WO2015006379A2 - Selection of cells for elimination - Google Patents

Abstract

Provided herein are molecular automaton system for elimination of a target cell. Some embodiments include modules specific for a target cell having a first cell surface marker and a second cell surface marker. Some embodiments include modules specific for a target cell having a first cell surface marker but not a second cell surface marker.

Description

TITLE OF INVENTION

SELECTION OF CELLS FOR ELIMINATION

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. Provisional Application

Serial No. 61/843,892 filed 08 July 2014, which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable.

MATERIAL INCORPORATED-BY-REFERENCE

The Sequence Listing, which is a part of the present disclosure, includes a computer readable form comprising nucleotide and/or amino acid sequences of the present invention. The subject matter of the Sequence Listing is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Molecular automata are mixtures of molecules that undergo precisely defined structural changes in response to sequential interactions with inputs. Previously studied nucleic acid based automata include game-playing molecular devices (MAYA automata) and finite-state automata for analysis of nucleic acids with the latter inspiring circuits for the analysis of RNA species inside cells.

SUMMARY OF THE INVENTION

One aspect of the present disclosure provides a molecular automaton system for isolation, elimination, or treatment of a target cell. Some

embodiments include modules specific for a target cell having a first cell surface marker and a second cell surface marker. Some embodiments include modules specific for a target cell having a first cell surface marker but not a second cell surface marker. In some embodiments, a system includes a first target marker, a second target marker, a single stranded fifth oligonucleotide, and single stranded sixth oligonucleotide linked to an isolation agent, a cytotoxic agent, or a therapeutic agent.

In some embodiments, the first target marker includes (i) a first antibody specific for a first cell surface marker and (ii) a first double strand complex comprising a first oligonucleotide and a second oligonucleotide, the second oligonucleotide linked to the first antibody.

In some embodiments, the first target marker includes a second target marker including (i) a second antibody specific for a second cell surface marker and (ii) a second double strand complex comprising a third oligonucleotide and a fourth oligonucleotide, the fourth oligonucleotide linked to the second antibody.

In some embodiments, the system includes a single stranded fifth oligonucleotide and a single stranded sixth oligonucleotide linked to an isolation agent, a cytotoxic agent, or a therapeutic agent. In some embodiments, the first oligonucleotide has more complementarity for the fifth oligonucleotide than for the second oligonucleotide, such that when in proximity, the fifth oligonucleotide will disrupt the first double strand complex to form a single stranded second oligonucleotide and a third double strand complex comprising the first oligonucleotide and the fifth oligonucleotide. In some embodiments, the third oligonucleotide has more complementarity for the second oligonucleotide than for the fourth oligonucleotide, such that when in proximity, a single stranded second oligonucleotide will disrupt the second double strand complex to form a single stranded fourth oligonucleotide and a fourth double strand complex comprising the second oligonucleotide and the third oligonucleotide, the fourth double strand complex linked to the first antibody via the second oligonucleotide, and the single stranded fourth oligonucleotide linked to the second antibody. In some embodiments, the sixth oligonucleotide has sufficient complementarity to the single stranded fourth oligonucleotide to form a fifth double strand complex therewith, but has insufficient complementarity for the fourth oligonucleotide to disrupt the second double strand complex. In some embodiments, the system includes a sixth double strand complex comprising a sixth oligonucleotide and a seventh oligonucleotide, the sixth oligonucleotide linked to an isolation agent, a cytotoxic agent, or a therapeutic agent. In some embodiments, the first oligonucleotide has more

complementarity for the fifth oligonucleotide than for the second oligonucleotide, such that when in proximity, the fifth oligonucleotide will disrupt the first double strand complex to form a single stranded second oligonucleotide and a third double strand complex comprising the first oligonucleotide and the fifth oligonucleotide. In some embodiments, the third oligonucleotide has more complementarity for the second oligonucleotide than for the fourth

oligonucleotide, such that when in proximity, a single stranded second oligonucleotide will disrupt the second double strand complex to form a single stranded fourth oligonucleotide and a fourth double strand complex comprising the second oligonucleotide and the third oligonucleotide, the fourth double strand complex linked to the first antibody via the second oligonucleotide, and the single stranded fourth oligonucleotide linked to the second antibody. In some embodiments, the sixth oligonucleotide has more complementarity for the second oligonucleotide than for the seventh oligonucleotide, such that when in proximity, a single stranded second oligonucleotide will disrupt the sixth double strand complex to form a single stranded seventh oligonucleotide and a seventh double strand complex comprising the second oligonucleotide and the sixth oligonucleotide, the seventh double strand complex linked to the first antibody via the second oligonucleotide, and the single stranded fourth oligonucleotide linked to the second antibody. In some embodiments, the third oligonucleotide has more complementarity for the second oligonucleotide than the sixth oligonucleotide has for the second oligonucleotide, such that when in proximity, the sixth oligonucleotide cannot displace the third oligonucleotide from the fourth double strand complex comprising the second oligonucleotide and the third oligonucleotide.

Another aspect provides a method for isolating, eliminating, or treating a target cell with a molecular automaton system described herein. In some embodiments, the method includes contacting the first target marker, the second target marker, and a population of cells optionally comprising a target cell, the target cell comprising the first cell surface marker and the second cell surface marker, to form a marked cell; and contacting the single stranded fifth oligonucleotide and the single stranded sixth oligonucleotide linked to an isolation agent, a cytotoxic agent, or a therapeutic agent with the marked cell.

In some embodiments, the method includes contacting the first target marker, the second target marker, and a population of cells optionally comprising a target cell, the target cell comprising the first cell surface marker but not second cell surface marker, to form a marked cell; and contacting the single stranded fifth oligonucleotide and the sixth double strand complex linked to an isolation agent, a cytotoxic agent, or a therapeutic agent with the marked cell.

Other objects and features will be in part apparent and in part pointed out hereinafter.

DESCRIPTION OF THE DRAWINGS

Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

FIG. 1 is a series of spectra showing demonstrations of potential for practical applications. FIG. 1A shows magnetic separation of PBMCs based on results of a YESCD45YESCD3 automaton. Before the cascade (left panel), the mixture of cells is observed with different CD3+ status. After the cascade, cells were incubated with magnetic microbeads conjugated with anti-FITC antibodies (MiltenyiBiotec) and applied on a MACS Column (MiltenyiBiotec; isolated purity of preparation was >95%). The "pass through" fraction (blue line in middle panel) and magnetically labeled cells (red line in middle panel) were re-analyzed with different clones of aCD3 antibodies to confirm purity (right panel). FIG. 1 B shows YESCD3YESCD8 automaton was demonstrated in whole blood: Flow cytometry analysis with gating strategy shown (left and middle left panels); nucleated cells were gated based on staining their DNA with 7-AAD, with lymphocytes selected based on forward and side scatter; The histograms show two steps of the cascade as performed in blood, Cy5 fluorescence is used to show that first step was accomplished, while fluorescein is used to demonstrate that the seconds step was accomplished, as in FIG. 10). Lines on histograms: yellow - unlabeled blood sample; green - blood sample incubated for 15 min with aCD3 conjugated with duplex 1 »2-Cy5 and aCD8 conjugated with duplex 3·4; blue - same, but with F-5»6-Q added and also incubated for 15 min; red - subsequent addition of 0-Q.

FIG. 2 is a series of oligonucleotide sequences and fluorescence spectra showing the effect of mismatches (depicted by hashed lines) on unwanted fluorescent signal (fluorescein F) leak in a YES-YES cascade. Duplexes b»c, d»e, and f»g (one experiment with mismatches and one experiment without mismatches) were formed by combining 100 μΜ stock solutions of the respective single stranded oligonucleotides in PBS buffer (pH 7) and incubating at room temperature for 30 minutes (Note - strands b and g were added in slight excess). 1 μΙ (-50 μΜ) of the resulting f»g duplex solution was added to 120 μΙ of PBS buffer in a cuvette for fluorescence measurements (monitoring fluorescein and Cy5 channels). Then 1 μΙ of the remaining duplexes and lastly 2 μΙ (100 μΜ) of a was added at time points indicated.

FIG. 3 is a series of oligonucleotide sequences showing the use of mismatches to diminish signal 'leak'. FIG. 3A depicts unwanted double helix formation between oligonucleotide 2 and oligonucleotide 3. FIG. 3B depicts the addition of a mismatch to make the steps in formation of a double helix between oligonucleotide 2 and oligonucleotide 3 less energetically favorable, and hence reduce the amount of unwanted double helix formation. When no mismatches were present in full cascade experiments, unwanted leakage of fluorescence signal was more pronounced. When a mismatch(es) was strategically introduced, unwanted leakage was significantly diminished, see e.g., FIG. 2, for an example of experiment results.

FIG. 4 is a series of DNA sequences and drawings showing

oligonucleotide sequences. FIG. 4A shows oligonucleotide sequences where NB strand labels do not coincide with one another across different cascades. For example, strand (2) in the YES-YES cascade is not the same as strand (2) in the YES-YES-YES cascade. Also, color-coding does not coincide across different cascades. For example, strand (0) in the YES-YES cascade is not the same as strand (2) in the YES-YES-YES cascade. Hashed lines indicate mismatches. Letters "ab" represent an antibody conjugate to oligonucleotide. FIG. 4B-E show various forms of cascades considered in the initial design phase. FIG. 4B is the basis for current cascades. It was taken into consideration that oligonucleotide 3 should be transferred in-between markers on the same cell, i.e., without diffusion. FIG. 4C shows a design using a long oligonucleotide complex that gets slowly degraded (sequential strip-off) by sequential interactions with oligonucleotides on the surface of the cell. The design can possibly diffuse away from the cell and hit non-target cells (bystander effect). FIG. 4D shows a similar design as FIG. 4C, but the oligonucleotide may not leave the surface. This design would also involve a very long linear DNA complex, which could be less favored in later in vivo studies and sequence optimization. FIG. 4E shows a variant of FIG. 4A. But the variant was not pursued due to the possibility of diffusion of 2 from cell and strong bystander effect.

FIG. 5 is a size exclusion chromatograph spectrum showing the purification of rituximab-oligonucleotide conjugates. Sample "4" are products isolated from the reaction of 4 equivalents of oligo-BMH with one equivalent of sulfhydryl-rituxan. Sample "2.4" are products isolated from the reaction of 2.4 equivalents of oligo-BMH with one equivalent of sulfhydryl-rituxan. For other antibodies, 4 equivalents of oligonucleotide were used.

FIG. 6 is a series of drawings showing the design considerations for automata operating on cell surfaces. FIG. 6A is a schematic showing automata operating on a targeted, e.g., B cell with C45⁺CD20⁺ phenotype, and non- targeted, e.g., T cells with CD45⁺CD20^" phenotype. Oligonucleotide components (colored horizontal lines) attached to antibodies (Y-shaped structures) are brought together on some cells and not others (for example, aCD45-1 ·2 and aCD20-3»4 are together only on B cells), leading to a cascade of oligonucleotide transfers driven by an increase in complementarity. The transfers result in a unique oligonucleotide (4) being displayed only on targeted cells. FIG. 6B is a schematic showing a typical strand displacement reaction used in the automata: 0 + 1 ·2+ 3·4->0· 1 + 2·3 + 4, controlled via a sequential exposure of toeholds (T1 then T3): single-stranded oligonucleotide 0 displaces oligonucleotide 2 from its complex with 1 via toehold interactions (T-i).This generates a new toehold T₃ in strand 2 that can extend the reaction cascade by displacing oligonucleotide 4 from 3·4 to generate the next toehold T₅ on 4. T₅ can be used to extend the cascade to 5·6 (not shown) and so on (as indicated by double dotted arrows) or label the cell with 4. Without T₃, the cascade stops. FIG. 6C shows an example of oligonucleotide sequences used in the automata.

FIG. 7 is a series of drawings illustrating four examples of molecular automata for evaluation of cell surfaces.

FIG. 8 is a HPLC spectrum showing the anion exchange HPLC analysis/purification of rituxan-oligonucleotide conjugates carried out on a Shimadzu LC-20AB pump equipped with an SPD-M20A PDA detector using a Tosoh Biosciences TSKgel DEAE-NPR column, 4.6x50 mm (IDxL). Buffer A was composed of 20 mM TRIS, and buffer B, 20 mM TRIS/1 M NaCI, both adjusted to pH 7.2.). Ratios of oligonucleotide:antibody are arrowed above the respective peak, and were determined by comparing the UV absorbance 260 nm/280 nm ratio with standards made from non-conjugated oligonucleotide and antibody. Each peak was checked for activity via its performance in a YES-YES cascade, and it was found that all peaks were active with performance increasing as oligonucleotide:antibody ratio increased. For all cascade experiments, however, all oligonucleotide-antibody conjugates were purified by size-exclusion FPLC due to increased yield (see e.g., FIG. 2).

FIG. 9 is a series of size exclusion chromatograph traces showing the purification of antibody-DNA double helix conjugates. Initial fractions (e.g. for 3»4Rituxan, fractions 12, 13, and 14) were preferred for running cascade experiments due to results obtained in FIG. 3.

FIG. 10 is a series of drawings and spectra showing the demonstration of an automata assessing the presence of two cell surface markers. FIG. 10A shows a schematic representation of YESCD45YESCD20 automata with the reaction: 0 + 1 »2_aCD₄5 + 3»4_aCD2o+ 5·6 -> 0· 1 + aCD4s2»3 + _acD2o4»5 + 6 occurring on the cell surface: 1 is labeled with Cy5 and 0 labeled with a quencher (Q) for Cy5; 5 is labeled with fluorescein (F), and 6 labeled with a quencher (Q) for fluorescein. FIG. 10B shows flow cytometry monitoring of the YESCD45YESCD20 cascade (each dot represents the fluorescence signal level from a single cell at the time of measurement, with the dot density representing number of cells, shown as increasing from blue-through-red): time course of the cascade reaction on CD20⁺ B-cells. The left panel shows removal of Cy5-1 after the triggering reaction with 0 monitors the removal of 1 occurring on CD45⁺ cells. The right panel shows fluorescein-labeled 5 is taken up from solution by CD20⁺ B-cells - this is used for monitoring the acquisition of F-5 by 4 enabled by prior removal of 3 from 4. The addition of 5·6 (indicated by first red arrow) produces an immediate fluorescence increase on all cells due to non-complete quenching of fluorescein; the addition of 0 (indicated by the second red arrow) triggers the cascade and separation of the subpopulations of cells. FIG. 10C shows the monitoring of a cascade on individual subpopulations within PBMCs by using fluorescently labeled monoclonal antibodies with non-overlapping epitopes for identification of cell subpopulations (PerCP-CD45 antibody, clone 2D1 and Pacific Blue-CD20 antibody (clone 2H7). These results confirm that all

CD45⁺CD20⁺ cells (right gate, i.e., right box on bottom left panel) are labeled by automata (i.e., an increase was observed in fluorescein uptake from solution, cf. bottom middle and right panels) and that cells that are CD45⁺CD20^" (left gate, i.e., left box on bottom left panel) are not (upper left and central panels). It was observed that -0.5% of cells that are gated (box at the central up panel) as

CD45⁺CD20^" may react with a delay (upper right panel). Arrows have the same meaning as under FIG. 10B.

FIG. 1 1 is a series of drawings and flow cytometry fluorescence spectra demonstrating the operation of automata YESCD45YESCD3,

YESCD45(YESCD20ORYESCD3), and control YESCD3YESCD20. FIG. 1 1 A shows a schematic representation of YESCD45YESCD3. The reaction is 0 + 1 ·2_α0οΛ5⁺ 3»4CCD3⁺ 5·6 -> 0· 1 +aCD452»3 + aCD3 »5 + 6, where 1 is labeled with Cy5 and 0 labeled with a quencher for Cy5, and 5 is labeled with fluorescein and 6 labeled with a quencher for fluorescein. FIG. 1 1 B shows flow cytometry results for the cascade depicted in FIG. 1 1 A, i.e., monitoring the kinetics of the cascade reaction on CD3⁺T-cells - left panel - removal of Cy5-labeled 1 after triggering reaction with 0; and right panel - picking up of fluorescein-labeled 5 from solution by CD3⁺T-cells (events are arrowed, that is, addition of 5·6 is followed by addition of 0). FIG. 1 1 C shows flow cytometry results for YESCD3YESCD20 demonstrating no labeling of cells that do not have both markers, i.e. negative control (cf., FIG. 13). FIG. 1 1 D shows flow cytometry results for YESCD3YESCD8: positive control for panel FIG. 1 1 C. FIG. 1 1 E shows flow cytometry results for YESCD45(YESCD20ORYESCD3) selectively labeling two cell populations (B- and T- cells) (upper right-hand side) using an OR function.

FIG. 12 is a series of fluorescence signals in a scheme showing experiments on enriched B- and T-cells, as described in FIG. 10 (*mean fluorescence signal normalized to background after the addition of F-5»6 but before addition of trigger 0).

FIG. 13 is a series of fluorescence spectra showing the estimation of the bystander effects (cross-talk between different types of cells in mixture) on YESCD3YESCD20 (left column) with no cells positive for both of these markers and YESCD3(YESCD20ORYESCD8) with automata supposed to increase fluorescence only on CD8⁺ cells. The cells in the top panel were enriched as CD3⁺ and CD20⁺, exposed to conjugates, excess of reagents washed away, and then the cells were remixed before the reaction was triggered. Middle row panels show with cells (PBMCs), excess of conjugates were removed from cells by centrifugation and washing, while excess of reagents in solution was not removed with PBMCs in bottom. Individual traces: Red - unlabeled cells

("autofluorescence"); blue - PBMCs incubated at 4 °C for 20 minutes with anti- CD3 conjugated with duplex 1 »2-Cy5 and anti-CD20 (left column) and/or anti- CD8 (right column) conjugated with duplex 3·4; green trace - same as "blue", but with F-5»6-Q added to PBMCs with cells incubated at room temperature for an additional 5 min; black - subsequent addition of 0-Q at room temperature for 20 min before measuring (where F is fluorescein and Q is the respective quencher; oligonucleotide numbering references the two-step YES-YES cascade). Comparison of black and green traces allows us to assess cascades that occur between two cells, as opposed to cascades that occur only on one cell.

FIG. 14 is a series of flow cytometry images demonstrating a reverse direction cascade (YESCD20YESCD45) showing selective labeling of CD20⁺ cells and no leak to CD3⁺ cells that are also CD45⁺ (Double labeling experiment as well). Anti-CD45 and anti-CD20 antibodies coupled to different fluorophores and targeting different epitopes were used in this experiment to focus the observation on primarily B- and T- cells.

FIG. 1 5 is a series of drawings and spectra showing the demonstration of an automata assessing the absence of a cell surface marker. FIG. 1 5A shows a schematic representation of a YESCD8NOTCD45RA cascade protecting na^'ive CD8⁺CD45RA⁺ T-cells in which the CD45RA isoform prevents the targeting of CD8. This automaton works by /f cell is CD8 positive and /f cell is CD45RA positive (and CD45RO^neg) then reaction is 2 + 3»4_aCD8 + 5*»6*_aCD45RA + 5·6 -» 2·3 + 5*»4_acD8 + 6*_acD45RA + 5·6, resulting in no labeling (red trace on right panel of FIG. 15B), else, when cell is CD45RA^" (CD45RO isoform), the reaction is: 2 + 3»4_acD8+ 5·6 -> 2·3 + 5»4_aCD8 + 6. As a result, fluorescein is taken up from the solution in a simple YESCD8 response (blue trace on right panel of FIG. 15B). FIG. 15B shows the monitoring of the YESCD8NOTCD45RA cascade. The left panel shows time-course of cascade reaction on the surface of CD8⁺ T-cells from peripheral blood: Right panel, histograms (or frequency distributions) of memory CD8⁺T-cells responding to automata (upper gate/box on left panel; blue trace on right panel, CD8⁺CD45RO⁺ or CD45RA^") while naive CD8⁺ T-cells are being protected from automata (lower gate/box on left panel; red trace,

CD8⁺CD45R07CD45RA⁺). For gating strategy, see e.g., FIG. 16.

FIG. 16 is a series of images and spectra demonstrating

YESCD8NOTCD45RA. FIG. 16A shows the gating strategy during the analysis of a YESCD8NOTCD45RA cascade protecting na^'ive CD8⁺CD45RA⁺ T-cells. Anti- CD19 antibody was used to focus observation on CD19^" cells (B-cells are CD19⁺, while non-B cells, that is, largely T-cells in this sample, are CD19^"); then anti CD4 antibody was used to focus on CD4^" cells within CD19^" subpopulation, that means that population that was observed was mostly CD8⁺ (some NK cells were present as well) because CD8 and CD4 are mutually exclusive (on over >95% of cells) T-cells. FIG. 16B shows the distribution of CD45RA and

CD45RO on human lymphocytes.

FIG. 17 is a series of drawings and spectra demonstrating an automata assessing the presence of three markers (CD45, CD3, and CD8) on the surface of the cell. FIG. 17A shows individual antibodies are conjugated to components of the cascade (e.g., otcD45- with 1 ·2 complex, otcD3 with 3·4, and OCCDS with 5·6), while oligonucleotides are labeled with fluorescent dyes and quenchers to facilitate monitoring of multiple events in parallel (1 with Pacific Blue or PB, 3 with Cy5, 7 with fluorescein or F; 0, 2, and 8 with quenchers). FIG. 17B shows flow-cytometry monitoring in three colors of the state transition (y-axis:

fluorescence intensity, x-axis: time, with arrows showing events, i.e., the additions of oligonucleotides 7·8 and 0. In the final step (right panel - fluorescein), the separation of CD45⁺CD3⁺CD8⁺ from all other lymphocytes is clearly shown. Arrows have the same meaning as under FIG. 10B, except herein F-7»8-Q was added.

FIG. 18 is a series of images showing three-layer cascade on

lymphocytes. Top row shows gated from PBMCs based on light scattering signals. The middle row shows enriched CD8+. The bottom row shows CD4⁺ cells, as in FIG. 17.

FIG. 19 is an illustration of lineages from hemangioblast precursor cells that include lymphocytes.

FIG. 20 is an illustration of cascades on cell surfaces connected with "action modules". Three surface markers (α, β, γ) interact with targeting moieties (antibodies, their fragments, aptamers or peptide ligands). Oligonucleotides coupled to these moieties participate in reaction cascades on the cell surface (a1 +β2*3+γ5*4 -» α1 *2+β3*4+γ5), leading to a display of a new oligonucleotide (5) on the surface. This oligonucleotide can interact with drug delivery or imaging modules (D) leading to the elimination or labeling of targeted cells.

FIG. 21 is an illustration of oligonucleotide sequences used in strand exchange reaction cascades. In FIG. 21 A showing strand exchange reaction cascades 1 + 2*3 -> 1 *2 + 3, red toehold initiates the exchange, also leading to an irreversible (thermodynamically favorable) reaction. FIG. 21 B shows schematics of a typical basic strand displacement reaction in solution as in A. Single-stranded oligonucleotide 1 displaces oligonucleotide 3 from its complex with 2, based on a stronger complementarity of 1 *2 over 2*3. The reaction proceeds rapidly by toehold interactions (red To). The resulting single-stranded 3 can again react, extending the reaction cascade, e.g., with 4*5 complex (as shown in FIG. 21 C displacing the next oligonucleotide 5, in analogy to 1 displacing 3. Light blue To' is a new toehold being formed - without it there will be no further reaction in this embodiment. FIG. 21 C shows the principle of NOT cascades, also used for thresholding (NOT is equivalent to a very high threshold): NOT and thresholding are based on having two competitive displacement reactions, one with high, the other with low reaction rates. The one with higher reaction rates (or higher local concentration) would prevail, and stop the other one from happening (NOT). Different reaction rates can be based on different lengths (number of bases) of toeholds (J& vs. Jg in AQ VS. 4S, that are in 4s*5 and 46*5-R, with R as a tag). Reaction rates can be controlled based on toehold sizes over several orders of magnitude.

FIG. 22 shows an illustration of a cascade assessing presence of three markers on the surface of the cell; the three markers are: CD45, CD3 and CD8 (cf., main text). FIG. 22A shows individual antibodies are conjugated to components of the cascade (e.g., aCD45- with 2*3 complex, aCD3 with 4*5, and aCD8- with 6*7), while oligonucleotides are labeled with fluorescent dyes (2 with Pacific Blue or PB, 4 with Cy5, 8 with fluorescein or F; 1 , 3, and 9 with quenchers) and quenchers to facilitate monitoring. FIG. 22B, shows flow- cytometry monitoring in three colors of the cascades on cell surfaces (y: intensity of fluorescence, x - time, with arrows showing events, i.e. , the addition of oligonucleotides 8*9 and then 1 . With fluorescein the separation of CD8+ from other lymphocytes is seen. FIG. 23 is an illustration of a therapeutic module having a toxin conjugate. FIG. 23A shows YESCD8YESCD45RO cascade (I) targets memory T- cells. Toxin-6 is also coupled to fluorescein, in order to monitor the reaction. FIG. 23B shows YESCD8NOTCD45RO cascade (II) protecting memory T-cells; the competition between elements on the surface of the cell and in solution leads to the suppression of the signal.

FIG. 24 is an illustration of a Y ESCD45RAYESCD3 module.

FIG. 25 is a series of flow cytometry graphs showing separation of na^'ive T-cells with a Y ESCD45RAYESCD3 module. FIG. 25A shows flow-cytometry prior to separation. FIG. 25B shows that cells not exposed to module do not bind to magnetic beads. FIG. 25C shows cells exposed to the module bind to magnetic beads. FIG. 25D shows flow-cytometry of a sample of control cells not exposed to module and not exposed to magnetic beads prior to separation, where 36% are CD19/20 D3^", 59.1 % are T-cells, and 4.9% are B-cells. FIG. 25E shows flow-cytometry of a sample of control cells not exposed to module and exposed to magnetic beads, where 77.5% are CD19/20 D3^", 18.4% are T-cells, and 4.1 % are B-cells. FIG. 25F shows flow-cytometry of a sample of cells exposed to module and exposed to magnetic beads, where 1 .3% are CD19/20 D3^", 96.2% are T-cells, and 2.5% are B-cells. DETAILED DESCRIPTION OF THE INVENTION

The present disclosure is based, at least in part, on the discovery that molecular automata based on oligonucleotide strand-displacement cascades directed by antibodies can analyze cells by using their cell-surface markers as inputs. In short, a cascade of oligonucleotide transfers driven by an increase in complementarity is exploited between a series components (e.g., a first single stranded oligonucleotide; a first target specific antibody coupled to second oligonucleotide and third oligonucleotide). Such an approach can be

accomplished while minimizing or avoiding diffusion away from cells or cross- linking.

While strand displacement reactions are known (see e.g., Yurke et al.

2000 Nature 406, 605-608; Seelig et al. 2006 Science 314, 1585-1588; Qiau et al. 201 1 Nature 475, 368-372; Rinaudo et al. 2007 Nature Biotechnology 25, 795-801 ; Xie et al. 201 1 Science 333, 1307-131 1 ), each of these approaches are directed to solution-phase analysis and regulation of protein expression. Various embodiments of the present disclosure use logical operations unrelated to the logic gates of prior approaches or cascades can follow a different algorithm (e.g., avoid diffusion away from cells or cross-linking).

While a nanobox that opens as a result of interactions between aptamers open up DNA nano-objects are known (e.g., Brand et al 2010 J Immunol 185, 2285-2294), such an approach relies on an equilibrium process within structure switching aptamers (see Nutiu 2004 Chemistry 10, 1868-1876) and therefore a significant proportion of the nano-objects would open even in the absence of targeted cells. Furthermore, embodiments described herein provide for longer cascades, thresholds, protective "NOT" elements, or an intermediate resembling a proximity ligation process.

As shown herein, an output (e.g., a final output) of a molecular automaton that successfully completes its analysis can be the presence of a unique molecular tag on the cell surface of a specific subpopulation of lymphocytes within human blood cells. Such an approach can be used for a variety of markers and cell types. Various approaches described herein can overcome problems associated with proximity principles, such as bi-specific antibodies or proximity ligation reactions.

Labeling a narrow subpopulation within a much larger population of related cells can be problematic because of the need to specifically tag a particular cell type for the purpose of elimination, analysis or isolation, or imaging. The problem can be addressed in a direct manner for a targeted subpopulation of cells having a unique cell-surface marker against which antibodies can be raised. But, as best illustrated through an example of a cancer therapy with antibody-drug conjugates (ADCs), markers are most often shared with non-targeted cells and can lead to dose-limiting toxicities. To uniquely target cells that may not have a distinctive marker on their surfaces, a plurality of markers for a subpopulation of cells can be used in a Boolean (i.e., a logical combinatorial system that represents relationships between entities) manner. Molecular automata with structural changes (e.g., state transitions) can be coupled to the sequential recognition of a selected set of cell surface markers and can contract the set into a single tag and thus can provide a unique handle for the targeted cells. In other words, various molecular devices described herein can autonomously evaluate Boolean functions on a cell surface with a plurality of surface markers as inputs and a tag as an output.

In some embodiments, a molecular automata described herein can be used by transfecting oligonucleotides into cell lines, which can permit new operations on native cells.

Molecular automata In conventional cell analysis approaches using molecular robotics, complexity of individual nanoparticles is increased using self- assembly of DNA nanoobjects displaying multiple aptameric locks. Described herein is a potentially simpler alternative. Molecular automata described herein can interact with a cell surface to execute more complex programmable

(automata) functions, an approach that is conceptually similar to that of distributed robotics paradigms.

TARGET MARKERS

As described herein, a molecular automaton system for elimination of a target cell will generally include a plurality of target markers. A target marker can include a oligonucleotide specific for a strand-displacement cascade optionally coupled to an antibody specific for a cell surface marker of a target cell or optionally coupled to a therapeutic agent. In some target markers,

oligonucleotide specific for a strand-displacement cascade is not coupled to another molecule.

A cascade of oligonucleotide transfers driven by an increase in

complementarity is exploited between a series of target markers (e.g., a first single stranded oligonucleotide; a first target specific antibody coupled to second oligonucleotide and third oligonucleotide). Various combinations of target markers operating in various logical operations are described throughout the present disclosure.

In some embodiments, an elementary unit of a strand displacement reaction (oligonucleotides 1 and 2*3 and reaction: 1 + 2*3 -> 1 *2 + 3) can be extended, leading to a cascade, i.e., reactions of a type: 1 + 2*3 + 4*5 + 6*7 + 8*9 + ... -> 1 *2 + 3*4 +5*6 + 7*8 + 9 +. Structures of oligonucleotides can be optimized and targeted mismatches introduced so as to, for example, minimize background reaction rates (i.e., interactions without introducing 1 to initiate reactions) or off-target effects.

TARGET CELLS

As described herein, molecular automata based on oligonucleotide strand-displacement cascades directed by antibodies can analyze cells by using their cell-surface markers as inputs. As such, a target cell can be any cell having some unique combination (or absence) of cell surface markers not generally possessed by other cells, e.g., cells in the same or similar tissues.

Exemplary target cells include stem cells, leukocyte groups, granulocytes, monocytes, T lymphocytes, T helper cells, T regulatory cells, Cytotoxic T cells, lymphocytes, thrombocytes, and natural killer cells.

In some embodiments, a target cell can be a lymphocyte (see e.g., FIG. 19), NK cell, T-cell, or B-cell. CELL SURFACE MARKER

As described herein, cell-surface markers can serve as input targets for antibodies linked to nucleotides specific for strand-displacement cascades. As such, a cell surface marker can be any cell surface marker or combination thereof that appears (or does not appear) in a unique combination on the surface of a target cell of interest. Generally, a cell surface marker will have an identified antibody specific for such marker.

In some embodiments, a cell surface marker can be a cluster of differentiation (or a cluster of designation) (CD). CD is understood as a protocol for the identification and investigation of cell surface molecules providing targets for immunophenotyping of cells. In the art, a surface molecule is assigned a CD number once two specific monoclonal antibodies (mAb) are shown to bind to the molecule. Cell populations are usually defined using a "+" or a "-" symbol to indicate whether a certain cell fraction expresses or lacks a CD molecule. A combination of markers (e.g., CD markers) can provide for cell types with very specific definitions (e.g., within the immune system).

Exemplary CDs are as follows: stem cells, CD34+, CD31 -, CD1 17; all leukocyte groups, CD45+; Granulocyte, CD45+, CD1 1 b, CD15+, CD24+, CD1 14+, CD182+; Monocyte, CD45+, CD14+, CD1 14+, CD1 1 a, CD1 1 b, CD91 +, CD16+; T lymphocyte, CD45+, CD3+; T helper cell, CD45+, CD3+, CD4+; T regulatory cell, CD4, CD25, Foxp3; Cytotoxic T cell, CD45+, CD3+, CD8+; naive T-cell, CD45RA+, CD3+; B lymphocyte, CD45+, CD19+ or CD45+, CD20+, CD24+, CD38, CD22; Thrombocyte, CD45+, CD61 +; Natural killer cell, CD16+, CD56+, CD3-, CD31 , CD30, CD38.

In some embodiments, a cell surface marker include CD3, CD8, and

CD25.

OLIGONUCLEOTIDE

As described herein, a plurality of oligonucleotide molecules can be configured so as to result in a series of strand-displacement reactions.

Nucleotides specific for strand-displacement cascades can be as described herein. Generally, differences in complementarity between oligonucleotides can drive the strand-displacement reactions.

An oligonucleotide can include about 10 to about 100 nucleotides. For example, an oligonucleotide can include about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100. It is understood that ranges between each combination of the above recited values are included in the present application.

As described herein, differences in complementarity between oligonucleotide can drive a strand-displacement reaction. A difference in complementarity sufficient to drive a strand-displacement reactions can occur where two oligonucleotides have less than about 99% sequence identity. For example, s difference in complementarity sufficient to drive a strand- displacement reactions can occur where two oligonucleotides have less than about 99%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, or less sequence identity. It is understood that ranges between each combination of the above recited values are included in the present application.

An extension of one oligonucleotide when paired with another

oligonucleotide can create a "toe hold" sufficient to drive a strand-displacement reaction. A toe hold can include at least about 1 nucleotide. For example, a toe hold can include at least about 1 , about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 1 1 , about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21 , about 22, about 23, about 24, about 25, or more nucleotides. It is understood that ranges between each combination of the above recited values are included in the present application.

Presence of a toe hold can contribute to differences in complementarity between two oligonucleotides. Differences in complementarity can include mismatches along with length of two oligonucleotides, presence of one or more toe hold, or a combination thereof.

For example, in a system with two target markers (e.g., YesYes), a first double strand complex can include a first oligonucleotide and a second oligonucleotide, the second oligonucleotide linked to the first antibody. The first antibody binds to a cell surface marker for which it is specific. Introduction of a single stranded fifth oligonucleotide, where the first oligonucleotide has more complementarity for the fifth oligonucleotide than for the second oligonucleotide, can result in a strand displacement reaction in which the first oligonucleotide and the fifth oligonucleotide become paired leaving a single stranded second oligonucleotide. In the above system, a second double strand complex including a third oligonucleotide and a fourth oligonucleotide, the fourth oligonucleotide linked to a second antibody, can be introduced. The second antibody binds to a cell surface marker for which it is specific. Introduction of a single stranded sixth oligonucleotide, where the third oligonucleotide has more complementarity for the second oligonucleotide than for the fourth oligonucleotide, can result in a strand displacement reaction in which the third oligonucleotide and the second oligonucleotide become paired leaving a single stranded fourth oligonucleotide linked to the second antibody linked to the cell surface marker for which it is specific.

In the above system, a single stranded sixth oligonucleotide linked to a therapeutic agent can be introduced. The sixth oligonucleotide can have sufficient complementarity to the single stranded fourth oligonucleotide to bind thereto, but insufficient complementarity for the fourth oligonucleotide to disrupt the pairing of the third oligonucleotide and fourth oligonucleotide. In such case, the therapeutic agent is selectively bound to a target cell having two particular cell surface markers.

In some embodiments, an oligonucleotide itself is a therapeutic agent. For example, in the above described system, the fourth oligonucleotide can be an aptamer against toxin or cytotoxic cells (e.g., NK cells or T cells). Where the third oligonucleotide is transferred to the second oligonucleotide, the fourth oligonucleotide (now single stranded) can become active and acquire a toxin effect or attract a cytotoxic cell. Where an oligonucleotide itself is a therapeutic agent, some other oligonucleotides may not be needed. For example, where the fourth oligonucleotide is an aptamer, the sixth oligonucleotide or the seventh oligonucleotide may not be present.

The above explanation of a YesYes system, can be adapted to other logical systems according to the disclosure herein.

ANTIBODY

As described herein, an antibody can be conjugated to a oligonucleotide specific for strand-displacement cascades. Such a conjugate can form a target marker allowing binding to cell surface markers of a target cell. An antibody for use with systems described herein will generally be capable of being coupled to a nucleotide.

An antibody as used herein can specifically bind to a particular cell surface marker. As such, choice of a cell surface marker can be determined according to availability of an antibody specific thereto.

THERAPEUTIC AGENT

As described herein, a therapeutic agent can be coupled to a

oligonucleotide specific for strand-displacement cascades. Accordingly, at the termination of strand-displacement cascades, the therapeutic agent can be bound to the target cell by way of a target marker.

A therapeutic agent can be any agent known to have an effect on a target cell. A therapeutic agent for use with systems described herein will generally be capable of being coupled to a nucleotide. A therapeutic agent can be a cell toxin. Exemplary cells toxins include ribosome inactivating proteins (RIPs), such as saporin and gelonin. A therapeutic agent can be calicheamicin or maytansinoid (e.g., gemtuzumab zogamicin or Mylotarg). A therapeutic agent can be a cardiotonic steroid (e.g., bufalin or carbamate).

A therapeutic agent can be an aptamer (see e.g., Boltz et al. 201 1 J Biol

Chem 286(24), 21896-21905).

Other therapeutic agents for therapeutic modules described herein include (i) bi-specific antibodies used to destroy target cells by crosshnking them to T-cells (which are then, in the process, activated; e.g., CD19 on B-cells and CD3 on T-cells); or connect 5, once displayed on targeted cells, to an antibody conjugate displayed on T-cells; (ii) crosshnking with CD95) ("death receptor"); (iii) delivery of siRNA using aptamers; (iv) GDEPT vector delivery; (v) liposome targeting; and (vi) hypercrosslinking.

In some embodiments, a therapeutic agent can be added to or substituted with an imaging agent (e.g., quantum dot; radiolabel, such as PET SPECT, etc.; fluorescent label; or MRI agent).

MOLECULAR COUPLING

As described herein, antibodies can be coupled to nucleotides specific for strand-displacement cascades. Also as described herein, a therapeutic agent can be coupled to nucleotides specific for strand-displacement cascades.

Coupling, tagging, or linking molecules to oligonucleotides, antibodies, or proteins are well known in the art. Except as otherwise noted herein, therefore, the subject matter of the present disclosure can be carried out in accordance with such known processes.

Conjugation of an oligonucleotide and an antibody can be according to any method understood in the art. For example, coupling can be based on a disulfide bond reduction and coupling to maleimide-derivatized oligonucleotides (see e.g., Liu et al. 2010 Anal. Chem. 82, 5219-5226; Hermanson, "Bioconjugate Techniques", 2nd Edition, Elsevier Inc, Academic Press, New York (2008)). In some embodiments, coupling techniques can provide on average from 1 :1 to 1 :4 (antibody:oligonucleotide) conjugates, as determined by both ion exchange HPLC and UV/Vis comparison to standard mixtures.

As another example, coupling can be according to crosslinking protocols based on NHS-ester coupling to antibody, for which commercial kits are available. As another example, coupling can be according to biotinylated antibodies and streptavidin. In some embodiments, biotinylated antibodies and streptavidin coupling is used only for one step in a cascade, due to potential exchange of biotinylated oligonucleotides between antibodies.

An antibody can be coupled, tagged, or linked to an oligonucleotide.

Strepavidin can be used to cross-link an antibody to a biotinylated

oligonucleotide (see e.g., Example 1 ).

A beacon, tracer, or stain (e.g., fluorophore) can be coupled, tagged, or linked to an oligonucleotide, antibody, or protein. A toxin or drug can be coupled, tagged, or linked to an oligonucleotide, antibody, or protein.

A crosslink to other cells, toxins, or drugs (e.g., aptamer, antibody) can be coupled, tagged, or linked to an oligonucleotide, antibody, or protein. An aptamer can be coupled, tagged, or linked to an oligonucleotide, antibody, or protein. An antibody can be coupled, tagged, or linked to an oligonucleotide, antibody, or protein.

A nanoparticle or vesicle (e.g., liposome, micelle) can be coupled, tagged, or linked to an oligonucleotide, antibody, or protein. A nanoparticle or vesicle (e.g., liposome, micelle) carrying a toxin or drug can be coupled, tagged, or linked to an oligonucleotide, antibody, or protein.

CELL SEPARATION

As described herein, cells can be separated based on sequential recognition of a selected set of cell surface markers by molecular automata. After a molecular device described herein autonomously evaluates Boolean functions on a cell surface with a plurality of surface markers as inputs and a tag as an output, targeted cells can be separated according to their unique handle.

Cell separation based on surface antigens are known in the art. Except as otherwise noted herein, therefore, the process of the present disclosure can be carried out in accordance with such processes.

For example, a target cell having a unique handle resulting from use of sequentially recognized target markers described herein can be separated by flow cytometry (e.g., Fluorescence-activated cell sorting (FACS)). Flow cytometry is understood as a laser-based, biophysical technology employed in cell sorting or marker detection by suspending cells in a stream of fluid and passing them by an electronic detection apparatus. Flow cytometry can allow simultaneous multiparametric analysis of the physical and chemical

characteristics of up to thousands of particles per second. Use of flow cytometry to physically sort particles based on their properties (e.g., a unique handle resulting from sequentially recognized target markers described herein) so as to isolate or purify target cells is understood in the art. Data generated by a flow- cytometer can be plotted in a single dimension, to produce a histogram, or in two-dimensional dot plots or even in three dimensions. The regions on these plots can be sequentially separated, based on fluorescence intensity, by creating a series of subset extractions, termed "gates". Plots can be made on logarithmic scales. Because different fluorescent dyes' emission spectra can overlap, signals at the detectors can be compensated electronically as well as computationally, as understood in the art. Data accumulated using the flow cytometer can be analyzed using software, e.g., WinMDI, Flowing Software, or web-based

Cytobank, FCS Express, Flowjo, FACSDiva, CytoPaint (aka Paint-A-Gate), VenturiOne, CellQuest Pro, Infinicyt or Cytospec. Representative automated population identification methods include FLOCK in Immunology Database and Analysis Portal (ImmPort), FLAME in GenePattern and flowClust, in

Bioconductor.

FACS can provide a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. FACS can provide fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of target cells having a unique handle. In FACS, a cell suspension can be entrained in the center of a narrow, rapidly flowing stream of liquid. The flow can be arranged so that there is a large separation between cells relative to their diameter. A vibrating mechanism can cause the stream of cells to break into individual droplets. The system can be adjusted so that there is a low probability of more than one cell per droplet. Just before the stream breaks into droplets, the flow can pass through a fluorescence measuring station where the fluorescent character of interest of each cell is measured. An electrical charging ring can be placed just at the point where the stream breaks into droplets. A charge can be placed on the ring based on the immediately prior fluorescence intensity measurement, and the opposite charge can be trapped on the droplet as it breaks from the stream. The charged droplets can then fall through an electrostatic deflection system that diverts droplets into containers based upon their charge. In some systems, the charge can be applied directly to the stream, and the droplet breaking off retains charge of the same sign as the stream. The stream can then be returned to neutral after the droplet breaks off.

As another example, a target cell having a unique handle resulting from use of sequentially recognized target markers described herein can be separated using a Cytometric Bead Array (CBA). Compositions and methods of the present disclosure can be adapted for detection in accordance with CBA.

As another example, a target cell having a unique handle resulting from use of magnetic-activated cell sorting (MACS). MACS is understood to be a method for separation of various cell populations depending on their surface antigens (CD molecules). MACS separation can include incubating target cells having unique handles with magnetic nanoparticles coated with antibodies against a particular unique handle. This can causes the target cells to attach to the magnetic nanoparticles. A cell solution can be transferred on a column placed in a strong magnetic field. In some embodiments, the cells attached to the nanoparticles (having the unique handle) stay on the column, while other cells (not having the unique handle) flow through. According to such methods, the cells can be separated positively or negatively with respect to a unique handle.

A magnetic nanoparticle can be coated with an anti-fluorochrome antibody. A unique handle of a target cell can be fluorescent-labelled. The magnetic nanoparticle coated with an anti-fluorochrome antibody can be incubated with the fluorescent-labelled target cells (resulting from sequentially recognized target markers described herein) and provide for cell separation with respect to the unique handle.

As another example, a target cell having a unique handle resulting from use of sequentially recognized target markers described herein can be separated using Dynabeads. Dynabeads are superparamagnetic spherical polymer particles with a uniform size and a consistent, defined surface for the adsorption or coupling of various bioreactive molecules or cells. Such conventional materials can be adapted for a target cell having a unique handle resulting from use of sequentially recognized target markers described herein. MOLECULAR ENGINEERING

The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

The terms "heterologous DNA sequence", "exogenous DNA segment" or "heterologous nucleic acid," as used herein, each refer to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides. A "homologous" DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced.

Expression vector, expression construct, plasmid, or recombinant DNA construct is generally understood to refer to a nucleic acid that has been generated via human intervention, including by recombinant means or direct chemical synthesis, with a series of specified nucleic acid elements that permit transcription or translation of a particular nucleic acid in, for example, a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector can include a nucleic acid to be transcribed operably linked to a promoter.

A "promoter" is generally understood as a nucleic acid control sequence that directs transcription of a nucleic acid. An inducible promoter is generally understood as a promoter that mediates transcription of an operably linked gene in response to a particular stimulus. A promoter can include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter can optionally include distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.

A "transcribable nucleic acid molecule" as used herein refers to any nucleic acid molecule capable of being transcribed into a RNA molecule.

Methods are known for introducing constructs into a cell in such a manner that the transcribable nucleic acid molecule is transcribed into a functional mRNA molecule that is translated and therefore expressed as a protein product.

Constructs may also be constructed to be capable of expressing antisense RNA molecules, in order to inhibit translation of a specific RNA molecule of interest. For the practice of the present disclosure, conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001 ) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754).

The "transcription start site" or "initiation site" is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1 . With respect to this site all other sequences of the gene and its controlling regions can be numbered. Downstream sequences (i.e., further protein encoding sequences in the 3' direction) can be denominated positive, while upstream sequences (mostly of the controlling regions in the 5' direction) are denominated negative.

"Operably-linked" or "functionally linked" refers preferably to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a regulatory DNA sequence is said to be "operably linked to" or "associated with" a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably- linked to regulatory sequences in sense or antisense orientation. The two nucleic acid molecules may be part of a single contiguous nucleic acid molecule and may be adjacent. For example, a promoter is operably linked to a gene of interest if the promoter regulates or mediates transcription of the gene of interest in a cell.

A "construct" is generally understood as any recombinant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating nucleic acid molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecule has been operably linked.

A constructs of the present disclosure can contain a promoter operably linked to a transcribable nucleic acid molecule operably linked to a 3'

transcription termination nucleic acid molecule. In addition, constructs can include but are not limited to additional regulatory nucleic acid molecules from, e.g., the 3'-untranslated region (3' UTR). Constructs can include but are not limited to the 5' untranslated regions (5' UTR) of an mRNA nucleic acid molecule which can play an important role in translation initiation and can also be a genetic component in an expression construct. These additional upstream and downstream regulatory nucleic acid molecules may be derived from a source that is native or heterologous with respect to the other elements present on the promoter construct.

The term "transformation" refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing the transformed nucleic acid fragments are referred to as

"transgenic" cells, and organisms comprising transgenic cells are referred to as "transgenic organisms".

"Transformed," "transgenic," and "recombinant" refer to a host cell or organism such as a bacterium, cyanobacterium, animal or a plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome as generally known in the art and disclosed (Sambrook 1989; Innis 1995; Gelfand 1995; Innis & Gelfand 1999). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like. The term "untransformed" refers to normal cells that have not been through the transformation process.

"Wild-type" refers to a virus or organism found in nature without any known mutation.

Design, generation, and testing of the variant nucleotides, and their encoded polypeptides, having the above required percent identities and retaining a required activity of the expressed protein is within the skill of the art. For example, directed evolution and rapid isolation of mutants can be according to methods described in references including, but not limited to, Link et al. (2007) Nature Reviews 5(9), 680-688; Sanger et al. (1991 ) Gene 97(1 ), 1 19-123;

Ghadessy et al. (2001 ) Proc Natl Acad Sci USA 98(8) 4552-4557. Thus, one skilled in the art could generate a large number of nucleotide and/or polypeptide variants having, for example, at least 95-99% identity to the reference sequence described herein and screen such for desired phenotypes according to methods routine in the art.

Nucleotide and/or amino acid sequence identity percent (%) is understood as the percentage of nucleotide or amino acid residues that are identical with nucleotide or amino acid residues in a candidate sequence in comparison to a reference sequence when the two sequences are aligned. To determine percent identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum percent sequence identity. Sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) software is used to align sequences. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. When sequences are aligned, the percent sequence identity of a given sequence A to, with, or against a given sequence B (which can alternatively be phrased as a given sequence A that has or comprises a certain percent sequence identity to, with, or against a given sequence B) can be calculated as: percent sequence identity = ΧΛΊ 00, where X is the number of residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of residues in B. If the length of sequence A is not equal to the length of sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

Generally, conservative substitutions can be made at any position so long as the required activity is retained. So-called conservative exchanges can be carried out in which the amino acid which is replaced has a similar property as the original amino acid, for example the exchange of Glu by Asp, Gin by Asn, Val by lie, Leu by lie, and Ser by Thr. Deletion is the replacement of an amino acid by a direct bond. Positions for deletions include the termini of a polypeptide and linkages between individual protein domains. Insertions are introductions of amino acids into the polypeptide chain, a direct bond formally being replaced by one or more amino acids. Amino acid sequence can be modulated with the help of art-known computer simulation programs that can produce a polypeptide with, for example, improved activity or altered regulation. On the basis of this artificially generated polypeptide sequences, a corresponding nucleic acid molecule coding for such a modulated polypeptide can be synthesized in-vitro using the specific codon-usage of the desired host cell.

"Highly stringent hybridization conditions" are defined as hybridization at 65 °C in a 6 X SSC buffer (i.e., 0.9 M sodium chloride and 0.09 M sodium citrate). Given these conditions, a determination can be made as to whether a given set of sequences will hybridize by calculating the melting temperature (T_m) of a DNA duplex between the two sequences. If a particular duplex has a melting temperature lower than 65°C in the salt conditions of a 6 X SSC, then the two sequences will not hybridize. On the other hand, if the melting temperature is above 65 °C in the same salt conditions, then the sequences will hybridize. In general, the melting temperature for any hybridized DNA:DNA sequence can be determined using the following formula: T_m = 81 .5 °C + 16.6(logi₀[Na⁺]) + 0.41 (fraction G/C content) - 0.63(% formamide) - (600/I). Furthermore, the T_m of a DNA:DNA hybrid is decreased by 1 -1.5°C for every 1 % decrease in nucleotide identity (see e.g., Sambrook and Russel, 2006).

Host cells can be transformed using a variety of standard techniques known to the art (see, e.g. , Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001 ) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring

Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754). Such techniques include, but are not limited to, viral infection, calcium phosphate transfection, liposome-mediated transfection, microprojectile-mediated delivery, receptor-mediated uptake, cell fusion, electroporation, and the like. The transfected cells can be selected and propagated to provide recombinant host cells that comprise the expression vector stably integrated in the host cell genome.

Exemplary nucleic acids which may be introduced to a host cell include, for example, DNA sequences or genes from another species, or even genes or sequences which originate with or are present in the same species, but are incorporated into recipient cells by genetic engineering methods. The term "exogenous" is also intended to refer to genes that are not normally present in the cell being transformed, or perhaps simply not present in the form, structure, etc., as found in the transforming DNA segment or gene, or genes which are normally present and that one desires to express in a manner that differs from the natural expression pattern, e.g., to over-express. Thus, the term "exogenous" gene or DNA is intended to refer to any gene or DNA segment that is introduced into a recipient cell, regardless of whether a similar gene may already be present in such a cell. The type of DNA included in the exogenous DNA can include DNA which is already present in the cell, DNA from another individual of the same type of organism, DNA from a different organism, or a DNA generated externally, such as a DNA sequence containing an antisense message of a gene, or a DNA sequence encoding a synthetic or modified version of a gene.

Host strains developed according to the approaches described herein can be evaluated by a number of means known in the art (see e.g., Studier (2005) Protein Expr Purif. 41 (1 ), 207-234; Gellissen, ed. (2005) Production of

Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley- VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression

Technologies, Taylor & Francis, ISBN-10: 0954523253).

Methods of down-regulation or silencing genes are known in the art. For example, expressed protein activity can be down-regulated or eliminated using antisense oligonucleotides, protein aptamers, nucleotide aptamers, and RNA interference (RNAi) (e.g., small interfering RNAs (siRNA), short hairpin RNA (shRNA), and micro RNAs (miRNA) (see e.g., Fanning and Symonds (2006) Handb Exp Pharmacol. 173, 289-303G, describing hammerhead ribozymes and small hairpin RNA; Helene, C, et al. (1992) Ann. N.Y. Acad. Sci. 660, 27-36; Maher (1992) Bioassays 14(12): 807-15, describing targeting

deoxyribonucleotide sequences; Lee et al. (2006) Curr Opin Chem Biol. 10, 1 -8, describing aptamers; Reynolds et al. (2004) Nature Biotechnology 22(3), 326 - 330, describing RNAi; Pushparaj and Melendez (2006) Clinical and Experimental Pharmacology and Physiology 33(5-6), 504-510, describing RNAi; Dillon et al. (2005) Annual Review of Physiology 67, 147-173, describing RNAi; Dykxhoorn and Lieberman (2005) Annual Review of Medicine 56, 401 -423, describing RNAi). RNAi molecules are commercially available from a variety of sources (e.g. , Ambion, TX; Sigma Aldrich, MO; Invitrogen). Several siRNA molecule design programs using a variety of algorithms are known to the art (see e.g., Cenix algorithm, Ambion; BLOCK-iT™ RNAi Designer, Invitrogen; siRNA Whitehead Institute Design Tools, Bioinofrmatics & Research Computing). Traits influential in defining optimal siRNA sequences include G/C content at the termini of the siRNAs, Tm of specific internal domains of the siRNA, siRNA length, position of the target sequence within the CDS (coding region), and nucleotide content of the 3' overhangs.

FORMULATION

The agents and compositions described herein can be formulated by any conventional manner using one or more pharmaceutically acceptable carriers or excipients as described in, for example, Remington's Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21 st edition, ISBN: 0781746736 (2005), incorporated herein by reference in its entirety. Such formulations will contain a therapeutically effective amount of a biologically active agent described herein, which can be in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.

The formulation should suit the mode of administration. The agents of use with the current disclosure can be formulated by known methods for

administration to a subject using several routes which include, but are not limited to, parenteral, pulmonary, oral, topical, intradermal, intramuscular,

intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, and rectal. The individual agents may also be administered in

combination with one or more additional agents or together with other biologically active or biologically inert agents. Such biologically active or inert agents may be in fluid or mechanical communication with the agent(s) or attached to the agent(s) by ionic, covalent, Van der Waals, hydrophobic, hydrophilic or other physical forces.

Controlled-release (or sustained-release) preparations may be formulated to extend the activity of the agent(s) and reduce dosage frequency. Controlled- release preparations can also be used to effect the time of onset of action or other characteristics, such as blood levels of the agent, and consequently affect the occurrence of side effects. Controlled-release preparations may be designed to initially release an amount of an agent(s) that produces the desired

therapeutic effect, and gradually and continually release other amounts of the agent to maintain the level of therapeutic effect over an extended period of time. In order to maintain a near-constant level of an agent in the body, the agent can be released from the dosage form at a rate that will replace the amount of agent being metabolized or excreted from the body. The controlled-release of an agent may be stimulated by various inducers, e.g., change in pH, change in

temperature, enzymes, water, or other physiological conditions or molecules.

Agents or compositions described herein can also be used in combination with other therapeutic modalities, as described further below. Thus, in addition to the therapies described herein, one may also provide to the subject other therapies known to be efficacious for treatment of the disease, disorder, or condition.

THERAPEUTIC METHODS

Also provided is a process of treating a disease or disorder in a subject in need administration of a therapeutically effective amount of a therapeutic module described herein, so as to deliver a therapeutic agent to a cell having a particular combination of markers on the cell surface or to prevent delivery of a therapeutic agent to a cell having a particular combination of markers on the cell surface. Cascades on a cell surface, as described herein, can result in a unique oligonucleotide displayed on the targeted cell. In some embodiments, a therapeutic module interacts with such unique oligonucleotide to cause a therapeutic effect. In some embodiments of a therapeutic module in which an output of a biomolecular computing cascade will be coupled to a therapeutic effect, e.g., cell elimination.

In various embodiments, systems of biomolecules, e.g., mixtures of interacting proteins and nucleic acids, can become active or inactive based on analyses of their environments. In such systems, switching between an active state and an inactive state (e.g., between possible outcomes) can occur if certain sets of sensory inputs are present or absent in the environment. The dependence of these changes of states on inputs can be described through input-output correlation tables (e.g., "truth tables"). Thus can be achieved implementation of systems of cascaded reactions coupled to agents (e.g., antibodies) interacting with cell surface markers.

According to approaches described herein, a therapeutic module can be constructed in which an output of a biomolecular computing cascade can be coupled to a therapeutic effect, e.g., cell elimination. This can be achieved by, for example, triggering delivery of proteins or small molecule toxins, or by activating an enzyme involved in a conversion of a prodrug into a drug. Thus can be achieved inhibition of toxic effects on non-target cells bearing a protective signatures on the cell surface, and analysis of a plurality of cell surface markers (e.g., three cell surface markers)

Also according to approaches described herein, an imaging module can be constructed in which an output of a biomolecular computing cascade can be coupled to generation or amplification of various signals useful for imaging of events on the surface of targeted cells. For example, a cell-specific signal can be amplified through accumulation of Gd(lll) complexes (e.g., MRI contrast) or "light-up" fluorophores, radiolabels, or the like. For example, an imaging module can be used to determine mass of insulin producing β-cells in the pancreas in vivo. The mass of β-cells, the insulin producing component of endocrine pancreas, represents less than 0.005 % of the normal adult bodyweight.

Imaging of β-cell molecular targets can reveal quantitative information about β-cell mass and or function, which can be used, e.g., to monitor diabetes progression, assess therapeutic approaches related to proliferation and differentiation of endogenous β-cell progenitors, or appraise methods of preserving mature β-cell mass or track the function or viability of transplanted cells.

According to approaches described herein, a plurality of cell surface markers (e.g., three) can be selected such that only pancreatic endocrine cells would have all of them (e.g., LAT-1 , Glut 2, D2R, Kir6.1 , or GLPI RI) (i.e., other cells in other tissues would not have all markers) individual markers. A system described herein can be modularly built, i.e., a combination of parts having individual functions. In some embodiments, available functions can include one or more of (i) recognition (e.g., interactions with environment that can be transduced downstream); (ii) computing (e.g., a process through which inputs are correlated to outputs); and (iii) actions (e.g., possible output functions, including light-up property, capture tags, or therapeutic moieties). Modularity can allow change of behaviors in systems by mixing and matching varieties of functional modules, or adjusting them to targeted applications. Thus can be achieved combining different recognition and computing modules with "tagging" modules (e.g., fluorescein or biotin). In this way, autonomous biocomputing cascades can mark for isolation a subpopulation, such as a narrow

subpopulations of lymphocytes within complex mixtures of peripheral blood mononuclear cells (PBMCs) in vitro.

Therapeutic approaches based on cell-surface analysis as described herein can be useful in a disease or disorder that can benefit from selective elimination of a cell type based on the presence or absence of multiple cell- surface makers. Non-limiting examples include hematopoietic malignancies and autoimmune diseases.

For example, with respect to lymphocytes during hematopoiesis (i.e., blood cell generation), branches of the hematopoietic tree are characterized by the expression of markers on the cell surface. These markers (or antigens) include cluster of differentiation markers or "CD"(s) with associated numbers. Hematopoietic neoplasms express markers characteristic for their lineage and stages of differentiation and such markers are regularly used in

immunophenotyping.

While targeted delivery of a toxin to a tumor cell by appropriate

monoclonal antibodies (MAbs) can result in significant beneficial effects in patients in both hematopoietic malignancies and SLE, the dosage and potential efficacy of such a MAb-toxin immunoconjugates has been limited by the concurrent development of toxic effects in normal cells. A major factor in the action of these conventional immunoconjugates on normal cells is the fact that the surface markers recognized by every immunoconjugate are present on significant numbers of normal cells. Thus, despite significant progress in immunotherapy, immunosuppression, or myelosuppression is a particular problem in the immunoconjugate treatment of hematopoietic neoplasms because virtually every lymphocyte tumor surface marker is shared by some populations of normal lymphocytes.

In contrast, approaches described herein can selectively eliminate one population, preserve another population, or activate therapeutic modules in the presence or absence of a combination of multiple markers. A system

implementing a basic Boolean logic AND (YESYES) and NOT (YESNOT) operation can significantly improve the outcome of therapy, either by enhancing delivery to target cells, or by protecting non-target cells.

Methods described herein are generally performed on a subject in need thereof. A subject in need of the therapeutic methods described herein can be a subject having, diagnosed with, suspected of having, or at risk for developing a disease or disorder that can benefit from selective elimination of a cell type based on the presence or absence of multiple cell-surface makers. A

determination of the need for treatment will typically be assessed by a history and physical exam consistent with the disease or condition at issue. Diagnosis of the various conditions treatable by the methods described herein is within the skill of the art. The subject can be an animal subject, including a mammal, such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, and chickens, and humans. For example, the subject can be a human subject.

Non-limiting examples of recognized or emerging clinical needs addressable with a system described herein: chronic lymphocytic leukemia (CLL); cutaneous T-cell lymphoma (CTCL), examples of hematopoietic malignancies, and targeting B-cells in autoimmune diseases. Specifically: in CLL, selective elimination of pathogenic lymphocytes can be based on

YESCD19YESCD5 (a combination extremely rare on healthy lymphocytes). In CTCL, side-effects are a recognized problem that can be minimized by protecting healthy CD8⁺ cells by specifically targeting YESCD25NOTCD8 subpopulations. In autoimmune diseases, therapy based on the elimination of broad populations of lymphocytes (B- or T-cells) may have beneficial effects, but only with a concomitant harmful effect on the immune system. A system described herein can eliminate or substantially reduce individual subpopulations of lymphocytes, narrowing down eliminated subpopulations.

Generally, a safe and effective amount of a system described herein is, for example, that amount that would cause the desired therapeutic effect in a subject while minimizing undesired side effects. In various embodiments, an effective amount of a therapeutic agent described herein can substantially inhibit, slow the progress of, or limit the development of a disease or disorder that can benefit from selective elimination of a cell type based on the presence or absence of multiple cell-surface makers.

According to the methods described herein, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.

When used in the treatments described herein, a therapeutically effective amount of an agent described herein can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form and with or without a pharmaceutically acceptable excipient. For example, systems of the present disclosure can be administered, at a reasonable benefit/risk ratio applicable to any medical treatment, in a sufficient amount to substantially inhibit, slow the progress of, or limit the development of a disease or disorder that can benefit from selective elimination of a cell type based on the presence or absence of multiple cell-surface makers.

The amount of an agent or system described herein that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of agent contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses.

Toxicity and therapeutic efficacy of compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals for determining the LD₅o (the dose lethal to 50% of the population) and the ED₅₀, (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio LD50/ED50, where larger therapeutic indices are generally understood in the art to be optimal.

The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see e.g. , Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincott Williams & Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical Pharmacokinetics, 4^th ed., Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics, McGraw-Hill/Appleton & Lange, ISBN 0071375503). For example, it is well within the skill of the art to start doses of the composition at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for purposes of administration. Consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. It will be understood, however, that the total daily usage of the compounds and compositions of the present disclosure will be decided by an attending physician within the scope of sound medical judgment. Again, each of the states, diseases, disorders, and conditions, described herein, as well as others, can benefit from compositions and methods described herein. Generally, treating a state, disease, disorder, or condition includes preventing or delaying the appearance of clinical symptoms in a mammal that may be afflicted with or predisposed to the state, disease, disorder, or condition but does not yet experience or display clinical or subclinical symptoms thereof. Treating can also include inhibiting the state, disease, disorder, or condition, e.g. , arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof. Furthermore, treating can include relieving the disease, e.g. , causing regression of the state, disease, disorder, or condition or at least one of its clinical or subclinical symptoms. A benefit to a subject to be treated can be either statistically significant or at least perceptible to the subject or to a physician.

Administration of agent or system can occur as a single event or over a time course of treatment. For example, agent or system can be administered daily, weekly, bi-weekly, or monthly. For treatment of acute conditions, the time course of treatment will usually be at least several days. Certain conditions could extend treatment from several days to several weeks. For example, treatment could extend over one week, two weeks, or three weeks. For more chronic conditions, treatment could extend from several weeks to several months or even a year or more.

Treatment in accord with the methods described herein can be performed prior to, concurrent with, or after conventional treatment modalities for a disease or disorder that can benefit from selective elimination of a cell type based on the presence or absence of multiple cell-surface makers.

An agent or system can be administered simultaneously or sequentially with another agent, such as an antibiotic, an antiinflammatory, or another agent. For example, an agent or system can be administered simultaneously with another agent, such as an antibiotic or an antiinflammatory. Simultaneous administration can occur through administration of separate compositions, each containing one or more of an agent or system, an antibiotic, an antiinflammatory, or another agent. Simultaneous administration can occur through administration of one composition containing two or more of an agent or system, an antibiotic, an antiinflammatory, or another agent. An agent or system can be administered sequentially with an antibiotic, an antiinflammatory, or another agent. For example, an agent or system can be administered before or after administration of an antibiotic, an antiinflammatory, or another agent.

ADMINISTRATION

Agents and compositions described herein can be administered according to methods described herein in a variety of means known to the art. The agents and composition can be used therapeutically either as exogenous materials or as endogenous materials. Exogenous agents are those produced or

manufactured outside of the body and administered to the body. Endogenous agents are those produced or manufactured inside the body by some type of device (biologic or other) for delivery within or to other organs in the body.

As discussed above, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.

Agents and compositions described herein can be administered in a variety of methods well known in the arts. Administration can include, for example, methods involving oral ingestion, direct injection (e.g., systemic or stereotactic), implantation of cells engineered to secrete the factor of interest, drug-releasing biomaterials, polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g. , up to 30 μΐη), nanospheres (e.g., less than 1 μΐη), microspheres (e.g., 1 -100 μΐη), reservoir devices, a combination of any of the above, or other suitable delivery vehicles to provide the desired release profile in varying proportions. Other methods of controlled-release delivery of agents or compositions will be known to the skilled artisan and are within the scope of the present disclosure. Delivery systems may include, for example, an infusion pump which may be used to administer the agent or composition in a manner similar to that used for delivering insulin or chemotherapy to specific organs or tumors. Typically, using such a system, an agent or composition can be administered in

combination with a biodegradable, biocompatible polymeric implant that releases the agent over a controlled period of time at a selected site. Examples of polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and combinations thereof. In addition, a controlled release system can be placed in proximity of a therapeutic target, thus requiring only a fraction of a systemic dosage.

Agents can be encapsulated and administered in a variety of carrier delivery systems. Examples of carrier delivery systems include microspheres, hydrogels, polymeric implants, smart polymeric carriers, and liposomes (see generally, Uchegbu and Schatzlein, eds. (2006) Polymers in Drug Delivery, CRC, ISBN-10: 0849325331 ). Carrier-based systems for molecular or biomolecular agent delivery can: provide for intracellular delivery; tailor biomolecule/agent release rates; increase the proportion of biomolecule that reaches its site of action; improve the transport of the drug to its site of action; allow colocalized deposition with other agents or excipients; improve the stability of the agent in vivo; prolong the residence time of the agent at its site of action by reducing clearance; decrease the nonspecific delivery of the agent to nontarget tissues; decrease irritation caused by the agent; decrease toxicity due to high initial doses of the agent; alter the immunogenicity of the agent; decrease dosage frequency, improve taste of the product; or improve shelf life of the product.

SCREENING

Also provided are methods for screening.

The subject methods find use in the screening of a variety of different candidate molecules (e.g., potentially therapeutic candidate molecules).

Candidate substances for screening according to the methods described herein include, but are not limited to, fractions of tissues or cells, nucleic acids, polypeptides, siRNAs, antisense molecules, aptamers, ribozymes, triple helix compounds, antibodies, and small (e.g., less than about 2000 mw, or less than about 1000 mw, or less than about 800 mw) organic molecules or inorganic molecules including but not limited to salts or metals.

Candidate molecules encompass numerous chemical classes, for example, organic molecules, such as small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons. Candidate molecules can comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, and usually at least two of the functional chemical groups. The candidate molecules can comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.

A candidate molecule can be a compound in a library database of compounds. One of skill in the art will be generally familiar with, for example, numerous databases for commercially available compounds for screening (see e.g. , ZINC database, UCSF, with 2.7 million compounds over 12 distinct subsets of molecules; Irwin and Shoichet (2005) J Chem Inf Model 45, 177-182). One of skill in the art will also be familiar with a variety of search engines to identify commercial sources or desirable compounds and classes of compounds for further testing (see e.g. , ZINC database; eMolecules.com; and electronic libraries of commercial compounds provided by vendors, for example:

ChemBridge, Princeton BioMolecular, Ambinter SARL, Enamine, ASDI, Life Chemicals etc).

Candidate molecules for screening according to the methods described herein include both lead-like compounds and drug-like compounds. A lead-like compound is generally understood to have a relatively smaller scaffold-like structure (e.g., molecular weight of about 150 to about 350 kD) with relatively fewer features (e.g., less than about 3 hydrogen donors and/or less than about 6 hydrogen acceptors; hydrophobicity character xlogP of about -2 to about 4) (see e.g., Angewante (1999) Chemie Int. ed. Engl. 24, 3943-3948). In contrast, a drug-like compound is generally understood to have a relatively larger scaffold (e.g., molecular weight of about 150 to about 500 kD) with relatively more numerous features (e.g., less than about 10 hydrogen acceptors and/or less than about 8 rotatable bonds; hydrophobicity character xlogP of less than about 5) (see e.g., Lipinski (2000) J. Pharm. Tox. Methods 44, 235-249). Initial screening can be performed with lead-like compounds.

When designing a lead from spatial orientation data, it can be useful to understand that certain molecular structures are characterized as being "drug- like". Such characterization can be based on a set of empirically recognized qualities derived by comparing similarities across the breadth of known drugs within the pharmacopoeia. While it is not required for drugs to meet all, or even any, of these characterizations, it is far more likely for a drug candidate to meet with clinical successful if it is drug-like.

Several of these "drug-like" characteristics have been summarized into the four rules of Lipinski (generally known as the "rules of fives" because of the prevalence of the number 5 among them). While these rules generally relate to oral absorption and are used to predict bioavailability of compound during lead optimization, they can serve as effective guidelines for constructing a lead molecule during rational drug design efforts such as may be accomplished by using the methods of the present disclosure.

The four "rules of five" state that a candidate drug-like compound should have at least three of the following characteristics: (i) a weight less than 500 Daltons; (ii) a log of P less than 5; (iii) no more than 5 hydrogen bond donors (expressed as the sum of OH and NH groups); and (iv) no more than 10 hydrogen bond acceptors (the sum of N and O atoms). Also, drug-like molecules typically have a span (breadth) of between about 8A to about 15A.

KITS

Also provided are kits. Such kits can include an agent or composition described herein and, in certain embodiments, instructions for administration. Such kits can facilitate performance of the methods described herein. When supplied as a kit, the different components of the composition can be packaged in separate containers and admixed immediately before use. Components include, but are not limited to all or parts of a molecular automata system described herein. Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition. The pack may, for example, comprise metal or plastic foil such as a blister pack. Such packaging of the components separately can also, in certain instances, permit long-term storage without losing activity of the components.

Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately. For example, sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, sterile saline or sterile each of which has been packaged under a neutral non-reacting gas, such as nitrogen. Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal or any other material typically employed to hold reagents. Other examples of suitable containers include bottles that may be fabricated from similar substances as ampules, and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix.

Removable membranes may be glass, plastic, rubber, and the like.

In certain embodiments, kits can be supplied with instructional materials. Instructions may be printed on paper or other substrate, and/or may be supplied as an electronic-readable medium, such as a floppy disc, mini-CD-ROM, CD- ROM, DVD-ROM, Zip disc, videotape, audio tape, and the like. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet web site specified by the manufacturer or distributor of the kit.

Compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see, e.g. , Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001 )

Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studier (2005) Protein Expr Purif. 41 (1 ), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10:

0954523253).

Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term "about." In some embodiments, the term "about" is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

In some embodiments, the terms "a" and "an" and "the" and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term "or" as used herein, including the claims, is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

The terms "comprise," "have" and "include" are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as "comprises," "comprising," "has," "having," "includes" and "including," are also open-ended. For example, any method that "comprises," "has" or "includes" one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that "comprises," "has" or "includes" one or more features is not limited to possessing only those one or more features and can cover other unlisted features.

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.

Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.

EXAMPLES

The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure, and thus can be considered to constitute examples of modes for its practice.

However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

EXAMPLE 1: GENERAL PROCEDURES, METHODS, AND MATERIALS

The following example outlines the general procedures, methods, and materials used in the subsequent examples.

Briefly, oligonucleotides were coupled to antibodies, unless stated otherwise, in a two-step procedure: (i) DTT was used under conditions that reduce interchain disulfide bonds; (ii) oligonucleotides with maleimides at 5' ends were coupled to resulting sulfhydryls, and the products were purified using gel filtration. One biotinylated antibody was used in the NOTCD45RA cascade, in which cases streptavidin was used to cross-link it to biotinylated

oligonucleotides, and this procedure was performed directly on cells without purification of conjugates (negative controls with no streptavidin and no antibody were successfully run as well, in this case). Reagents were added to cell suspensions, and in all experiments involving PBMC's, reagents were removed from solution by centrifugation. In whole blood experiments (see e.g., FIG. 1 ), reagents were left in blood, to mimic in vivo applications.

EXAMPLE 2: ANTIBODY SELECTION

The following example describes the materials used as the antibodies for the following examples.

Anti-human CD3 (clone HIT3a); anti-human CD8 (clone SK1 ); anti-human CD45 (clone HI30); anti-human CD45RA (clone H1100), were commercially supplied by Biolegend. Anti-human CD20 Rituxan (Rituximab) was commercially supplied by Genentech.

EXAMPLE 3: OLIGONUCLEOTIDE MATERIALS AND DESIGN METHODS

The following example describes the materials and methods used in the sequence design of the oligonucleotides described in the following examples.

All oligonucleotides were commercially manufactured by Integrated DNA

Technologies Inc. (Coralville, IA), with HPLC purification, and used as received, except for Pacific Blue dye modified oligonucleotides were commercially manufactured by Invitrogen™ (Life Technologies Corporation). The following 3' and 5' modifications were used: thiol modifier C6 S-S; Biotin-TEG; Iowa

Black®FQ; Iowa Black®RQ; Fluorescein dye 6-FAM™ (NHS ester); or Cy5™. Cascade sequences were designed to have minimal (ideally none) secondary structure, and to have minimal non-desired base-pairing with any other sequence in the cascade. This was achieved using the software NUPACK. (see e.g., FIG. 2)

Two nucleotides were added to the 3' ends of sequences for strands 3 and 5 in the YES-YES and YES-NOT cascade and to strands 5 and 7 in the YES-YES-YES cascade, to inhibit unwanted strand invasion by the 5'-ends of sequences 2 and 4, and 4 and 6, respectively. By optimizing the operation of the cascades in solution, signal 'leakage' was minimized by addition of mismatches (see e.g., FIG. 2 and FIG. 3).

EXAMPLE 4: SAMPLE STAINING

The following example describes the materials used for staining samples.

Samples were stained with CD4 APCCy7, CD19 PerCp Cy5.5

(eBioscience), CD45RO Pacific Blue and CD45RA PE (BioLegend). Further materials used in the following examples include: 7-aminoactinomycin-D (7-AAD) (Sigma) and was used in final concentration 2 μg/ml, 1 x BD FACS Lysing Solution (BD Biosciences), ImmunoPure Streptavidin (ThermoScientific), and anti-FITC MicroBeads (MiltenyiBiotec GmbH).

EXAMPLE 5: CELL CULTURE

The following example describes the materials and methods used in cell culture.

Peripheral blood mononuclear cells (PBMC) were isolated on a Ficoll- Paque Plus (GE HealthCare) gradient from whole blood or buffy coat obtained from NYC Blood center. Cells were washed and stained in PBS (Sigma) buffer, supplemented with 2% FBS (Gibco), and 1 .2 mM MgCI₂ (Ambion).

EXAMPLE 6: FLOW CYTOMETRY

The following example describes the materials and methods used in flow cytometry.

FACSCanto (Becton Dickinson) flow cyto meter with 405 nm, 488 nm, and 633 nm excitation wavelengths was used for flow cytometry measurements. Instrument setup was performed by using CST beads. Fluorescence

compensation was performed by single staining using anti-mouse Ig, k/Negative Control (FBS) Compensation Particles Set (BD CompBeads, BD Biosciences). Amplifier settings for forward scatter and side scatter were used in linear mode and for fluorescence channels, logarithmic mode was used. Events were gated based on the forward scatter versus side scatter and fluorescence intensities versus time. Kinetics experiments were recorded for around 30 minutes. Cells analysis was performed at the rate of 2 μ I per min. The fluorescence intensities of each event were measured using 530 (30 nm band pass), 660 nm (20 nm band pass) and 450 nm (50 nm band pass), 585 nm (42 nm band pass), 780 nm (60 nm band pass) filters, respectively. The data was transferred and analyzed with FlowJo software version 9.4.1 1 .

EXAMPLE 7: SYNTHESIS OF ANTIBODY-OLIGONUCLEOTIDE CONJUGATES

The following example describes the synthetic procedure of Rituxan- oligonucleotide conjugate and general synthesis for an antibody-oligonucleotide conjugates.

Part 1 - Activated (Si) oligonucleotide (26 nmoles, 200 μΙ from 130 μΜ stock) was combined with excess 1 ,6-bismaleimidohexane (BMH) (200 μΙ of 1 .44 mg/802 μΙ in DMSO, i.e. 1300 nmoles {50-fold excess}). The reaction mixture (50% DMSO) was incubated at room temperature for 1 hour and then split in two and each half precipitated with cold ethanol (1 .5 ml_) by leaving at -20 °C for 45 mins. The precipitate was separated by centrifugation and the pellet washed twice with cold ethanol and dried in vacuo. The dried pellets were resuspended in water and applied to a NAP5 desalting column (GE Healthcare) to remove any remaining traces of free BMH. The eluent was frozen and lyophilized.

Part 2 -The following was carried out using aseptic techniques. 700 μΙ of rituximab (1 .4 mg, 9.7 nmoles) (1000 mg/500 mL, 5% dextrose, 0.01 % NaN₃) was buffer exchanged with 0.1 M sodium phosphate pH 8.0 buffer containing 1 mM EDTA via Zeba desalting column ("2 ml_", Pierce). DTT (10 mM stock) was added to the resulting solution to give a final DTT concentration of 0.1 mM. The reaction mixture was incubated at 37 °C overnight (22 hrs). Unreacted DTT was removed using two subsequent Zeba desalting columns, eluting with PBSE (PBS with 5 mM EDTA), pH 6.8. The final concentration of rituximab was 9.3 uM determined by UV-vis (ε280 nm = 1.7 mU(mgXcm) i.e. 240,000 M^" cm^"1). Using Ellman's reagent it was determined that there were on average six sulfhydryl groups per antibody (i.e. reduction of 3 disulfide bonds). The activated antibody was kept on ice.

For anti-human CD3, CD8, and CD45, the antibodies were buffer exchanged with 0.1 M TRIS, pH 8.0 and DTT added to give a concentration of 5 mM, then incubated at 37 °C for 30 mins. (S2) Theses antibodies were then purified and characterized as above.

Part 1 and Part 2 products were then combined, for example, 1 nmole of activated oligonucleotide (4.3 μΙ) was added to 0.25 nmoles of activated rituximab (27 μΙ) i.e. 4: 1 oligo:antibody (see e.g., FIG. 4 for results). For coupling double helical DNA to the antibody, a slight excess of complementary strand was added to the activated oligonucleotide from Part 1 with incubation for 30 mins, then this combined with Part 2 (see e.g., FIG. 3 for results). Purification was carried out by size exclusion chromatography on a Superdex 200 10/300 GL column (GE Healthcare) with an Akta purifier system (GE Healthcare).

EXAMPLE 8: 3-STEP AND 2-STEP CASCADE PROTOCOL

The following example describes the protocol for the preparation and analysis of the 3-step cascade, YESCD8YESCD3YESCD4, and 2-step cascades.

A mixture of the antibody-oligonucleotide conjugates were incubated with 1.5x10⁶ cells at a final concentration of 0.1 μΜ (or 7.5 μg antibody/ml), each duplex in a final volume of 100 μΙ, on ice for 20 minutes. After incubation the cells were washed twice with 2.5 ml of cold buffer on 300xg for 5 minutes at +4 °C (Eppendorf Centrifuge 5804 R). The pellet was resuspended in 400 μΙ of buffer and then run on a FacsCanto (BD Bioscience) flow cytometer to measure Fluorescence intensity vs Time. Duplex 5·6 and trigger 0 were added to a final concentration of 0.5 μΜ in real time during measurement.

EXAMPLE 9: YESCD8YESCD3 CASCADE IN WHOLE BLOOD

The following example describes the protocol for the preparation and analysis of YESCD8YESCD3 cascade in whole blood.

Antibody conjugated duplexes 3*4_aCD8 and 1 »2_aCD3 were added to 300 μΙ of whole blood cells at a final concentration of 1 .5 μg of antibody/ml and incubated for 15 minutes at room temperature. Afterwards, 4 μΙ of duplex 5·6 was added to a final concentration of 0.5 μΜ, incubated 15 minutes, followed by trigger 0 at a final concentration of 1 μΜ and incubated for a further 15 minutes at room temperature. To examine the result of the cascade reaction, red blood cells were lysed with 1 x BD FACS Lysing Solution in the dark at RT for 30 minutes. During flow cytometric analysis, nucleated cells were gated based on 7- aminoactinomycin-D (7AAD) staining.

EXAMPLE 10: YESCD8NOTCD45RA CASCADE

The following example describes the protocol for the preparation and analysis of YESCD8NOTCD45RA cascade.

aCD45RA was conjugated to duplex 5*»6*via biotin-streptavidin coupling (assembly in situ method without purification of conjugates). Specifically, 1 .5 million PBMCs were incubated with 0.5 μg of biotinylated anti-human antibody

CD45RA in a final volume of 100 μΙ of cold buffer for 20 minutes on ice. Then, the cells were washed twice with 2.5 ml of cold buffer by centrifugation (300xg, 5 minutes, at 4 °C). The next incubation was performed with 0.5 μg of

ImmunoPure Streptavidin in a final volume of 100 μΙ of cold buffer, for 20 minutes on ice. The cells were then washed by centrifugation as above and biotinylated duplex 5*»6*_biotin was added at a final concentration of 0.1 μΜ in a final volume of 100 μΙ of cold buffer for 20 minutes on ice. The cells were then washed twice as described above. Next, a final concentration of 0.1 μΜ of

3»4_acD8 was added together with aCD4 and aCD19 antibodies and the cells were incubated for 20 minutes on ice. After incubation, the cells were washed twice by centrifugation as described above, and resuspended in a total volume of 400 μΙ of buffer. During flow cytometric analysis, duplex 5·6 and trigger 2, at a final concentration 0.5 μΜ and 1 μΜ respectively, were added in real time at room temperature.

The biotin coupling method was not used on more than one antibody, because it was observed, in this case, a noticeable exchange of oligonucleotides between two biotinylated antibodies (5-20%), which makes data analysis more difficult and results less clear-cut (i.e., the observation could need to be

"corrected").

EXAMPLE 11: MAGNETIC BEADS SEPARATION OF CELLS (YESCD45YESCD3)

The following example describes the separation of cells with magnetic beads.

3»4CCD3 was attached to the PBMC cell surface via biotin-streptavidin, as previously described. PBMCs were then incubated with 1 »2_acD45 (0.1 μΜ) for 20 minutes on ice. The cells were washed twice by centrifugation (300xg, 5 minutes, at 4 °C) and incubated with 0.5 μΜ oligonucleotide duplex 5·6 at room temperature for 5 minutes. Cells were washed and trigger 0 (1 μΜ final concentration) was added and incubated for 20 minutes. Afterwards, cells were washed with 15 ml of buffer and incubated with Anti-FITC Micro beads

(MiltenyiBiotec GmbH) as described in the kit protocol.

EXAMPLE 12: ISOLATION AND ENRICHMENT OF SUBPOPULATIONS OF T- AND B- , CDS™⁵-, AND CD4^P0S- CELLS.

The following example describes the isolation and enrichment of subpopulations of T- and B-, CD8^pos-, and CD4^pos- cells.

Isolation of subpopulations of T- and B-, CD8^pos-, and CD4^pos-cells was performed using Pan T Cell Isolation Kit I I, B Cell Isolation Kit II, CD8+ T Cell Isolation Kit (all from MiltenyiBiotec) and Negative Selection Human CD4+ T Cell Enrichment Kit (StemCell Technologies). The enrichment was accomplished precisely as described in the original kits protocols.

To assess the purity of the enriched T- and B-, CD8^pos-, and CD4^pos- subpopulations, cells were stained with the following fluorochrome - conjugated antibodies: CD4 Pacific Blue (eBioscience, clone OKT4), CD8 PECy7

(BioLegend, cloneSKI ), CD20 APC (eBioscience, clone 2H7), CD3 PE

(eBioscience, clone UCHT1 ), CD45 Pacific Orange (Invitrogen, clone 2D1 ).

EXAMPLE 13: CONCENTRATION DETERMINATION BY UV-VIS SPECTROSCOPY

The following example describes the procedure for determining the concentration of oligonucleotides by UV-vis spectroscopy.

For example, absorption coefficients for rituximab at 280 nm is 240,000 M^" cm^"1 , and at 260 nm is 126,000 M^" cm^"1. Absorption coefficient for two-step cascade strand (4) oligonucleotide is 480,000 M^" cm^"1.

By adding 1 , 2, 3, 4, 5, 6, and 7 equivalents of oligonucleotide to one equivalent of rituximab, the following A260 nm/A280 nm ratios were found: 1 .12, 1.33, 1 .41 , 1 .46, 1 .50, 1 .52, and 1 .54 respectively (see e.g., FIG. 5).

For the "4" sample, the 260/280 ratio was found to be 1 .52, and for the "2.4" the 260/280 ratio was 1.38, corresponding to an average of 6.0

oligonucleotides per rituximab for the "4" sample, and 2.9 oligonucleotides per rituximab for the "2.4" sample (see e.g., FIG. 5). Therefore, the absorption coefficient for the "4" sample is (126,000 + 6x480,000)M^" cm^"1 = 3,010,000 M^" cm^"1 , and using Beer's Law, the concentration of '6: 1 ' conjugate in the sample is 3.14 μΜ, which implies an absolute oligonucleotide concentration of 19 μΜ (see e.g., FIG. 5). Analogously, the concentration of '2.9: 1 ' conjugate (abs coeff. = 1 ,520,000 M^" cm^"1) in the sample "2.4" is 1 .2 μΜ, which implies an absolute oligo concentration is 3.4 μΜ (see e.g., FIG. 5).

EXAMPLE 14: BLOOD CELLS AS TARGETS FOR MOLECULAR AUTOMATA

The following example describes the targets used for molecular automata. Blood cells were chosen as targets for molecular automata, as they are the most exhaustively studied examples of cells with lineages and stages of differentiation defined by the presence or absence of multiple cell surface markers. Blood cells are commonly characterized by flow cytometry via different levels of expression of multiple cell surface markers known as Clusters of Differentiation or CDs, with CD45s, CD20, CD3, and CD8 used as examples herein. Here the basic design principles for automata that can tag lymphocytes with targeted markers characteristic for B-cells (i.e., CD45⁺CD20⁺cells) in the presence of CD45⁺CD20^"cells (e.g., CD45⁺CD3⁺' T-cells) is shown (see e.g., FIG. 6). EXAMPLE 15: PROGRAM EXECUTION

The following example describes the automata program execution. The "program" (conditional sequential transitions) that an automaton can execute on the surfaces of lymphocytes can be defined by a set of antibodies against markers M, directing cascades of chemical reactions on cell surfaces (see e.g., FIG. 6, FIG. 7 with CD20 and CD45 as Mis).

The well-established antibodies targeting CD markers (aCD45, aCD45RA, aCD20 (Rituximab), aCD3, and aCD8) were used as antibodies against markers, Mi. All of these antigens are present at between 80 and 200 thousand copies per cell surface on targeted subpopulations of lymphocytes, ensuring strong signal when measured by flow cytometry. These antibodies were conjugated with a set of partially complementary oligonucleotides (1 ·2, 3·4, and 5·6) optimized to execute, when triggered with oligonucleotide 0, modified strand-displacement cascades (see e.g., FIG. 2, FIG. 3, FIG. 4, FIG. 5, FIG. 6B, FIG. 6C, FIG. 8, FIG. 9).

Once turned on, such an automaton would ask a series of questions regarding the presence on the same cell surface of different markers via oligonucleotide transfers enabled by sequential exposure of new toeholds (cf. FIG. 4B), executing 'if YES M then proceed' or 'if NorMj then proceed' functions. EXAMPLE 16: AUTOMATA EVALUATION OF TWO SURFACE MARKERS

The following example describes the demonstration of the ability of the automata to evaluate two surface markers.

First, the ability for automata to evaluate two surface markers (see e.g., FIG. 10A for YESCD45YESCD20 experiment, functionally equivalent to

CD45ANDCD20) and to selectively label one targeted subpopulation within peripheral blood mononuclear cells (PBMCs - a mixture of various lymphocytes, monocytes, and macrophages) was tested.

All possible automata that could read combinations of two out of three markers, CD45 (a marker of nucleated hematopoietic cells), CD20 (a B-cell marker), and CD3 (a pan-T-cell marker) were constructed. Two of these automata are capable of successful completion of their

"program":YEsCD45YEsCD20 would operate (label) only on B-cells (see e.g., FIG. 10A) and YESCD45YESCD3 would operate only on T-cells (see e.g., FIG. 1 1 ). The third possible two-step automaton, YESCD3YESCD20 is a negative control, because no subpopulations in this example display these two markers simultaneously. The operation of these automata is equivalent to asking: "Is this cell a nucleated hematopoietic cell?" (YESCD45) followed by, in the case of the first automaton, "Is this a nucleated hematopoietic cell from a B-cell lineage?" (YESCD20) and, in the case of the second automaton, "Is this nucleated hematopoietic cell from the T-cell lineage?" (YESCD3). In all these automata, if both questions are answered positively in a row, the reaction performed, given here on an example of B-cells, will be: 0 + 1 »2_ACD45+ 3*4_aCD2o^ 0*1 +aCD4s2»3 + aCD2o4, with targeted subpopulations displaying a newly uncovered single- stranded oligonucleotide, 4. This one marker can then be said to contain the same information as traditional multicolor labeling with the same antibodies that were used in construction of automata and that would otherwise be used to characterize the immunological phenotype of these cells (e.g., CD45⁺CD20⁺). Additionally, a system was set up so the output oligonucleotide would interact with a solution phase label such as: _acD2o4+F-5»6->_acD2o4*5-F+ 6 (where F is a fluorescent signal from fluorescein when not quenched by 6), and the response of targeted cells to the cascade could be directly analyzed by flow cytometry (YESCD45YESCD20-> F) within a heterogenous population of cells. In order to assess the full operation of automata, 1 was labeled with Cy5, so both its removal and subsequent acquisition of fluorescein by _kCD2O4 on the cell surface could be monitored simultaneously in real time.

Experiments showed the first two automata successfully labeled only surfaces of either B- (CD45⁺CD20⁺) or T- (CD45⁺CD3⁺) cells (see e.g., FIG. 10A, FIG. 10B, FIG. 1 1 , FIG. 12). Each outcome was confirmed three or more times on individual human blood samples and monitored by multicolor flow cytometry. From these same components an automaton was also made that could label the surfaces of both B- and T-cells by using 3»4_aCD2o and 3»4_aCD3 in the same solution (cf., FIG. 1 1 E); a possible presentation of this automaton is that it is demonstrating an OR function, as in YESCD45(YESCD20ORYESCD3). After the successful demonstrations of these automata in mixtures of cells (PBMCs), it was also confirmed that the automata worked on enriched cell subpopulations with correct marker combinations (B- or T-cells), and that all cells that were

CD45⁺CD20⁺(or CD3⁺) were labeled anti-CD45 and anti-CD20 (or anti-CD3) antibodies were used; cells that were negative in one of these markers were not labeled (see e.g., FIG. 12). EXAMPLE 17: CONTROL AUTOMATA

The following example describes the control experimental protocol.

Various controls were studied in further detail in automata that are not supposed to provide an answer or cascades that could occur only between markers on separate cells (between two subpopulations). Using the third possible two-step automaton as described above, YESCD3YESCD20, no labeling was observed within the time-frame of the experiment, indicating that T- cells are not observably exchanging elements with B-cells either through diffusion or through direct physical contact of cells (see e.g., FIG. 13). T- and B-cells were separated, labeling the former with 1 »2_-aCD3, the latter with 3*4_-aCD2o- Upon remixing the cells, no crosstalk between different lineages was observed, within the detection limits of the flow cytometer (these are also negative controls for a direct 0 + 3·4 reaction; see e.g., FIG. 13). Finally, it was demonstrated that automata YESCD20YESCD45, with the inverted order of assessing the cell, worked without labeling any CD45⁺CD20^"cells (see e.g., FIG. 14). All of these experiments demonstrate low noise in the automata in the absence of an excess of elements in the solution-phase (i.e., they demonstrate minimal tagging of cells via diffusion or by direct contact between cells). In order to estimate the effects of washing away excess of antibody conjugates, automata YESCD3YESCD20 and YESCD3 (YESCD20 OR YESCD8) were studied without prior removal of the excess components from the solution. In both cases a visible change was observed in the fluorescence of non-target cells, albeit several-fold weaker than in the case of targeted cells (see e.g., FIG. 13).

The structures comprising these two-step automata were adjusted to enable an 'if NOTM, then proceed' function (NOTM,, see e.g., FIG. 15, FIG. 16), i.e., automata labeling cells with fluorescent oligonucleotides only in the absence of a CD marker. During the differentiation of T-cells, from na^'ive to memory, there is a transition in expression of two different isoforms of CD45 (CD45RA and CD45RO) and an automaton assessing the presence of isoforms of CD45 on CD8⁺T-Cells was created, with one of the isoforms inhibiting the cascade (CD45RA). The automaton YESCD8NOTCD45RA consisted of 3»4_aCD8 and

5*»6*„cD45RA triggered by 2 in the presence of solution-phase F-5»6. All cells that strongly responded to the automaton, by acquiring F-5 from solution-phase, strongly expressed CD45RO, that is, they were CD45RA^" cells (see e.g., FIG. 2B, FIG. 16B). This was in contrast with CD8⁺CD45RA⁺ T-cells, namely

CD45RO^" or CD45RO^DIM, which were hindered in acquiring F-5 due to competition with 5* from CD45RA in proximity to CD8-displaying 4, instead forming 5**4_KCD8 (see e.g., FIG. 15, FIG. 16). It should be noted that the 'if NOTM, then proceed' function is currently limited by the ratio of levels of expression of individual markers on the cell surface (at least until a threshold function is introduced). EXAMPLE 18: 3-STEP CASCADE YESCD45YESCD3YESCD8

The following example describes the establishment of the three step cascade. The previous examples describe, among other things, the

establishment of three types of transitions that could be used to build larger automata, YES ,, NOT ,, and OR (the last function consisting of adding to the cells two antibodies conjugated to identical oligonucleotide components). As an example of the feasibility of building more complex automata from these simple transitions, an automaton with a three-step cascade was built, evaluating the presence of up to three markers, and executing on the cell surface

YESCD45YESCD3YESCD8 (the third question: "Is this nucleated hematopoietic cell of T-Cell lineage a CD8 positive cell?", thus separating helper from cytotoxic T- cells). In this automaton, the surface of CD8⁺ cells enabled the following reaction: 0 + 1 »2_ACD45 + 3»4_ACD3 + 5»6„CD8 + 7·8 - 0· 1 + _acD4s2»3 + _acD3 »5 + aCD86»7 + 8. The labeling scheme allowed for the monitoring of each step in this cascade via flow cytometry in real time (see e.g., FIG. 17B, FIG. 18). This automaton was successfully demonstrated on targeted cells, with changes in fluorescence of cells being fully consistent with changes in distances between various components upon each step in the cascade (the first step is monitored by the removal of Pacific Blue, second by the drop in Cy5 due to quenching, and third by the acquisition of fluorescein from solution).

EXAMPLE 19: APPLICATIONS OF AUTOMATA

The following example describes applications of automata. Here, the automata are tested under conditions that could lead to applications. It was demonstrated that: (1 ) isolation with a purity equivalent to a standard isolation protocol fluorescein-labeled cells after a YESCD45YESCD3 automaton; where a standard method for isolation of cells was used (see e.g., FIG. 1A, using anti- fluorescein antibody conjugated to magnetic beads) and (2) an automaton (using YESCD3YESCD8) can function in whole blood, such that it was possible to simply add automata components to the mixture all together prior to triggering the reaction (see e.g., FIG. 1 B). The former demonstration was important, because it showed that there is no detectable decrease in purity of isolated cells between a single step automaton-based procedure {in situ cascade) and the standard separation protocol based on individual separation steps for each CD marker. The latter demonstration also established that blood components did not interfere with the cascades. Together with demonstrations that interactions via solution-phase information transfer do not represent major pathways in labeling cells (see e.g., FIG. 13), this example shows automata can be sued for labeling and eventually eliminating cells in vivo, depending on the pharmacokinetic properties of conjugates.

The above examples have established that a combination of antibodies and oligonucleotide-based reaction cascades can operate as molecular automata to assess the presence or absence of cell surface markers on living human cells.

EXAMPLE 20: THERAPEUTIC MODULES WITH RIPS

This example describes therapeutic modules that interact with unique oligonucleotides displayed on a targeted cell.

Models of cell targeting with toxins are based on results described above, e.g., YESYES and YESNOT (protective) cascades based on CD8⁺ T-Cells and CD45RO/RA isoforms (CD45RA is mostly located on naive T cells and CD45RO is located on memory T cells). Further demonstration occurs by targeting lymphocyte subpopulations with direct mechanistic implications in animal models. Thus is demonstrated in vivo elimination of specific subpopulations of lymphocytes in rats, and results in animal models can be monitored with imaging approaches described herein. Cytotoxicity is assessed ex vivo with the FMCA assay and cell proliferation with the MTT assay.

Two toxin-delivery module ribosome inactivating proteins (RIPs), saporin and gelonin, are used to target lymphocytes and cancer cells. For saporin, it is estimated that 1 ,000 binding events of its conjugate on a cell surface is sufficient to cause cell death. Although gelonin is about ~ 6-10-fold less toxic, it has some significant practical advantages. It is less costly; it is readily available on a larger scale; it has been previously conjugated to oligonucleotides; and used to eliminate specific subsets of lymphocytes with immunotoxins. It was also confirmed that gelonin can be readily conjugated to oligonucleotides at approximately 1 :1 ratio. The conjugate to 8 showed low cytotoxicity (>10 μΜ), unless it was delivered directly across the cell membrane (e.g., via lipofectamine); in this case toxicity became low-to-sub nanomolar.

Toxin-carrying modules contain gelonin or saporin conjugated to an oligonucleotide 5 displayed on CD8. Elimination of subsets of CD8⁺ cells are compared via four possible cascades for three markers (TABLE 1 ), CD8, CD45RO, and CD45RA (CD45 isoforms RO and RA are mostly exclusive, although there are some minor mixed populations, cf., Figure 4A).

TABLE 1

Each of YESCD8YESCD45RO (I) and YESCD8NOTCD45RO (II),

YESCD8YESCD45RA (III) and YESCD8NOTCD45RA (IV) are tested with delivery of toxin module (T in table). Aside from standard assays, proof of activity of RIPs (i.e., ability to halt protein synthesis) is confirmed by a radioactive glycine uptake assay.

One scheme for cascade I is provided in FIG. 23A, with toxin being conjugated to 6 as in 6^*7 conjugate. In two-step cascades, lengths of oligonucleotides can be minimized (down to 30) and mismatches that were introduced to prevent non-specific interaction with downstream elements in three-and-more-step cascades can be eliminated. RNAse's triggered degradation of RNA can be used to start a cascade so as to simplify a therapeutic procedure by reducing the number of injections. The cascade can be triggered by a cleavage of an RNA loop.

One scheme for a protective cascade (e.g., II) is given in FIG. 23B. This is a cascade in which CD8⁺ cells are protected by the presence of

CD45RO; thus, the uptake of the toxin from solution is minimized by the faster competing reaction from the cell surface. This cascade can be optimized by adjusting toehold regions and by optimizing loads of antibody-oligonucleotide conjugates.

Additional demonstrations in animal models include CD4⁺CD25⁺ Vbeta (18+7+8.6) T_reg or CD4⁺CD45RC⁺ T_c cells. Additional demonstrations in clinical models include CLL lymphocytes targeted via YESCD19YESCD5. ]

EXAMPLE 21: THERAPEUTIC MODULES WITH CARDIOTONIC STEROIDS

Cardiotonic steroids are known for inhibiting digitalis-sensitive isoforms of Na+,K+ ATPases in the cell membranes of human cells, inducing

differentiation and causing apoptosis and cytolysis in a variety of human tumor cell lines, with EC50's ranging from 380 pM to 10 nM. Using several established tumor cell lines (e.g. Jurkat, U-937, CCRF-CEM, PLC/PRF5), these reports were confirmed, i.e., that bufalin is cytotoxic at EC50 < 10 nM.

The severe toxicity of cardiotonic steroids with serum concentrations above the 5-10 nM range has precluded their administration to humans in tumoricidal doses. However, cardiotonic steroids have been used

therapeutically for centuries and human subjects without cardiac disease can easily tolerate total digoxin body stores of 2.5 μιηοΐββ and serum digoxin concentrations of 2 nM. Unlike other potent low MW compounds (such as calicheamicin and maytansine), cardiotonic steroids have been well

characterized pharmacologically and pharmacokinetically, are readily measured in blood, and have specific Fab antidotes which can also be used to promote the excretion of drug being released from targeted cells. As such, reaction cascades allow sufficient local (effective) tumor concentrations, while keeping concentrations of drug in serum and in normal tissues below the established toxic range.

Also demonstrated is bufalin esters and carbamates at the C3 hydroxyl group can control cytotoxicity.

cleavage

While ordinary esters (e.g. succinamate) are reasonably cytotoxic (-10-20 nM), sterically hindered carbamates and esters were found to be three orders of magnitude less cytotoxic. This result support that bufalin analogs with well- adjusted steric hindrance can be used as prodrugs or in conjugates.

EXAMPLE 22: PREVENTING GRAFT VERSUS HOST DISEASE

This example describes two step cascades for elimination of na^'ive T- cells.

Naive T-cells are CD3 positive, CD45RA positive (CD3⁺CD45RA⁺). Many β-cells are CD45RA⁺. All other T-cells are CD3⁺.

A two step YESCD45RAYESCD3 cascade is depicted in FIG. 24. FIG. 25 Flow cytometry was used to analyze and isolate various cell samples exposed or not to magnetic beads or the YESCD45RAYESCD3 module. Control (no magnetic beads, no module) is shown in FIG. 25A. Control (magnetic beads, no module) is shown in FIG. 25B. Cells exposed to module and magentic beads are shown in FIG. 25C. FIG. 25D shows flow-cytometry of a sample of control cells not exposed to module and not exposed to magnetic beads prior to separation, where 36% are CD19/20 D3^", 59.1 % are T-cells, and 4.9% are B-cells. FIG. 25E shows flow-cytometry of a sample of control cells not exposed to module and exposed to magnetic beads, where 77.5% are CD1 9/20 D3^", 1 8.4% are T- cells, and 4.1 % are B-cells. FIG . 25F shows flow-cytometry of a sample of cells exposed to module and exposed to magnetic beads, where 1 .3% are CD1 9/20^" CD3^", 96.2% are T-cells, and 2.5% are B-cells.

As shown above, the YESCD45RAYESCD3 module can provide for isolation of na^'ive T-cells. Such an approach can be used for depleting T-cells from an allograft so as to prevent attack of recipient tissues (see generally, Anderson et al. 201 3 Biol Blood Marrow Transplant 1 9, 1 85-1 95).

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Claims

Claim 1 . A molecular automaton system for isolation, elimination, or treatment of a target cell comprising a first cell surface marker and a second cell surface marker:

(a) a first target marker comprising

(i) a first antibody specific for the first cell surface marker and

(ii) a first double strand complex comprising a first oligonucleotide and a second oligonucleotide, the second oligonucleotide linked to the first antibody;

(b) a second target marker comprising

(i) a second antibody specific for the second cell surface marker and

(ii) a second double strand complex comprising a third oligonucleotide and a fourth oligonucleotide, the fourth oligonucleotide linked to the second antibody;

(c) a single stranded fifth oligonucleotide;

(d) a single stranded sixth oligonucleotide linked to an isolation agent, a cytotoxic agent, or a therapeutic agent;

wherein,

the first oligonucleotide has more complementarity for the fifth oligonucleotide than for the second oligonucleotide, such that when in proximity, the fifth oligonucleotide will disrupt the first double strand complex to form a single stranded second oligonucleotide and a third double strand complex comprising the first oligonucleotide and the fifth oligonucleotide;

the third oligonucleotide has more complementarity for the second oligonucleotide than for the fourth oligonucleotide, such that when in proximity, the single stranded second oligonucleotide will disrupt the second double strand complex to form a single stranded fourth oligonucleotide and a fourth double strand complex comprising the second oligonucleotide and the third

oligonucleotide, the fourth double strand complex linked to the first antibody via the second oligonucleotide, and the single stranded fourth oligonucleotide linked to the second antibody; and

the sixth oligonucleotide has sufficient complementarity to the single stranded fourth oligonucleotide to form a fifth double strand complex therewith, but has insufficient complementarity for the fourth oligonucleotide to disrupt the second double strand complex.

Claim 2. A molecular automaton system for isolation, elimination, or treatment of a target cell comprising a first cell surface marker but not a second cell surface marker:

(a) a first target marker comprising

(i) a first antibody specific for the first cell surface marker and

(ii) a first double strand complex comprising a first oligonucleotide and a second oligonucleotide, the second oligonucleotide linked to the first antibody;

(b) a second target marker comprising

(i) a second antibody specific for the second cell surface marker and

(ii) a second double strand complex comprising a third oligonucleotide and a fourth oligonucleotide, the fourth oligonucleotide linked to the second antibody;

(c) a single stranded fifth oligonucleotide;

(d) a sixth double strand complex comprising a sixth oligonucleotide and a seventh oligonucleotide, the sixth oligonucleotide linked to an isolation agent, a cytotoxic agent, or a therapeutic agent;

wherein,

the first oligonucleotide has more complementarity for the fifth oligonucleotide than for the second oligonucleotide, such that when in proximity, the fifth oligonucleotide will disrupt the first double strand complex to form a single stranded second oligonucleotide and a third double strand complex comprising the first oligonucleotide and the fifth oligonucleotide; the third oligonucleotide has more complementarity for the second oligonucleotide than for the fourth oligonucleotide, such that when in proximity, the single stranded second oligonucleotide will disrupt the second double strand complex to form a single stranded fourth oligonucleotide and a fourth double strand complex comprising the second oligonucleotide and the third

oligonucleotide, the fourth double strand complex linked to the first antibody via the second oligonucleotide, and the single stranded fourth oligonucleotide linked to the second antibody;

the sixth oligonucleotide has more complementarity for the second oligonucleotide than for the seventh oligonucleotide, such that when in proximity, the single stranded second oligonucleotide will disrupt the sixth double strand complex to form a single stranded seventh oligonucleotide and a seventh double strand complex comprising the second oligonucleotide and the sixth

oligonucleotide, the seventh double strand complex linked to the first antibody via the second oligonucleotide, and the single stranded fourth oligonucleotide linked to the second antibody; and

the third oligonucleotide has more complementarity for the second oligonucleotide than the sixth oligonucleotide has for the second oligonucleotide, such that when in proximity, the sixth oligonucleotide cannot displace the third oligonucleotide from the fourth double strand complex comprising the second oligonucleotide and the third oligonucleotide.

Claim 3. A method for isolating, eliminating, or treating a target cell with the molecular automaton system of claim 1 , comprising:

contacting the first target marker, the second target marker, and a population of cells optionally comprising the target cell, the target cell comprising the first cell surface marker and the second cell surface marker, to form a marked cell; and

contacting the single stranded fifth oligonucleotide and the single stranded sixth oligonucleotide linked to the isolation agent, the cytotoxic agent, or the therapeutic agent with the marked cell.

Claim 4. A method for isolating, eliminating, or treating a target cell with the molecular automaton system of claim 2, comprising:

contacting the first target marker, the second target marker, and a population of cells optionally comprising the target cell, the target cell comprising the first cell surface marker but not second cell surface marker, to form a marked cell; and

contacting the single stranded fifth oligonucleotide and the sixth double strand complex linked to the isolation agent, the cytotoxic agent, or the therapeutic agent with the marked cell.

Claim 5. The system or method of any one of claims 1 -4, wherein the target cell comprises a stem cell, a leukocyte group, a granulocytes, a monocyte, a T lymphocyte, a T helper cell, a T regulatory cell, a cytotoxic T cell, a na^'ive T cell, a lymphocyte, a thrombocyte, or a natural killer cell.

Claim 6. The system or method of any one of claims 1 -5, wherein the target cell comprises an NK cell, a T-cell, or a B-cell.

Claim 7. The system or method of any one of claims 1 -6, wherein the target cell is a stem cell and the first cell surface marker or the second cell surface marker is selected from the group consisting of CD34+, CD31-, and

CD1 17;

the target cell is a leukocyte group and the first cell surface marker or the second cell surface marker is CD45+;

the target cell is a granulocyte and the first cell surface marker or the second cell surface marker is selected from the group consisting of CD45+, CD1 1 b, CD15+, CD24+, CD1 14+, and CD182+;

the target cell is a monocyte and the first cell surface marker or the second cell surface marker is selected from the group consisting of CD45+, CD14+, CD1 14+, CD1 1 a, CD1 1 b, CD91 +, CD16+; the target cell is a T lymphocyte and the first cell surface marker or the second cell surface marker is selected from the group consisting of CD45+ and CD3+;

the target cell is a T helper cell and the first cell surface marker or the second cell surface marker is selected from the group consisting of CD45+, CD3+, and CD4+;

the target cell is a T regulatory cell and the first cell surface marker or the second cell surface marker is selected from the group consisting of CD4, CD25, and Foxp3;

the target cell is a Cytotoxic T cell and the first cell surface marker or the second cell surface marker is selected from the group consisting of CD45+, CD3+, and CD8+;

the target cell is a na^'ive T-cell and the first cell surface marker or the second cell surface marker is selected from the group consisting of CD45RA+ and CD3+;

the target cell is a B lymphocyte and the first cell surface marker or the second cell surface marker is selected from the group consisting of CD45+, CD19+ or CD45+, CD20+, CD24+, CD38,and CD22;

the target cell is a Thrombocyte and the first cell surface marker or the second cell surface marker is selected from the group consisting of CD45+ and CD61 +; or

the target cell is a Natural killer cell and the first cell surface marker or the second cell surface marker is selected from the group consisting of CD16+, CD56+, CD3-, CD31 , CD30, and CD38.

Claim 8. The system or method of any one of claims 1 -7, wherein the first oligonucleotide, the second oligonucleotide, the third oligonucleotide, the fourth oligonucleotide, the fifth oligonucleotide, the sixth oligonucleotide, or the seventh oligonucleotide comprise about 10 to about 100 nucleotides.

Claim 9. The system or method of any one of claims 1 -8, wherein the first oligonucleotide, the second oligonucleotide, the third oligonucleotide, the fourth oligonucleotide, the fifth oligonucleotide, the sixth oligonucleotide, or the seventh oligonucleotide comprise about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 nucleotides.

Claim 10. The system or method of any one of claims 1 -9, wherein more complementarity comprises about 1 %, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 1 1 %, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21 %, about 22%, about 23%, about 24%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60%

Claim 1 1 . The system or method of any one of claims 1 -10, wherein a double strand complex comprises a pair of oligonucleotides having a difference in nucleotide number selected from the group consisting of about 1 , about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 1 1 , about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21 , about 22, about 23, about 24, and about 25 nucleotides; and

the difference in nucleotide number creates a toe hold sufficient to drive a strand-displacement reaction.

Claim 12. The system or method of any one of claims 1 -1 1 , wherein the target cell is isolated according to flow cytometry, fluorescence-activated cell sorting (FACS), or magnetic-activated cell sorting (MACS).