yellow) are
added to a solution of A+B leading to an aggregation of A
and B particles. Presence of 10 nM of input DNA molecules
(yellow) with three mutations (A+B+Y3), two mutations
(A+B+Y2) and one mutation (A+B+Y) failed to produce
any aggregations; and all arranged according to at least some
embodiments described herein.

DETAILED DESCRIPTION OF THE
INVENTION

In the following Detailed Description, reference is made
to the accompanying drawings, which form a part hereof. In
the drawings, similar symbols typically identify similar

components, unless context dictates otherwise. The illustra- 5

tive embodiments described in the detailed description,
drawings, and claims are not meant to be limiting. Other
embodiments may be utilized, and other changes may be
made, without departing from the spirit or scope of the
subject matter presented herein. It will be readily understood
that the aspects of the present disclosure, as generally
described herein, and illustrated in the Figures, can be
arranged, substituted, combined, separated, and designed in
a wide variety of different configurations, all of which are
explicitly contemplated herein.

It will be understood that any compound, material or
substance which is expressly or implicitly disclosed in the
specification and/or recited in a claim as belonging to a
group or structurally, compositionally and/or functionally
related compounds, materials or substances, includes indi-
vidual representatives of the group and all combinations
thereof.

S

40

60

4

The present oligonucleotides can be any type of oligo-
nucleotide, either naturally-occurring or artificial. Typically,
the oligonucleotide is DNA or RNA.

In one embodiment, an isothermal molecular amplifica-
tion approach is provided by which a molecule of interest
(e.g., DNA with a specific sequence as an analyte molecule,
or referred to also as an input oligonucleotide molecule
(yellow) or “Input-Oligonucleotide”) may be amplified to
produce output molecules (i.e., “Stored-Input-Oligonucle-
otide”) with the same or different oligonucleotide (e.g.,
DNA) sequence through a reaction that may be induced
between specifically designed and oligonucleotide function-
alized particles, namely protected particles A (i.e., “Particle-
A”) and storage particles B (i.e., “Particle-B”). As a result,
more output oligonucleotide molecules (yellow), of the
same sequence as the input molecules (yellow), may be
produced than the input oligonucleotide molecules (yellow)
originally added. The molecule of interest may be a targeted
or analyte input DNA molecule and may be designated as
yellow strands. The input molecule may be a single stranded
oligonucleotide (e.g., DNA) sequence, e.g., DNA with a
length of between about 10 to 120 bases. Input DNA
molecules (yellow) may be added to initial or partially
deprotected particles A. A mixture of protected particles A
comprising oligonucleotide origami and storage particles B
can be prepared, formulated and/or provided.

Initial or Deprotected Particles a (i.e., Deprotected “Particle-
A’)

Initial or deprotected nanoparticles or particles A may be
functionalized with first sequence oligonucleotide (e.g.,
DNA) strands and second sequence oligonucleotide (e.g.,
DNA) strands. First sequence and second sequence strands
may be complementary in sequence to one another. The first
sequence strand may be designated as red strands (or “Oli-
gonucleotide-1”) and the second sequence strand may be
designated as green strands (or “Oligonucleotide-2”). Thus,
second sequence strand (green strands) may be complemen-
tary in sequence with first sequence strands (red strands.)
First sequence strands and second sequence strands (i.c.,
green and red strands) may partially hybridize to form
“Duplex 1-2”. Typically, Oligonucleotide-1 is grafted to
Particle-A.

Partially hybridized first sequence strands and second
sequence strands green and red strands) may facilitate
binding of oligonucleotide (e.g., DNA) origami to initial or
deprotected particles A. Oligonucleotide origami may pro-
tect initial or deprotected particles A from reacting to form
aggregates or binding with targeted particles B. When
oligonucleotide origami binds to initial or deprotected par-
ticles A they are transformed to protected particles A.
Protected Particles A (i.e., Protected “Particle-A”)

The design of protected nanoparticles or particles A may
render them nonreactive and protected from aggregation
with other particles, namely storage particles B (i.e., “Par-
ticle-B”). The design of protected particles A may be ren-
dered reactive with the addition of an input oligonucleotide
molecule (yellow). The protected particle A when reacted
with the input oligonucleotide molecule (yellow) (i.e., “Oli-
gonucleotide-Y”) may become partially or fully deprotected
and amplification may be enabled. The amplification reac-
tion may proceed until the deprotected particles A are
consumed/reacted and no more release is possible (i.e.,
substantially all or all stored input oligonucleotide mol-
ecules are released and the reaction ends). Fully deprotected
particles A cannot bind to input oligonucleotide molecule
(yellow).

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US 11,866,775 B2

5

The protected particles A may have a shell or a shield of
oligonucleotide origami. Oligonucleotide origami may act
as a protective shield (“umbrella” or “blocking plates”) for
initial or deprotected designed particles A transforming them
to protected particles A. Blocking plates may comprise
oligonucleotide (e.g., DNA) origami. Oligonucleotide ori-
gami may be bound to initial or deprotected designed
particles A. In some embodiments, oligonucleotide origami
may be planar and have a shape that is rectangular, round or
other designed 2-dimensional constructs, or may be a 3-di-
mensional construct, such as, for example, polyhedron
shaped. Planar oligonucleotide origami plates may be bound
to initial designed nanoparticles or deprotected particles A
may act as a protective shield/shell “umbrella” or “blocking
plates”). The oligonucleotide origami may be a coating on
particles A.

Oligonucleotide origami may provide steric shielding
against hybridization between oligonucleotide strands (e.g.,

first sequence (red) strands, i.e., “Oligonucleotide-1”) bound 2

to particles A and oligonucleotide strands bound to other
particles (e.g., storage particles B). Specifically, oligonucle-
otide origami may provide steric shielding against hybrid-
ization of third sequence (purple or “Oligonucleotide-3”)
strands on storage particles B and first sequence (red or
“Oligonucleotide-1”) strands on initial or deprotected par-
ticles A. Such a design of the shell may prevent aggregation
of particles A with, for example, storage particles B in the
solution when no input DNA molecule (yellow or “Input-
Oligonucleotide”) is present. The oligonucleotide origami
shape can be designed by one skilled in the art to maximize
the particle A shielding or optimize other reaction param-
eters (e.g. DNA hybridization with input DNA).

Storage Particles B (i.e., “Particle-B”)

Storage nanoparticles or particles B (i.e., Particle-B) may
be functionalized with third sequence strands. The third
sequence strands may be grafted onto storage particles B.
The third sequence strands may be designated as purple
strands (or “Oligonucleotide-3”). Third sequence (purple)
strands may form a duplex with first sequence (red) strands
of initial or deprotected particles A, i.e., when particles A are
not protected by oligonucleotide origami. Such duplex is
referred to as “Duplex 1-3.” Storage particles B may also
have stored input DNA molecules, designated as yellow
strands hybridized partially on them, e.g., a hybridization
between “Oligonucleotide-3” and “Stored-Input-Oligo-
nucleotide” to form “Duplex 3-Y.” Stored input oligonucle-
otide molecules (yellow) partially hybridized on storage

particles B may have the same or different oligonucleotide 5

sequence as input oligonucleotide molecule (yellow). If it is
a different sequence, then there may be a portion or part that
is different and a portion or part that is the same. For
example, less than about 50%, about 40%, about 30%, about
20, about 10% or about 5% can be different.
Amplification Reaction

The reaction between specifically engineered and oligo-
nucleotide-functionalized nanoparticles, namely partially
deprotected particles A (i.e., “Particle-A”) and storage par-
ticles B (i.e., “Particle-B”), may result in amplification of a
certain oligonucleotide (e.g., DNA) sequence of stored input
DNA molecules (yellow) on storage particles B if they
match a targeted or analyte input DNA molecule (yellow)
and a cascade reaction is induced. On the other hand, if the
oligonucleotide sequence of stored input DNA molecules on
storage particles B do not match the targeted input oligo-
nucleotide molecule, no reaction may take place. No reac-

a

8

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8

3

40

4

35

2
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B

6

tion may occur without addition of a targeted or analyte
input DNA molecule to partially deprotected particles A and
storage particles B.

An input oligonucleotide molecule (yellow) (¢.g., a single
stranded DNA sequence, “Input-Oligonucleotide”) may
induce an amplification reaction between certain designed
initial or partially deprotected particles A (“Particle-A”) and
storage particles B (“Particle-B”). The reaction may produce
and result in a release of new output DNA molecules
(yellow) stored on storage particles B (“Stored-Input-Oli-
gonucleotide”). When input oligonucleotide molecules (yel-
low) are added to protected particles A, the protected par-
ticles A may be deprotected. When protected particles A are
partially deprotected, an amplification or cascade reaction
may produce output oligonucleotide molecules (yellow) in a
much larger number/amount than the input oligonucleotide
molecules (yellow) that were added to start the reaction. In
turn, the released new output DNA molecules may act as
input DNA molecule and promote one or more follow-on
(second, third, and so on) reaction(s) and new additional
output DNA molecules (species) of the same sequence (T1)
or different sequence (I2) may be produced and released.
The cascade reaction may repeat until protected particles A
are fully deprotected.

As used herein amplification refers to a number of output
oligonucleotide (e.g., DNA) molecules that is larger than
input oligonucleotide molecules. The amplification by the
present reaction may be by a factor of 100-200, 100-300,
100-400, or 100-500 and larger factors depending on the
details of the desired nanoparticle design, i.e. it size,
sequence length, environmental parameters etc. The ampli-
fication factor (i.e., ratio of output number of molecules to
input number of molecules) may depend on the size of
nanoparticles A and B (about 10-20 nm), design of the shell
and protection shield (DNA origami), and reaction param-
eters (e.g., salt, particle concentration, DNA, temperature).
The amplification reaction may occur without temperature
ramping. The operational temperature for the “amplifier”
may be determined by nanoparticle design, as would be
known to a skilled artisan, so it may be referred to as an
isothermal amplification. The operational temperature may
be room temperature.

The input molecules (yellow) are capable of deprotecting
a subset of the protected particles A partially by removing
one or more ‘umbrella’ oligonucleotide (e.g., DNA) origa-
mis (“blocking plates”). The protected particles A may be
deprotected by release of blocking plates. After particles A
are deprotected, a duplex between third sequence (purple)
strands on particles B and first sequence (red) strands on
particles A may be energetically favorable (i.e., “Duplex
1-3”). Deprotected particles A and storage particles B may
bind when first sequence (red) and third sequence (purple)
strands are able to hybridize. Multiple binding of particles A
and B may result in particle aggregation rather than isother-
mal amplification.

For example, after adding 0.5 nM input DNA (yellow)
molecules, the reaction between partially deprotected par-
ticles A and storage particles B (having stored input (yellow)
strands partially hybridized on it) may be activated. Stored
input DNA (yellow) strands on storage particles B may be
hybridized with third sequence (purple) strands that are
grafted on. The inter-particle reaction between partially
deprotected particles A and storage particles B may produce
and release new output molecules (yellow) of the same or
different type (that have been stored on the particles B). In
turn, the newly released output oligonucleotide molecules
(yellow) may further promote inter-particle reaction that

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US 11,866,775 B2

7

may lead to further releases of output oligonucleotide mol-
ecules (yellow). Thus, amplification of the input DNA
molecules (yellow) in the cascade of “chain”-like reactions
may be obtained. (The amount of input DNA molecule
(yellow) may be, for example, about 0.5 nM to about 1000
nM.)

As depicted in FIG. 1(a), the energy landscape of the
present isothermal oligonucleotide molecule amplification
system may relate to a transition from a higher energy
metastable state to a lesser energy configuration to a lowest
energy state. This transformation through the energy states
from a high energy state to low energy state may be via a
sequential toehold mediated strand displacement reaction by
input oligonucleotide molecule (yellow). The input oligo-
nucleotide molecules (strands) may be utilized to overcome
an initial energy barrier, or may be utilized to activate the
amplification reaction through for example, deprotection
(ie., release) of blocking plates, made out of oligonucleotide
origami.

In one embodiment, input molecules and output mol-
ecules may be the same and amplification of the same type
(noted as ‘T1*) may occur. The amplification reaction may
be activated through deprotection (i.e., release of blocking
plates made out of oligonucleotide origami) of particles A
and the reaction may feed itself: additional strands of output
molecules may be released while A and B particles begin to
aggregate due to binding of oligonucleotide on their sur-
faces. The reaction may end when substantially all or all
initial particles A are reacted and output molecules (the same
type as the input molecule, but larger amount than the input
molecules) are released.

In one embodiment, multiplication of other predefined
molecules may be achieved, whereby an input molecule
‘TI’ may result in the release of output molecules ‘T2’
(stored on storage particles B), and the amount of ‘T2’ may
be larger than ‘T1’. The amplification factor may be defined
as a ratio of the number/concentration of output molecules
to the number/concentration of input molecules, i.e. [T2]/
[Tl].

Another Embodiment

In one embodiment, a method for amplifying an Input-
Oligonucleotide is provided. The Input-Oligonucleotide can
be of any length. Typically, the Input-Oligonucleotide is
about 5 to about 200 bases, or about 10 to about 120 bases.
Typically, the Input-Oligonucleotide is an unknown sample
of oligonucleotides that is to be identified.

The method includes the use of a plurality of particles.

The particles can be any type of particle to which oligo- 5

nucleotides can be grafted. Typically, the particles can have
diameters in the nanometer to micron range. For example,
nanoparticle can range from about 5 to about 100 nm in
diameter. Typically, the particles are metallic or semicon-
ducting. Examples of suitable particles include gold (Au),
silver (Ag), copper (Cu), platinum (Pt), palladium (Pd) and
combinations thereof. A plurality of such particles is func-
tionalized with oligonucleotides. Particles which are func-
tionalized with the same oligonucleotides have the same
designation.

For example, a plurality of particles, designated as Par-
ticle-A, can be functionalized with a plurality of Oligonucle-
otide-1 and a plurality of Oligonucleotide-2. Typically, Oli-
gonucleotide-1 is grafted onto Particle-A. There are about 25
to about 400 Oligonucleotide-1 attached per about 5 nm to
about 40 nm diameter of Particle-A. Typically, there are
about 200 Oligonucleotide-1 attached per about 20 nm

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diameter of Particle-A. Oligonucleotide-1 is hybridized to
Oligonucleotide-2 to form Duplex 1-2. Typically, Duplex
1-2 is formed by a partial hybridization.

Oligonucleotide origami is attached to Duplex 1-2 to
render Particle-A non-reactive (i.e., protected). The origami
can be any size which protects Particle-A. The origami is
2-dimensional or 3-dimensional, for example, the origami
can be rectangular. A typical size of the origami is about 5
to about 100 nm per about 20 nm of particle diameter, for
example, about 50 nm in length per about 20 nm diameter
of Particle-A. Due to selective hybridization between Oli-
gonucleotide-1 and Oligonucleotide-2, Particle-A may bind
to several origami structures. Typically, there can be about 2
to about 10 origami per about 5 nm to about 40 nm diameter
of Particle-A. For example, there can be about 3 to about 4
origami per about 20 nm diameter of Particle-A.

Additionally, a plurality of particles, designated as Par-
ticle-B, can be functionalized with a plurality of Oligonucle-
otide-3 and a plurality of Stored-Input-Oligonucleotide.
Typically, Oligonucleotide-3 is grafted onto Particle-B.
Typically, there are about 25 to about 400 Oligonucleotide-3
attached per about 5 nm to about 40 nm diameter of
Particle-B. For example, there can be about 200 Oligonucle-
otide-3 attached per about 20 nm diameter of Particle-B.
Oligonucleotide-3 is hybridized to Stored-Input-Oligonucle-
otide to form Duplex 3-Y. Typically, Duplex 3-Y is formed
by a partial hybridization. Typically, the hybridization does
not take place at the ends of the oligonucleotide strands; that
is, hybridization takes places within the strands, referred to
as “inter-strand” hybridization.

In one embodiment, at least a portion of the sequences of
Input-Oligonucleotide and the Stored-Input-Oligonucleotide
are identical. For example, the sequences are at least about
50%, about 60%, about 70%, about 80%, about 90%, about
95%, about 97%, about 98%, about 99% or 100% identical.

The Input-Oligonucleotide, Particle-A and Particle-B are
mixed. The mixing can occur in any order. Typically, the
reaction is under isothermal conditions. For example, the
whole reaction can occur at about room temperature. Typi-
cally, the relative amount of Particle-A:Particle-B is about
10:1 to about 1:10, e.g., about 1:1. Typically, the relative
amount of Input-Oligonucleotide to Particle-A is at least
about 1:4. The amount of Input-Oligonucleotide to Par-
ticle-A can be in vast excess, e.g., about 1000:4

Typically, the Input-Oligonucleotide is added to Par-
ticle-A first. Once mixed, a cascade reaction occurs so that:
i) the Input-Oligonucleotide hybridizes with Oligonucle-
otide-2 to form Duplex Y-2, thereby removing Oligonucle-
otide-2 and origami from Particle-A, and exposing Oligo-
nucleotide-1, ii) exposed Oligonucleotide-1 hybridizes with
Oligonucleotide-3 to form Duplex 1-3, thereby releasing
Stored-Input-Oligonucleotide, and iii) step (i) reoccurs with
the Stored-Input-Oligonucleotide replacing the Input-Oligo-
nucleotide until the reaction terminates. Typically the reac-
tion terminates because the Stored-Input-Oligonucleotide
has all been released. By the reaction, the Input-Oligonucle-
otide is amplified by the release of the Stored-Input-Oligo-
nucleotide. Typically, the Input-Oligonucleotide is amplified
by a factor of about 50 to about 1000.

The reaction can occur efficiently because the melting
temperature (Tm) of Duplex Y-2 is greater than the Tm of
Duplex 1-2; and the Tm of Duplex 1-3 is greater than the Tm
of Duplex 3-Y. For example, the Tm of Duplex Y-2 is at least
about 5% greater or at least about 20% greater than the Tm
of Duplex 1-2. The Tm of Duplex 1-3 is at least about 5%
greater or at least about 20% greater than the Tm of Duplex
3-¥.

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In another embodiment, a method for identifying an
Analyte-Oligonucleotide is provided. The method comprises
providing a plurality of Particle-A and a plurality of Particle-
B. Particle-A is functionalized with Oligonucleotide-1 and
Oligonucleotide-2, Oligonucleotide-1 and Oligonucle-
otide-2 are hybridized to form Duplex 1-2, and have
attached oligonucleotide origami. The origami renders Par-
ticle-A non-reactive. Oligonucleotide-1 is typically grafted
onto Particle-A. Particle-B is functionalized with Oligo-
nucleotide-3 and Target-Oligonucleotide. Typically, Oligo-
nucleotide-3 is grafted onto Particle-B. Oligonucleotide-3
and Target-Oligonucleotide are partially hybridized to form
Duplex 3-Y. The Analyte-Oligonucleotide, Particle-A and
Particle-B are mixed, in any order. If the sequences of the
Analyte-Oligonucleotide and the Target-Oligonucleotide are
substantially identical (e.g. 99% or 100% identical), a cas-
cade reaction occurs so that: i) the Analyte-Oligonucleotide
fully hybridizes with Oligonucleotide-2 to form Duplex Y-2,
thereby removing Oligonucleotide-2 and origami from Par-
ticle-A, and exposing Oligonucleotide-1, ii) exposed Oligo-
nucleotide-1 partially hybridizes with Oligonucleotide-3 to
form Duplex 1-3, thereby releasing Target-Oligonucleotide,
and iii) step (i) reoccurs with the Target-Oligonucleotide
replacing the Analyte-Oligonucleotide. The Analyte-Oligo-
nucleotide is identified as being the Target-Oligonucleotide
if Particle-A and Particle-B aggregate in the reaction mix-
ture.

In another embodiment, a sensor apparatus for identifying
an Analyte-Oligonucleotide is provided. The apparatus com-
prises a plurality of Particle-A, and a plurality of Particle-B.
Particle-A is functionalized with Oligonucleotide-1 and Oli-
gonucleotide-2. Oligonucleotide-1 and Oligonucleotide-2
are hybridized to form Duplex 1-2. Oligonucleotide Origami
is attached to Duplex 1-2. The origami renders Particle-A
non-reactive. Oligonucleotide-1 is typically grafted onto
Particle-A. Particle-B is functionalized with Oligonucle-
otide-3 and Target-Oligonucleotide. Typically, Oligonucle-
otide-3 is grafted onto Particle-B. Oligonucleotide-3 and
Target-Oligonucleotide are hybridized to form Duplex 3-Y.
An Analyte-Oligonucleotide is mixed with Particle-A and
Particle-B, in any order. If at least a portion of the sequence
of Analyte-Oligonucleotide is identical to the Target-Oligo-
nucleotide, a cascade reaction occurs so that: i) the Analyte-
Oligonucleotide fully hybridizes with Oligonucleotide-2 to
form Duplex Y-2, thereby removing Oligonucleotide-2 and
origami from Particle-A, and exposing Oligonucleotide-1,
ii) exposed Oligonucleotide-1 partially hybridizes with Oli-
gonucleotide-3 to form Duplex 1-3, thereby releasing Tar-
get-Oligonucleotide, and iii) step (i) reoccurs with the Tar-

get-Oligonucleotide replacing the Analyte-Oligonucleotide. 5

The Analyte-Oligonucleotide is identified as being the Tar-
get-Oligonucleotide if there is an aggregation of Particle-A
and Particle-B in the reaction mixture.

EXAMPLES
Example 1

Begin with two 20 nm gold nanoparticles (AuNP),
namely AuNP A (initial or deprotected particles A) and
AUuNP B (storage particles B), where each may be function-
alized with ‘(red strand)’ first sequence strands and ‘(purple
strand)’ third sequence strands of DNA, respectively. Red
(first sequence) strands may be grafted on AuNP A and
purple (third sequence) strands may be grafted on AuNP B.
There may be an approximately 200 first sequence (red)
strands on AuNP A and approximately 200 third sequence

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(purple) strands on AUNP B. AuNP A, in addition to the first
sequence (red) strand, may be functionalized with a second
sequence (green) strand. Part of the second sequence (green
strand) of DNA may contain a sequence portion (5' AAT
CGC ATG C 3'—underlined) which may be complementary
to the part of first sequence (red) strand of DNA sequence (5'
GCA _TGC_GAT T 3'—underlined). First sequence and
second sequence (green and red) strands of AuNP A may be
partially hybridized. Due to selective hybridization between
the first sequence and second sequence (red and green)
strands particle (AUNP) A may bind to several origami
structures as shown in FIG. 1(6)(i). Particle (AuNP) A may
be protected by the origami structures.

On the other hand, in FIG. 1(a), AuNP B functionalized
with the third sequence (purple) strand, has a part of the third
sequence ‘purple strand’ that may contain a sequence (5'
GCT GCT GTA A 3'—underlined) which may be comple-
mentary to a part of a yellow (input) strand of DNA
sequence (5' TTA CAG CAG C 3'—underlined) of an input
molecule. The yellow (input) strand may have a specific
inter strand hybridization with the third sequence (purple)
strand. The specific inter strand hybridization may allow
input molecule (yellow) to bind to storage particle (AUNP)
B as shown in FIG. 1(4)(ii).

DNA replacement reaction induced aggregation and iso-
thermal amplification of DNA input molecule (yellow input
strand) is shown in FIG. 1(4). First, in FIG. 1(6)(i), input
DNA molecule (yellow strand) may fully hybridize with
second sequence (green) strand (Tm~64.4° C.) of particle
(AuNP) A (initial or protected particle). Partially hybridized
second sequence and first sequence (green and red) strands
(Tm~44.8° C.) may be displaced. This displacement may
lead to deprotection of first sequence (red) strands grafted on
now deprotected particle (AuNP) A (former initial or pro-
tected particles). The origami structures bound to initial or
protected particle (AuNP) A may be released. First sequence
(red) strands of deprotected particle (AuNP) A may be
exposed.

The exposure of first sequence (red) strands of partially
deprotected particle (AuNP) A may lead to a further DNA
replacement induced aggregation reaction as shown in FIG.
1(6)(ii). At this point, partially complementary stored input
molecule (yellow strand) and third sequence (purple) strands
(Tm~42.3° C.) on storage particle (AuNP) B may be dis-
placed by partially deprotected AuNP A and exposed first
sequence (red) strands on partially deprotected particle
(AuNP) A forming a first sequence (red) and third sequence
(purple) strand duplex (Tm~64.2° C.). Subsequent release of
multiple output molecules (yellow strands) and aggregation
of partially deprotected particles (AUNPs) A and storage
particles B may occur as shown in FIG. 1(b)(iii).

The formation of the duplex between third sequence
(purple) strands and first sequence (red) strands may be
energetically favorable and if these two strands are able to
hybridize then partially deprotected particles A and storage
particles B may bind (i.e., aggregate). Multiple binding of
particles A and particles B may result in particle aggregation
rather than isothermal amplification. Bare particles A and
particles B functionalized with first sequence (red) strands
and third sequence (purple) strands respectively may aggre-
gate upon mixing at room temperature as first sequence (red)
and third sequence (purple) strands may hybridize with each
other.

Input molecules (yellow (input) strands) of DNA may be
added in an amount of 0.5 nM to the solution containing
origami protected or initial particles A and stored input
molecules (yellow strands) hybridized with third sequence

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Bl

(purple) strands grafted on particle B. The amount of stored
input molecules (yellow strands) may be 0.5 nM to 1000
oM. A 0.5 nM or greater amount of yellow (input) strands
may lead to a cascade of reactions and may produce and
release a larger number of output molecules of DNA (yellow
strands) than input strands (yellow) as delineated in FIG.
1(6).

As shown in FIG. 1(a), there may be a higher energy of
hybridization between input DNA molecules (yellow)

strands and second sequence (green) strands on protected :

particles A as compared to second sequence (green) strands
and first sequence (red) strands. Selective hybridization
between second sequence (green) strands and first sequence
(red) strands on some protected particles A may be inter-
rupted. One or more of the DNA origami bound to protected
particles A may be ejected from particles A. The removal of
DNA origami from protected particles A or from the vicinity
of protected particle A may lead to the activation of several

first sequence (red) strands on deprotected particle A which 2

were previously dormant when under the protected particles
A that had “umbrella” shielding of the DNA origami.

As shown in FIG. 1(4)(i), the activated first sequence
(red) strands on partially deprotected particle A may hybrid-
ize with third sequence (purple) strands of storage particle B.
Stored input molecules (yellow strands) from storage par-
ticles B in the solution may be released. As shown in FIG.
1(6)(iii), released output molecules (yellow strands) may in
turn release more DNA origami shields on remaining pro-
tected particles A, and that may promote a cascade of
deprotection and hybridization events between partially
deprotected particles A and storage particles B. The outcome
of the avalanche may be a liberation of output molecules of
single-stranded (ss) yellow output strands triggered by a
small amount of input molecules (yellow strands) thereby
achieving an amplification of the input molecule (yellow
strand). The cascade reaction may be irreversible.

Example 2—Fabrication and Characterization of
AuNP-Origami Constructs (Protected Particles A)

AuNP-Origami constructs were fabricated by mixing 20
nm AuNPs A and rectangular origami together (several per
particle) by mixing and letting origami hybridize on initial
or deprotected particles A.

After purification by known methods such as for example
gel electrophoresis, atomic force microscopy (AFM) mea-
surements were carried out to characterize origami-AuNP

conjugates (protected particles A). An AFM image is shown 5

in FIG. 2(6). The height profile extracted from AFM imaging
shows several DNA origami structures with approximately
1.5 nm height that are connected to an approximately 18 nm
height spherical AuNP (particles A) in high yield. There are
very few unbound DNA origami found in the area of view
suggesting serial purification was successful. In FIG. 2(c) a
histogram of number of origami bound to one particle A is
shown and the distribution fits a Gaussian profile. On an
average 3-4 origami structures were found to be connected
to one 20 nm AuNP A. The hydrodynamic diameter of the
construct was measured to be 200 nm with broad size
distribution using Dynamic Light Scattering (DLS) shown
as black trace in FIG. 3(d) which is significantly larger than
that of only origami (70 nm) and the AuNP A (30 nm).
Amplification experiments were conducted with these as
prepared AUNPs with AuNP-origami constructs (protected
particles A).

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Amplification Experiments:

An experiment was conducted in a test tube at room
temperature by mixing particles (AuNPs) A protected with
origami (protected AUNP-A) with storage particles B having
stored input (yellow) strands on it (AUNP-B-Y). Particles A
and B are in a 1:1 ratio, where Y represents third sequence
(purple) strands hybridized with stored input (yellow)
strands on storage particle B. The concentration (amount) is
2.1 nM each of particle A and particle B. The outcomes of
the experiments were verified by two methods, DLS mea-
surements and steady state fluorescence spectroscopy. These
inter-particle hybridization reactions were completed within
one hour as shown in FIG. 4(c), as verified by in situ
fluorescence and DLS measurements.

DLS (Dynamic Light Scattering) Measurements.

The ensemble hydrodynamic diameter was measured of a
1:1 mixture with protected particle A (protected AuNP-A)
and storage particle B having stored input (yellow) strands
on it (AuNP-B-Y) [where Y denotes third sequence (purple)
strand hybridized with stored (yellow) strands on storage
particle B where stored (yellow) strands have the same or
different DNA as yellow input strands]. The concentration is
2.1 nM for each of protected particle A and storage particle
B and is shown by trace (b) in FIG. 3(d). The ensemble
hydrodynamic diameter exhibited similar size distribution
for only protected AuNP A size distribution except there is
a shoulder at lower size due to the presence of smaller
AuNP-B-Y (hydrodynamic diameter approximately 30 nm).
The ensemble hydrodynamic diameter did not change over
time for up to several hours validating a premise of steric
protection of protected particle A from reaction with storage
particle B (i.e., protected particles A were protected against
aggregation).

To the approximately 2 nM solution of each protected
particle A and storage particle B, different amounts of input
molecules (yellow strands) were added and kept at room
temperature for 12 hours. Addition of 1 (1000 nM) input
molecule (yellow strand) was found to lead to aggregation of
aggregates of partially deprotected particles A with storage
particles B with sizes shown by trace (c3) in FIG. 3(d). The
amount of input molecule (yellow strand) was sequentially
decreased by orders of magnitude and found that 100 nM
(trace (c2)) and 10 nM (trace (cl)) concentration of input
molecule (yellow strands) leads to aggregations of partially
deprotected particles A with storage particles B as shown by
and trace respectively. The concentration of input molecule
(yellow strand) decreased to 0.5 nM was found to lead to
visible aggregation of partially deprotected particles A with
storage particles B. However, the system without any input
molecule (yellow strand) added remained dispersed without
signs of aggregations between protected particles A with
storage particles B. Thus, the DLS measurements showed
that the presence of 0.5 nM amount of yellow input strand
(which can be considered miniscule) leads to a cascade of
strand displacement reactions, which, in turn, leads to aggre-
gation of partially deprotected particles A and storage par-
ticles B, and the release of output molecules (yellow strands)
greater than input molecules (yellow strands) i.e. amplifi-
cation of input molecules (yellow strands). In order to gain
a quantitative picture of this amplification process, a fluo-
rescence enhancement assay described in the following
section was developed.

Fluorescence Measurements:

In order to obtain a quantitative understanding of the
number of output molecule (yellow DNA strands) released
during the aggregation of partially deprotected particles A
and storage particles B, Cy5 labeled yellow DNA was used.

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CyS has excitation maximum at approximately 650 nm and
emission maximum at approximately 670 nm. The metal
nanoparticle surface is in close proximity to the bound
fluorophores and typically exhibits a quenched fluorescence.
However, when the fluorophores are displaced from the
metal surface proximity, the fluorescence is recovered back
to the intrinsic fluorescence value. This method was used to
quantify the amount of output molecules (yellow DNA
strands) released from the cascade reaction. The number of
stored input molecules (yellow strands) carried by storage
particle B was estimated. Known 500 times excess of
fluorophore modified yellow input strand was added to a
known concentration in nanomolar range depending on
particular analysis of storage particle B, for example, in the
1 to 10 nM range. Although 500 times of Cy5-yellow DNA
(Cy5) was added, it was found that approximately 200 times
remained in the solution as estimated by UV-Vis and fluo-
rescence measurements of the supernatant. These results
indicated that there are approximately 300 copies of stored

input molecules (yellow DNA strands) on a single storage 2

particle B. To figure out the quenching effect of 20 nm
AuNPs on the Cy5, a strand displacement assay was carried
out. Different amounts of excess of first sequence (red)
strands on partially deprotected particles A, which is fully
complementary to the input molecules (yellow input strands)
were added to the AuNP-Y-CyS (labeled storage particle B)
solution.

FIGS. 4(a) through (d) show results from a fluorescence
enhancement assay for the determination of the number of
DNA released. Due to high thermodynamic stabilization of
duplex formation between first sequence (red) and third
sequence (purple) strands, excess first sequence (red) strands
can chase off input molecule (yellow strands) in the solution
creating a separation between the fluorophore and AuNP
shown in the upper panel of FIG. 4(a). The change in steady
state fluorescence change is shown in FIG. 4(a). In the
presence of 300 times excess yellow input strands it is
observed that the fluorescence signal of Cy5 gets amplified
by approximately 1.85 times the initial intensity. In FIG.
4(c), the relative fluorescence intensity change is plotted
with the increasing amount of the first sequence (red) DNA
strand added. The plot shows steady increase of the fluo-
rescence up to 200 times excess of first sequence (red) DNA
strands. As first sequence (red) DNA amount increases no
change of intensity was observed, and this suggests that the
bound yellow-Cy5 DNA input strands were removed by the
excess of first sequence (red) DNA strands. The intercept of
the black dotted line with x-axis in FIG. 4(c), corresponds to
the number of stored input molecule (yellow DNA) per

particle. This observation further verifies that there are 5

approximately 300 stored input molecules (yellow DNA
input strands) grafied on storage particle B.

Fluorescence enhancement assay was carried out to inves-
tigate how many yellow-Cy5 strands were released during
the aggregation of partially deprotected particles A and
storage particles B in the absence or presence of 0.5 nM.
input molecules (yellow DNA strands). During the aggre-
gation process involving AuNPs A and B, all of the comple-
mentary DNAs are not necessarily fully hybridized. Thus, a
certain percentage of output molecules (yellow strands)
were released, as shown in the upper panel FIG. 4(5). The
amount of the output molecules (yellow DNA strands)
released could be estimated from the fluorescence signal
change in the solution. The observed change was 1.2-1.4
times that of the initial intensity. The dotted lines in FIG.
4(c) indicate the number of released DNA, based on the
fluorescence signal change. This result suggested that

0

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15-22% of the yellow-CyS input strands were released from
the storage particles B during the aggregation process. The
amplification factor was calculated as a number of output
molecules (yellow strands) released (output) per one input
molecule (yellow input strand) as 200-300, as shown in
Table 1.

TABLE 1

Amplification factor is the ratio of amount of output
strand released to input strand (or a number of output strands
pet one input strand) for the experimental realizations.

Cone. of Cone. of Conc. of = ~Output
Particle Particle Input DNA
A B DNA Released Amplification
(mM) (nM) (mM) (mM) Factor
24 24 as 300%,1542.1 191
24 24 Os 300%,2242.1 277

Molecular Selectivity for Amplification Reaction:

The robustness of the present molecular amplification
approach in the presence of slightly different input mol-
ecules was investigated. This investigation tested the selec-
tivity of amplification (i.e., only targeted or analyte input
molecules are amplified) and how the system can withstand
the interference from other non-targeted molecules. The
point mutations in DNA can produce very similar molecular
species and may not be distinguishable from the original
DNA. These mutations may relate to the origin of genetic
disorders or may be the cause for cancer. Robustness of
amplification against sequence mutations may contribute
counterfeit detection, chemical safety, bio-sensing, DNA
nanotechnology, information-computing and other applica-
tions. Improved detection sensitivity of targeted molecules
may be useful in bio-sensing, DNA nanotechnology, infor-
mation-computing, counterfeit detection, chemical safety
and other applications.

The present amplification system was tested in the pres-
ence of the modified input molecules (yellow strands) with
1, 2 and 3 point mutations. The point mutations are replace-
ment of a single base nucleotide with another randomly
chosen base (from the other three bases). Three sequences
were selected to mimic the 1, 2 and 3 point mutations shown
in FIG. 5. At low concentrations of 10 nM, it was observed
that the mutated input molecules (mutated yellow strands)
are not able to initiate the cascade reaction, as evident from
the absence of aggregations confirmed by no change in the
DLS hydrodynamic diameter (FIG. 5). This experiment
illustrates that the present amplification method is capable of
discriminating the signal from closely related signals with
near or at 100% specificity. Thus the present amplification
system can be utilized with other intricate amplification
schemes and circuits (analogues to electronics) that can
perform complex operations with an input molecular signal
for a variety of applications.

Additional Embodiments

Tn one embodiment, a method is provided for isothermal
molecular amplification comprising formulating a mixture
of protected particles A comprising DNA origami and of
storage particles B, where protected particles A do not react
with storage particles B, where storage particles B are
hybridized with stored input DNA molecule (yellow) having
an input sequence, and where stored input DNA molecule
(yellow) has the same input sequence as or has a portion of
input sequence that is the same as an input DNA molecule

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(yellow); adding the input molecule (yellow) to the mixture:
releasing DNA origami from some of the protected particles
A and deprotecting some of the protected particles A; iii.
released origami reacting with input DNA molecule (yel-
low); deprotected particles A reacting with storage particles
B and inducing release of stored input DNA molecule
(yellow) from storage particle B as an output DNA molecule
(yellow); and released output DNA molecule (yellow) mix-
ing with some of the remaining protected particles A creat-
ing a cascade reaction of ii. through iv. repeating until stored
input DNA molecules are released and deprotected particles
A are fully deprotected. In one embodiment, protected
particles A are functionalized with first sequence (red)
strands and second sequence (green) strands. In one embodi-
ment, protected particles A are functionalized with first
sequence (red) strands and second sequence (green) strands
that are complementary in sequence with one another. In one
embodiment, partially hybridized first sequence (red)
strands and second (green) strands permit binding of origami

to protect particles A. In one embodiment, protected par- 2

ticles A are functionalized with first sequence (red) strands
and second sequence (green) strands and wherein input
molecule Y fully hybridizes with second sequence (green)
strands. In one embodiment, partially hybridized first
sequence (red) strands and second sequence (green) strands
permit binding of origami to protect particle A, wherein
input molecule (yellow) fully hybridizes with second
sequence (green) strands and displaces partially hybridized
first sequence (red) strands and second sequence (green)
strands, and wherein origami is released, protected particles
A are deprotected and first sequence (red) strands are
exposed. In one embodiment, protected particles A are
functionalized with first sequence (red) strands and second
sequence (green) strands, and storage particles B are fune-
tionalized with third sequence (purple) strands, wherein
deprotecting particle A exposes first sequence (red) strands,
and wherein exposed first sequence (red) strands form a
duplex with third sequence (purple) strands. In one embodi-
ment, protected particles A are functionalized with first
sequence (red) strands and second sequence (green) strands,
and storage particles B are functionalized with third
sequence (purple) strands, wherein deprotecting protected
particles A exposes first sequence (red) strands, and wherein
exposed first sequence (red) strands form a duplex with third

S

a

8

w

40

16

sequence (purple) strands, and deprotected particles A bind
with storage particles B. In one embodiment, storage par-
ticles B are functionalized with third sequence (purple)
strands. In one embodiment, storage particles B are func-
tionalized with third sequence (purple) strands, and third
sequence (purple) strands form a duplex with first sequence
(red) strands of deprotected particle A. In one embodiment,
storage particles B are functionalized with third sequence
(purple) strands and wherein third sequence (purple) strands
are complementary in sequence with part of stored input
molecule (yellow). In one embodiment, partially hybridized
third sequence (purple) strands of storage particles B and
stored input molecule Y permit binding of stored input
molecule (yellow) to storage particle B. In one embodiment,
the invention is a DNA output molecule produced by the
aforementioned methods. In one embodiment, the present
invention provides a sensor apparatus for detecting targeted
molecules comprising one or more protected particles A,
storage particles B and input DNA molecules wherein
protected particles A comprise first sequence (red) strands
and second sequence (green) strands functionalized thereon,
storage particles B comprise third sequence (purple) strands
grafted thereon and stored targeted DNA molecule (yellow)
partially hybridized thereon, protected particles A, depro-
tected by input DNA molecules, react with storage particles
B by a duplex between third sequence (purple) strands and
first sequence (red strands), and input DNA molecules have
a matching DNA sequence with stored targeted DNA (yel-
low) molecules and output DNA molecules (yellow).

While various aspects and embodiments have been dis-
closed herein, other aspects and embodiments will be appar-
ent to those skilled in the art. The various aspects and
embodiments disclosed herein are for purposes of illustra-
tion and are not intended to be limiting, with the true scope
and spirit being indicated by the following claims.

INCORPORATION OF SEQUENCE LISTING

Incorporated herein by reference in its entirety is the
Sequence Listing for the application. The Sequence Listing,
is disclosed on a computer-readable ASCII text file titled,
“BSA17-02_IP2016-016-01_sequence_listing.txt”, created
on Aug. 7, 2018. The BSA17-02_IP2016-016-01_
sequence_listing.txt file is 2 KB in size.

SEQUENCE LISTING

<160> NUMBER OF SEQ ID Nos: 4
<210>
<211>
<212>
<213>
«220>
<223>

SEQ ID NO1

LENGTH: 10

TYPE: DNA

ORGANISM: Artificial Sequence
FEATURE:

OTHER INFORMATION: DNA Fragment

<400> SEQUENCE: 1

aategeatge

<210>
«211>
<212>
<213>
«220>
<223>

SEQ ID NO 2

LENGTH: 10

TYPE: DNA

ORGANISM: Artificial Sequence
FEATURE:

OTHER INFORMATION: DNA Fragment

<400> SEQUENCE: 2

10

Page 17

US 11,866,775 B2

17

18

-cont inued

geatgegatt

<210>
<21l>
<212>
«213>
«220>
«223>

SEQ ID NO 3

LENGTH: 10

TYPE: DNA

ORGANISM: Artificial Sequence
FEATURE:

OTHER INFORMATION: DNA Fragment

<400> SEQUENCE: 3

getgetgtaa

<210>
<21l>
<212>
«213>
«220>
«223>

SEQ ID NO 4

LENGTH: 10

TYPE: DNA

ORGANISM: Artificial Sequence
FEATURE:

OTHER INFORMATION: DNA Fragment

<400> SEQUENCE: 4

ttacageage

10

10

10

The invention claimed is:
1. A method for amplifying a molecular signal of an
Input-Oligonucleotide, the method comprising

a) providing a plurality of Particle-A protected by oligo-
nucleotide origami, wherein Particle-A is functional-
ized with a plurality of Oligonucleotide-1, wherein a
sequence in Oligonucleotide 1 complementary to a
sequence in a plurality of Oligonucleotide 2 hybridize
to form Duplex 1-2, and sequence in Oligonucleotide 2
complementary to a sequence in oligonucleotide ori-
gami hybridize;

b) providing a plurality of Particle-B, wherein Particle-B
is functionalized with a plurality of Oligonucleotide-3,
wherein a sequence in Oligonucleotide-3 complemen-
tary to a sequence in a plurality of Stored-Input-
Oligonucleotide hybridize to form Duplex 3-Y; and

c) mixing the Input-Oligonucleotide, plurality of origami
protected Particle-A and plurality of Particle-B, in any
order, wherein a cascade reaction occurs so that:

i) sequence in the Input-Oligonucleotide complementary
to sequence in Oligonucleotide-2 hybridize to form
Duplex Y-2, thereby removing Oligonucleotide-2 and
the oligonucleotide origami, and exposing Oligonucle-
otide-1 on Particle A,

ii) sequence in Oligonucleotide-1 complementary to
sequence in Oligonucleotide-3 hybridize to form
Duplex 1-3, thereby releasing the Stored-Input-Oligo-
nucleotide hybridized to the sequence in Oligonucle-
otide-3,

iii) sequence in the Stored-Input Oligonucleotide released
in ii) complementary to sequence in Oligonucleotide-2
hybridize to form Duplex Y-2, thereby removing Oli-
gonucleotide-2 and the oligonucleotide origami, and
exposing Oligonucleotide-1 on Particle A to hybridize
to complementary sequence in Oligonucleotide-3 to
form Duplex 1-3, thereby releasing the Stored-Input-
Oligonucleotide hybridized to the sequence in Oligo-
nucleotide-3, and,

iv) where (iii) reoccurs until the cascade reaction termi-
nates, when Particle A are fully deprotected.

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50

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2. The method of claim 1, wherein the cascade reaction
terminates by a first occurrence of either complete depro-
tection of origami protected Particle A or the release of all
Stored-Input-Oligonucleotide from Duplex 3-Y.

3. The method of claim 1, wherein Duplex Y-2 has a Tm
20% greater than the Tm of Duplex 1-2.

4. The method of claim 1, wherein Duplex 1-3 has a Tm
20% greater than the Tm of Duplex 3-Y.

5. The method of claim 1, wherein the Input-Oligonucle-
otide is DNA or RNA.

6. The method of claim 1, wherein the Particle-A and the
Particle-B are nanoparticles from about 5 to about 100 nm
in diameter.

7. The method of claim 6, wherein the nanoparticles are
selected from the group consisting of Au, Ag, Cu, Pt, Pd and
combinations thereof.

8. The method of claim 1, wherein the Input-Oligonucle-
otide consists of about 5 to about 120 bases.

9. The method of claim 1, wherein there are about 25 to
about 400 Oligonucleotide-1 attached per about 5 nm to
about 40 nm diameter of Particle-A.

10. The method of claim 1, wherein there are about 25 to
about 400 Oligonucleotide-3 attached per about 5 nm to
about 40 nm diameter of Particle-B.

11. The method of claim 1, wherein the oligonucleotide
origami is 2-dimensional or 3-dimensional.

12. The method of claim 1, wherein the oligonucleotide
origami is rectangular, round or polyhedron.

13. The method of claim 1, wherein the oligonucleotide
origami is rectangular.

14. The method of claim 1, wherein there are about 3 to
about 4 origami per about 20 nm diameter of Particle-A.

15. The method of claim 1, wherein the cascade reaction
is under isothermal conditions.

16. The method of claim 1, wherein the cascade reaction
is under room temperature.

17. The method of claim 1, wherein the released Stored-
Input-Oligonucleotide is measured.

18. The method of claim 17, wherein the amplified
molecular signal of the released Stored-Input-Oligonucle-
otide is measured.