WO2010138960A2 - Methods and systems for single-molecule rna expression profiling - Google Patents

Abstract

In general, the invention features methods and systems for expression profiling of RNA from a sample, e.g., a single cell, based on the simultaneous measurement of continuous incorporation of fluorogenic nucleotides into complementary nucleic acids. Methods for profiling RNA (e.g., of prokaryotic, eukaryotic, or viral origin) expression in a sample may include disposing, in each of a plurality of optionally sealed microreactors, a mixture in solution phase comprising a single copy of a target RNA (or DNA copy thereof) from a sample (e.g., a single cell), a nucleic acid replicating catalyst, and a mixture of nucleotides having a first nucleotide with a first label that is substantially non-fluorescent until after incorporation into a nucleic acid based on complementarity to the target; allowing continuous template-dependent replication of the targets in the microreactors; sequencing the targets in the microreactors by detecting in real time the individual incorporation of the first nucleotide during template-dependent replication by monitoring fluorescence emission resulting from the first label; and determining from the sequencing the level of expression of RNA in the sample.

Description

METHODS AND SYSTEMS FOR SINGLE-MOLECULE RNA EXPRESSION PROFILING

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No. 61/182,208, filed May 29, 2009, which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION The invention relates to the fields of RNA expression profiling and single- molecule detection.

Molecular biology, genetics, and genomics have advanced through the emergence of three major technologies: DNA sequencing, PCR, and DNA microarrays. In recent years, Sanger sequencing has been replaced by a new generation of DNA sequencing techniques, some with single-molecule sensitivities. This development has recently led to RNAseq, which has made gene expression profiling by DNA microarrays essentially obsolete because direct sequencing of cDNA offers much better dynamic range and sensitivity. For DNA sequencing and gene expression profiling, PCR has been used to date for DNA amplification before detection. However, PCR is prone to amplification bias in systems-wide analyses, i.e., some sequences are amplified more than others when random primers are used. In addition, PCR distorts the overall mRNA distribution and cannot generally be applied to individual cells. Single cell and single molecule gene expression profiling is needed to allow for measurement of differences in anatomical and temporal expression. Current gene expression techniques only determine the mean expression levels, while analysis of many single cells can reveal the mRNA distribution. Gene expression profiling of many individual cells with current techniques is also cost prohibitive. Moreover, liver and skin cells from the same individual have identical genes but very different mRNAs because of distinctly different gene expression. Additionally, it is well recognized that gene expression of individual cells is stochastic, i.e., cells are difficult to synchronize and hence exhibit different phenotypes even under identical conditions. Gene expression of an individual cell also exhibits temporal fluctuations due to the small number of macromolecules involved, and these stochastic events can cause phenotypic changes in time. Many mRNAs exist in low copies in a single cell. Current expression profiling technology relies on PCR, which introduces significant noise when amplifying, and which is incapable of accurately quantifying copy numbers below -100. To count rare transcripts, digital PCR can be employed, but it is very difficult and expensive to implement at the single cell, full-transcriptome level.

One single-molecule sequencing system employs discontinuous sequencing-by-synthesis method. This approach involves the sequential detection of base-labeled nucleotides incorporated by DNA polymerase. Because the substrate and polymerase must be flowed in and then washed out for each incorporation step, this method consumes large amounts of reagents, increasing overall cost.

Another method eliminates a number of these difficulties by conducting real-time, asynchronous sequencing using terminal-phosphate labeled nucleotides. This method employs a zero-mode waveguide (ZMW), a ~50-nm- diameter metallic structure which allows the investigation of enzyme dynamics at high substrate concentration by reducing the fluorescence probe volume to the zeptoliter scale. By immobilizing a single DNA polymerase molecule in the bottom of a ZMW, only fluorescently tagged molecules that enter this waveguide and bind to the polymerase are detected, eliminating the overwhelming background fluorescence from substrates in solution. Because the nucleotides are labeled on the phosphate moiety, the fluorescent label is naturally cleaved away upon incorporation of the base, leaving native DNA behind. Because this technique is sensitive to nucleotide binding rather than incorporation, the accuracy of the technique is limited, requiring repeated sequencing of the same DNA. Finally, the production of ZMWs requires the precision machining of nanometer-scale structures in metals, increasing the cost and complexity of the sequencing instrument.

Accordingly, there is a need for new methods for RNA expression profiling, especially for single cells.

SUMMARY OF THE INVENTION

In general, the invention features methods and systems for expression profiling of RNA from a sample, e.g., a single cell, based on the simultaneous measurement of continuous incorporation of fluorogenic nucleotides into complementary nucleic acids.

In one aspect, the invention features a method for profiling RNA (e.g., of prokaryotic, eukaryotic, or viral origin) expression in a sample by disposing, in each of a plurality of optionally sealed microreactors, a mixture in solution phase comprising a single copy of a target RNA (or DNA copy thereof) from a sample (e.g., a single cell), a nucleic acid replicating catalyst, and a mixture of nucleotides having a first nucleotide with a first label that is substantially non- fluorescent until after incorporation into a nucleic acid based on complementarity to the target; allowing continuous template-dependent replication of the targets in the microreactors; sequencing the targets in the microreactors by detecting in real time the individual incorporation of the first nucleotide during template-dependent replication by monitoring fluorescence emission resulting from the first label; and determining from the sequencing the level of expression of RNA in the sample. The method does not require any nucleic acid amplification. Alternatively, the method further includes the step of producing a DNA copy from the target RNA, and the DNA copy is then sequenced as a surrogate for the RNA.

The method may also include the steps of lysing the sample, e.g., a single cell or fewer than 10 cells, prior to delivery to the microreactors and optionally separating RNA from other components of the lysate prior to sequencing. The method may also include the step of isolating the sample, e.g., a single cell or fewer than 10 cells, from a larger mass of cells, e.g., a tissue. An exemplary method for isolation is laser microdissection.

When RNA is sequenced, a preferred nucleic acid replicating catalyst is a reverse transcriptase. Other suitable catalysts are described herein. The method may also include identifying the RNA sequence from a database, e.g., by matching a partial sequence obtained from the method with a known full sequence in the database. In certain embodiments, the sequencing only identifies between 10 and 50 nucleotides of at least one target in the microreactors. In other embodiments, the method sequences sufficient nucleotides to determine any transcriptional variation of at least one target in the microreactors.

In certain embodiments, the mixture in solution phase further includes an activating enzyme that renders the first label fluorescent, typically after incorporation by template-dependent replication. The first label may also be photobleached, or otherwise destroyed, after detection. The first label may be a phosphate label that is cleaved from the first nucleotide during replication. In further embodiments, the mixture of nucleotides further includes second, third, and fourth nucleotides, including a second, third, or fourth label that is substantially non-fluorescent until incorporation of the nucleotide into the nucleic acid based on complementarity to the target nucleic acid. The steps of template-based replication and detection may be repeated, e.g., to provide greater accuracy in the sequence determination. In other embodiments, the target is immobilized on a surface of the microreactor or a bead disposed in the microreactor. Methods of the invention may also include determining the morphological state of the sample or the point in the cell cycle when profiling occurs.

In a related aspect, the invention features a system capable of performing the methods described herein. In one example, the system includes a plurality of microreactors, e.g., 100,000 and 1,000,000, that are each capable of holding a mixture in solution phase of a single copy of a target RNA or a DNA copy thereof, a nucleic acid replicating catalyst, and a mixture of nucleotides, at least one of which comprises a label that is substantially non- fluorescent until after incorporation of at least one nucleotide into a nucleic acid based on complementarity to the target nucleic acid; a fluorescent microscope for imaging the microreactors to sequence targets in the microreactors by detecting in real time in each microreactor the incorporation of an individual, labeled nucleotide during template-dependent replication of the single copy of the target by monitoring fluorescence from the label resulting from incorporation of the nucleotide; a fluidic delivery system connected to the microreactors and including an inlet for a sample (e.g., a single cell or fewer than 10 cells), a reservoir comprising a lysing reagent, a reservoir comprising a reagent for purifying RNA from a cellular lysate, a chamber for contacting the sample with the lysing reagent and/or the reagent for purifying RNA, and a pump for delivering single nucleic acid molecules to the microreactors.

The system may further include a reservoir of reagents for producing a DNA copy of the RNA; a laser microdissector capable of isolating a single cell or fewer than 10 cells from a sample, wherein the dissector is coupled to the inlet in the fluidic delivery system; a light source capable of photobleaching the label after detection; and/or a temperature controller for regulating the temperature of the microreactors. In other embodiments, the excitation source of the fluorescent microscope is capable of photobleaching the label.

The microreactors may include poly(dimethyl siloxane) (PDMS) and/or a control layer, pressurization of which conformally seals the microreactors against a flat surface, wherein the system further includes a pressure source.

Components that are in contact with sample and/or reagents may be readily separable from the rest of the system to allow for disposal, cleaning, or sterilization.

It will also be understood that any fluidic connections necessary to carry out the methods described herein will be present in the system, e.g., contacting various reagents with cells, lysate, or nucleic acid. Delivery of single target molecules to microreactors may precede any step for purification or production of a DNA copy. Appropriate lysing and purifying reagents are known in the art.

By a "microreactor" is meant a vessel having a volume such that a light microscope can detect a freely diffusing fluorophore using a sensitive photon detector, e.g., capable of detecting a single molecule.

By "fluorogenic" or "substantially non-fluorcsccnt" is meant not emitting a significant amount of fluorescence at a given wavelength until after a chemical reaction has occurred. By "sequencing" a nucleic acid is meant identification of one or more nucleotides in, or complementary to, a target nucleic acid. Sequencing may include determination of the individual bases in sequence, determination of the presence of an oligonucleotide sequence, or determination of the class of nucleotide present, e.g., member of A-T, A-U, or G-C pair, or purine base or pyrimidine base.

By sequencing that occurs "continuously" is meant a sequencing by synthesis that results in the generation of a single complementary nucleic acid, e.g., of 10, 25, 100, 300, 1000, or 10,000 base pairs. The phrase does not imply that the sequencing occurs at a constant rate. In addition, replication may occur as a result of catalysis by different copies of a catalyst, i.e., a single enzyme molecule need not catalyze synthesis of the entire complementary nucleic acid. By "detecting in real time" is meant detecting light emitted from a label after incorporation of a labeled nucleotide into a nucleic acid but prior to incorporation of a subsequent labeled nucleotide. By "incorporation" of a nucleotide into a nucleic acid is meant the formation of a chemical bond, e.g., a phosphodiester bond, between the nucleotide and another nucleotide in the nucleic acid. For example, a nucleotide may be incorporated into a replicating strand of DNA via formation of a phosphodiester bond. Other types of bonds may be formed if non- naturally occurring nucleotides are employed. By "nucleotide" is meant a natural or synthetic ribonucleosidyl, 2'- deoxyribonucleosidyl radical, 2'-O-methyl ribonucleosidyl, Locked Nucleic Acid, peptide nucleic acid, glycerol nucleic acid, morpholino nucleic acid, or threose nucleic acid connected, e.g., via the 5', 3' or 2' carbon of the radical, to a phosphate group and a base. The nucleotide may include a purine or pyrimidine base, e.g., cytosine, guanine, adenine, thymine, uracil, xanthine, hypoxanthine, inosine, orotate, thioinosine, thiouracil, pseudouracil, 5,6- dihydrouracil, and 5-bromouracil. The purine or pyrimidine may be substituted as is known in the art, e.g., with halogen (i.e., fluoro, bromo, chloro, or iodo), alky] (e.g., methyl, ethyl, or propyl), acyl (e.g., acetyl), or amine or hydroxyl protecting groups. In certain embodiments when DNA is being sequenced, the nucleotides employed are dATP, dCTP, dGTP, and dTTP. In other embodiments when RNA is being sequenced, the nucleotides employed are ATP, CTP, GTP, and UTP. A target DNA sequence can also be sequenced with riboside bases using RNA polymerase, and a target RNA sequence can also be sequenced with deoxyriboside bases using reverse transcriptase. The term includes moieties having a single base, e.g., ATP, and moieties having multiple bases, e.g., oligonucleotides.

By "nucleotide replicating catalyst" is meant any catalyst, e.g., an enzyme, that is capable of producing a nucleic acid that is complementary to a target nucleic acid. Examples include DNA polymerases, RNA polymerases, reverse transcriptases, ligases, and RNA-dependent RNA polymerases. Other features and advantages of the invention will be apparent from the following drawings, detailed description, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1 A-IB: A) Excitation and emission spectra for DDAO and resorufin. The excitation wavelength is indicated with a vertical line. B) Microscope diagram for a two-color single-molecule fluorescence microscope with single color laser illumination and EM-CCD camera. A wide field epifluorescence or total internal reflection fluorescence (TIRF) excitation is used to achieve multiplexed detection of polymerase activity on many nucleic acid molecules with two different color channels. The fluorescence signal is collected in the epi-direction by the same high numerical aperture objective (Nikon 60^χ, 1.45 NA) that delivers the laser light to the sample. Custom-built detection optics placed past the tube lens of the microscope (Nikon TE-2000 Eclipse) split the collected fluorescence into two spectral channels and image the two colors onto two halves of a frame transfer, electron-multiplied charge- coupled device (EM-CCD) camera (Cascade 512B, Roper Scientific) that is capable of video-rate imaging of hundreds of polymerase reactions at the single turnover level because of its frame-rate, high quantum efficiency, and on-chip multiplication gain. Given the excitation and emission wavelengths of DDAO and resorufin (Figure IA), these two dyes can be excited by a dye laser at 560 nm and provide spectrally distinct emissions, which can be easily separated onto the two halves of the camera. Figures 2A-2B: A) The work flow of mRNA processing from a single cell for sequencing. B) A PDMS microfluidic system for processing mRNA from a single cell. The pressure controlled valves allow fluid handling and delivery of the mRNA fragments to the microreactors for sequencing.

Figures 3A-3B: A) SEM image of PDMS microreactors cast from a silicon master, which was generated with standard photolithography. B) Valve-based sealing of microreactors. A control layer allows for reversible sealing of the microreactors upon application of pressure.

Figure 4: Bulk measurements for fluorogenic reverse transcription of a primed, poly-rU homopolymer RNA molecule by Superscript II reverse transcriptase (MMLV-RT RNaseH- mutant).

Figures 5A-5B: Microscope experiment showing reverse transcriptase activity on RNA templates with fluorogenic nucleotides. A) Streptavidin- coated polystyrene beads are mixed with an excess amount of RNA molecules (UC heteropolymer) that are annealed with biotinylated DNA primers and trapped in PDMS microreactors. B) Microreactors were supplied with dGTP- resorufin, dATP-DDΛO, shrimp alkaline phosphatase, and Superscript® III. Top: Bright field image of 5-μm-diameter microreactors shown in circles. Some reactors were occupied with one or two beads (black dots), and the others were empty. Bottom: Fluorescence image of microreactors (resorufm channel). After initial background fluorescence was bleached, resorufin signal built-up after 5 minutes. Only the reactors that had beads showed fluorescence.

DETAILED DESCRIPTION OF THE INVENTION

We provide a method for multiplex sequencing of individual RNA molecules, e.g., using a reserve transcriptase, that employs fluorogenic nucleotide substrates. The invention also features the integration of a single- molecule fluorogenic sequencer with microfluidic devices that process, isolate, and deliver RNA from samples, e.g., a single lysed cell. Preferably, the sequencing occurs directly during the synthesis of cDNA. The sequencing of RNA from a sample, e.g., a single cell, is useful for expression profiling, and the invention allows for the simultaneous determination of substantially all RNA molecules present in the sample or substantially all of a type of RNA molecule, e.g., mRNA, present in the sample. Expression profiling may be used to count the number of sequences present in a sample and/or to determine transcriptional variations of RNAs. Since the genome of many cells is already known, sequence counting may rely on the sequencing of a few bases, e.g., 10, 15, 25, or 30, to identify the RNA molecule. Longer sequence read-lengths may also be employed in sequence counting or to determine transcriptional variation in closely related RNAs. Any type of RNA may be profiled using the methods of the invention, including mRNA, tRNA, rRNA, and other non- coding RNAs, such as siRNA, miRNA, pri-miRNA, pre-miRNA, piRNA, snRNA, and snoRNA. The invention can be employed in conjunction with eukaryotic or prokaryotic cells or viruses.

Many single cells can be analyzed using the invention, revealing both the mean expression levels and distribution within the population. Such technology allows digital quantification of the gene expression uncontaminated by amplification noise and bias introduced by PCR. Such single cell/single molecule RNAseq would not only lead to new biological discoveries, but powerful medical diagnostics as well. For example, the invention allows for analysis of transcriptomes within a heterogeneous cell population; identification of allele-specific and strand-specific gene expression in a particular cell detecting point mutations in transcripts; identification of alternative splicing events and different isoforms of transcripts in eukaryotic cells; discovery of new classes of RNA with low abundance; and analysis of differential expression among cells from a gross tissue sample.

Advantages of the invention include: 1 ) No PCR amplification bias and noise is introduced.

2) A read length of ~25 bases with moderate accuracy is sufficient to identify an mRNA in a known genome and small, non-coding RNAs, such as siRNA, miRNA, pri-miRNA, pre-miRNA, piRNA, snRNA, and snoRNA.

3) Longer read-lengths offer redundancy in identifying a given mRNA and the prospect of detecting allele-specific gene expression and isoforms associated with splice variants. Direct sequencing of mRNA also provides the genomic sequence of the coding regions of DNA.

4) True digital read out with single copy sensitivity, allowing the detection of a single copy of a particular RNA from a single cell, opening the door for discovering new RNAs with low abundance.

5) High dynamic range of expression level. Simply counting the number of times that an RNA sequence occurs gives the expression level of a particular gene.

6) The total volume used for each reaction is less than 1 fL, and no flow is needed during sequencing, allowing for low reagent costs.

7) Sample preparation is significantly simplified compared to any existing techniques, further cutting the cost and allowing fast turnaround time.

8) Parallel detection of numerous RNA targets, e.g., 100,000 reaction chambers can be simultaneously observed with a standard fluorescence microscope. The invention does not require high processivity, as an excess of replicating catalyst may be employed; does not require flow of reagents; can be used with background free detection, by photobleaching (or otherwise eliminating) a label after detection; and is not subject to false binding error, as the invention only detects incorporation of nucleotides and not mere hybridization to a template strand.

Expression Profiling

Expression profiling is accomplished by sequencing individual RNA molecules (or DNA copies thereof), which may then be counted, identified, or otherwise analyzed. Identification of RNA, e.g., mRNA, may occur by determining the nucleic acid sequence of the entire molecule or a unique portion thereof. For example, for known genomes, sequencing of a few bases is sufficient for identification of the RNA. Such partial sequences may be compared with various RNA databases, e.g., mRNA databases, for identification according to the invention.

The invention employs sequencing by synthesis and detects the incorporation of an individual nucleotide, e.g., including a single base or multiple bases, into a nucleic acid during replication. The nucleotides that are incorporated are labeled, and, as a result of incorporation, the label is rendered able to emit light, e.g., by cleavage from the incorporated nucleotide (e.g., when bound to the terminal phosphate of a nucleotide). The sequential incorporation of individual nucleotides is then measured in real time by detecting the emitted light. Tens of thousands of bases on a single nucleic acid can be read continuously with high speeds up to 10-100 bp/sec, although a few bases are sufficient in certain embodiments.

A portion of the nucleotide, e.g., pyrophosphate, is typically cleaved as a result of incorporation, and the label is typically bound to the cleaved portion, i.e., does not form part of the nucleic acid after incorporation. The label may or may not be immediately fluorescent upon cleavage from the nucleotide, as discussed below. Various chemical mechanisms that may be involved in rendering a label fluorescent include acid and base catalyzed reactions and other catalytic processes described herein. In another example, a label is quenched or otherwise rendered non-emitting by proximity to the nitrogenous base of a nucleotide or a moiety associated with the base. For high fidelity, rapid nucleic acid sequencing, the generation of a fluorophore is typically closely coupled in time with the incorporation of a fluorogenic label into a nucleic acid, by a nucleic acid replicating catalyst. In a preferred embodiment, nucleotides are incorporated by the nucleic acid replicating catalyst at a rate of approximately 1 per second, which allows rapid generation of the fluorophore, optical excitation and detection of the fluorophore, and subsequent bleaching (see, e.g., US 7,125,671 and US 7,041,812).

When each nucleotide is added to the synthesized strand, the nucleotide added is preferably identified. One method of determining the identity of a particular nucleotide is to attach a different, distinguishable label to each nucleotide being added, typically A, T, C, and G, or A, U, C, and G. By detecting which of the labels is added at a given point in synthesis, the corresponding nucleotide added can be identified, and, when present, the sequence of a target nucleic acid can be determined, by virtue of its complementary nature. Methods for detecting four or more optically distinguishable labels are well known in the art.

Alternatively, fewer labels may be employed. Two labels may be employed when a target double stranded nucleic acid or a single stranded nucleic acid and its complement are sequenced. In this example, one of A and T (or U) is labeled, and one of C and G is labeled. Another example of two- label detection is to label one nucleotide with a first label and the other three nucleotides with another label.

Binary sequencing may also be employed in which two bases, such as A and T (or U), are labeled with one label, while the other two bases, such as G and C, are labeled with a second label. A subsequent sequencing in which the label in one of each pair is changed, e.g., A and G are labeled with one label, and T (or U) and C are labeled with the other, may be employed to obtain base specificity.

One label may be employed, where the other three nucleotides are not labeled but are kept at lower concentrations, where the time, and therefore position, between the detection of each label is determined. Subsequent experiments using the same label with a different nucleotide can be used to provide the remaining sequence information.

When more than one labeled nucleotide is employed, each label may become light emitting as a result of different mechanisms. For example, one label may require additional chemical reaction after cleavage from the synthesized nucleic acid, while a second label may become light emitting upon cleavage without additional reaction.

Sequencing may also be performed using ligase, in which oligonucleotides hybridized adjacent to one another on a template strand are ligated together. Each oligonucleotide employed may be uniquely labeled. Oligonucleotides having the sequence complementary to a region of repeated sequence may be added sequentially using the methods of the invention, and the number of repeats determined by the number of oligonucleotides ligated. Many proteins and enzymes require metallic co-factors such as divalent metal cations (Mg²⁺, Mn²⁺, Zn²⁺, etc.). For example, magnesium ions may be required for nucleic acid polymerase and alkaline phosphatase activity; manganese ions may be required to enhance the ability of the nucleic acid polymerase to incorporate modified nucleotide substrates (as described in US 7,125,671 and Tabor S., Richardson CC, Proc. Natl. Acad. ScL USA, 1989, 86, 4076-4080); and zinc ions may be required for alkaline phosphatase activity. The presence of metal ions at high concentrations can complicate protein-protein interactions, protein-nucleic acid interactions, and surface passivation. In addition, divalent cations can destabilize polyphosphate compounds. Buffer components such as ammonium sulfate and chelating agents can be used to tune intermolecular interactions and control the effective concentration of metal ions. Many nucleic acid polymerizing replicating catalysts also require a reducing environment to perform optimally. There are many classes of reducing agents such as thiols (such as 2-mercaptoethanol or dithiothreitol) and phosphines (such as tris(2-carboxyethyl)phospinc (TCEP)), which are compatible with physiological buffers. An individual sequencing reaction may be controlled by controlling the introduction of Mg or Mn ions, nucleotides, and other co-factors necessary to effect replication. Other methods for controlling replication include changing the temperature or introducing or removing substances that promote or discourage complex formation between the target and catalyst. The catalyst or target may also be rendered inoperative to end sequencing, e.g., through denaturation or cleavage.

Expression profiling may also include determining the morphological state or point in the cell cycle for a sample, e.g., a single cell, being profiled. Such determination can be made using visual or chemical analysis of the sample as is known in the art.

Nucleic Acids and Nucleotides

Expression profiling according to the invention typically employs RNA, e.g., mRNA, isolated from a sample, e.g., a single cell. Although unnecessary for the present invention, RNA in a sample may be converted to DNA (as either a sense or antisense copy), which is then used as an RNA surrogate in expression profiling. The RNA may be from any source, e.g., of prokaryotic, eukaryotic, or viral origin and from a single or multicellular organism. Any type of RNA may be profiled according to the invention. For example, the invention may be used to profile the expression of mRNA and/or other RNAs described herein in bacteria, e.g., E. coli and Mycobacterium spp., or human cells, e.g., cancer cells. The invention may be employed in conjunction with a single cell or a few cells (e.g., 5 or 10 cells in a population). Single cells (or small numbers of cells) can be isolated from a sample using well known techniques, ranging from dilution to laser microdissection. The invention allows for the expression profiling of substantially the entire transcriptome of a cell, e.g., at least 70%, 80%, 90%, 95%, or even 99% of RNA, e.g., mRNA, expressed in a given cell. The invention further allows for the simultaneous sequencing of the individual RNA molecules in a cell.

Nucleotides used in sequencing may be naturally occurring or synthetic, e.g., synthetic ribonucleosidyl, 2'-deoxyribonucleosidyl, Locked Nucleic Acid, peptide nucleic acid, glycerol nucleic acid, morpholino nucleic acid, or lhreose nucleic acid connected, e.g., via the 5', 3', or 2' carbon of the radical, to a phosphate group and a base. The nucleotide may include a purine or pyrimidine base, e.g., cytosine, guanine, adenine, thymine, uracil, xanthine, hypoxanthine, inosine, orυtate, thioinosine, thiouracil, pseudouracil, 5,6- dihydrouracil, and 5-bromouracil. The purine or pyrimidine may be substituted as is known in the art, e.g., with halogen (i.e., fluoro, bromo, chloro, or iodo), alkyl (e.g., methyl, ethyl, or propyl), acyl (e.g., acetyl), or amine or hydroxyl protecting groups. In certain embodiments, the nucleotides employed are dATP, dCTP, dGTP, and dTTP, e.g., when reverse transcriptase is employed with mRNA templates. In other embodiments, the nucleotides employed are ATP, CTP, GTP, and UTP. Ribosides may be employed for sequencing DNA, e.g., when DNA-dependent RNA polymerase is employed. Ribosides may be employed for sequencing RNA, e.g., when RNA-dependent RNA polymerase is employed.

Preferably, the methods of the invention produce a nucleic acid that is complementary to the target RNA, e.g., mRNA, (or DNA copy thereof) and that includes only naturally occurring nucleotides, i.e., the label is removed during incorporation. Alternatively, nucleotides may include a moiety that is retained in the synthesized nucleic acid. Such moieties are preferably present on fewer than all of the labeled nucleotides employed, e.g., only one, two, or three, to minimize disruption of replicating catalyst activity.

Stability of RNA secondary structure may impact the read length and nucleotide incorporation speed of a nucleic acid replicating catalyst. Increased reaction temperatures, other denaturing conditions, and catalysts with improved ability to transit stable secondary structures may be used to allow sequencing in the presence of any RNA prone to secondary structure formation.

Nucleic Acid Replicating Catalysts Typically, the invention employs direct expression profiling from RNA and therefore employs a nucleic acid replicating catalyst for use with an RNA template, e.g., a reverse transcriptase. Other nucleic acid replicating catalysts may also be employed, e.g., RNA polymerases, ligases, and RNA-dependent RNA polymerases. Exemplary reverse transcriptases include AMV reverse transcriptase, MMLV reverse transcriptase, Superscript® 1, Superscript® II, Superscript® III, and HIV- 1 reverse transcriptase. Exemplary RNA polymerases include T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, and E. coli RNA polymerases. Exemplary ligases are known in the art. Exemplary RNA-dependent RNA polymerases are known in the art. IfRNA in the sample is converted to a DNA copy prior to expression profiling, a replicating catalyst for use with a DNA template will be employed. Nucleic acid replicating catalysts for use with a DNA template include DNA polymerases and ligases. Exemplary DNA polymerases include E. coli DNA polymerase I, E. coli DNA polymerase I Large Fragment (Klenow fragment), Klenow fragment (exo-), Sequenase™, phage T7 DNA polymerase, T4 DNA polymerase, Phi-29 DNA polymerase, Phi-29 (exo-) DNA polymerase, and thermophilic polymerases (e.g., Thermus aquaticus (Taq) DNA polymerase, Thermusflavus (TfI) DNA polymerase, Thermus Thermophilus (Tth) DNA polymerase, Thermococcus litoralis (TIi) DNA polymerase, Pyrococcus furiosus (Pfu) DNA polymerase. Vent™ DNA polymerase, or Bacillus stear other mophilus (Bst) DNA polymerase, Therminator™, Therminator II™). Other suitable DNA polymerases are known in the art.

Catalysts may bind to a target at any appropriate site as is known in the art. Multiple copies of the replicating catalyst may be present. If a particular catalyst molecule disassociates from the template strand, another catalyst molecule may bind and continue replication without affecting the sequencing function.

Detection The invention employs a multiplexed mode, where the sequences of multiple RNAs, e.g., mRNAs, are determined simultaneously, e.g., using a wide field of view detector such as a charge-coupled device (CCD) or multiple detectors. Incorporation of an individual nucleotide during the sequencing reaction is detected by detecting the light emitted from its corresponding label by any appropriate method. For fluorescent labels, one or more excitation sources may be employed, depending on the nature and number of labels. Methods for single molecule detection are known in the art. Examples are conventional fluorescence microscopy, total internal reflection fluorescence microscopy, or parallel confocal microscopy (Lundquist et al. Optics Letters. 2008 33(9) 1026-1028). An exemplary system for detection of fluorescence is shown in Figure IB.

Integrated Sample Preparation

The invention may employ integrated fluid delivery systems for sample preparation (see, e.g., U.S. Patent No. 6,352,838 or Toriello et al., Proc. Natl. Acad. Sci.,2008 105( 51), 20173-20178). In certain embodiments, RNA is purified from crude biomaterials (such as blood, tissue, etc.). Fluid delivery systems useful with the invention include those with elements for introducing a sample, e.g., a single cell; contacting the sample with a lysing reagent to release the intracellular contents; contacting the lysed sample with purifying reagents; and delivering purified RNA to the individual microreactors. The invention may also integrate elements for the separation of single cells from larger samples, e.g., using laser microdissection. An exemplary fluidic system is shown in Figure 2B. Purification of eukaryotic mRNA may be based on capture of poly- A tails of mRNA, e.g., using poly-T oligonucleotides on the surface of the device or a bead. Other components of the lysate can then be washed away from the captured mRNA. The purified mRNA is then delivered to microreactors under conditions biased towards delivery of a single molecule to a given microeactor. Delivery of mRNA may occur while the mRNA is bound to a bead, or the mRNA may first be released from the capture surface prior to delivery. mRNA in crude lysate may also be captured in microreactors prior to purification. For prokaryotes, rRNA can be removed with capture beads, and then microfluidic capillary electrophoresis can be performed on the remaining RNA to separate mRNA from tRNA, protein, and DNA. Polycystronic mRNA may be sheared (e.g., using a divalent cation), and poly(A) polymerase I may be used to add poly- A tails onto the 3' ends of the mRNA. Polyadenylated prokaryotic mRNA is then delivered to microreactors for detection. PoIy-A tails also provides a uniform sequence for any primer necessary for sequencing. Figure 2A details methods for purification of mRNA and an integrated device for purification. Methods for purifying other types of RNA are known in the art.

Microreactors

The invention employs microreactors to isolate single RNA (or DNA) molecules and the reagents used for sequencing in a confined volume, e.g., 0.0001 fL to 1000 fL, although larger volumes are possible. Confinement of the fluorogenic labels by the microreactor allows for localized signal detection of nucleotide incorporation. In addition, small, sealed reaction chambers reduce the autohydrolysis rate for fluorogenic substrates and eliminate crosstalk between sequencing reactions. RNA, activating catalyst, or replicating catalyst may be immobilized within the microreactor, although the methods of the invention do not require immobilization. Methods for immobilizing nucleic acids or catalysts are well known in the art and include biotin-streptavidin, antibody-antigen interactions, covalent attachment, or attachment to complementary nucleic acid sequences. In certain embodiments, it is preferred for the RNA to be immobilized, e.g., on a bead or to the microreactor, to allow for repeated sequencing.

1 ! Materials that are useful in forming the microreactors include glass, glass with surface modifications, silicon, metals, semiconductors, high refractive index dielectrics, crystals, gels, lipids, and polymers (e.g., poly(dimethyl siloxane). Mixtures of materials may also be employed. Microreactor arrays are preferably created using polydimethylsiloxane

(PDMS) soft lithography (Figure 3A). The PDMS array can be sealed against a flat, PDMS layer which was previously spun-coated onto a glass coverslip, or sealed against a treated glass surface. Use 0.60 micron diameter reactors with 0.2 micron diameter edge-to-edge spacing provides more than 300,000 reaction chambers in a 500 x 500 micron field of view, of which -100,000 will have a single nucleic acid molecule that generates a usable read (due to Poisson statistics). This field-of-view can be accommodated by standard high-NA oil immersion objectives. Electron beam lithography can be used to create a mold of a microreactor array, from which PDMS replicas can be produced. Other materials for microreactor fabrication include polytetrafluoroethylene, perfluoropolyethers, and parylene. Additionally, lipid vesicles can be generated using standard lipid extrusion techniques (Okumus et al. Biophys. J. 2004, 87(4), 2798-2806) and used to confine the reaction. Another method of generating microreactors is the creation of an emulsion of the reaction mixture in an immiscible solvent such as mineral oil or silicon oil. These and other methods for manufacturing microreactors are known in the art, e.g., U.S. Patent Nos. 7,081,269, 6,225,109, 6,225,109, and 6,585,939.

Single molecules of RNA (or DNA) can be delivered to a microreactor using methods known in the art. One method for delivery is to provide a dilute solution of nucleic acid so that each microreactor, on average, holds less than one molecule. Using this approach some microreactors will have no target nucleic acid, some will have a single target nucleic acid, and a very small number will have more than one. The same strategy may be employed to attach a single molecule of nucleic acid to a bead or to the lid of a microreactor. Fluorophores and fluorogenic labels are preferably trapped in the microreactor during the course of a sequencing. If either the generated fluorophore or the fluorogenic-label escapes the reactor, then information regarding the sequencing of the nucleic acid may be lost. Microreactors are preferably manufactured from materials that prevent or reduce diffusion of fluorophores, evaporation of water, and nonspecific absorption of proteins. Alternatively, microreactors are treated to prevent or reduce such diffusion, evaporation, and nonspecific absorption. For example, PDMS can be treated with fluorinated fluid (e.g., Fluorinert FC-43 and FC-770, 3M) to seal the small holes in PDMS. In addition, in order to prevent the nonspecific binding of proteins on the surfaces of microreactors, a self-assembled polyethylene glycol brush can be generated on the surface. For example, PDMS is treated with an amorphous fluoropolymer, e.g., CYTOP (perfluoro( 1 -butenyl vinyl ether) homocyclopolymer) from Asahi Glass Co., by spincoating and baking at 75 ⁰C for 15 minutes and 145 °C for 15 min. Then the CYTOP is coated with a poly(ethylene oxide)-poly (propylene oxide) block copolymer, e.g., Pluronic F- 108, which spontaneously forms a polyethylene glycol brush on the surface of the microreactor because of hydrophobic interactions of the poly(propylene glycol) portion of the copolymer.

Microreactors may or may not have lids to enclose the reaction mixture. When a lid is employed, the nucleic acid may be immobilized on it. The lid can be sealed by conformal pressure, adhesives, and other bonding techniques known in the art. An exemplary process for sealing microreactors made from PDMS (or other elastomeric materials) is shown in Figure 3B. This process employs valve technology known in the art (linger, M.A. et al. 2000. Science, 288, 113-116; Jung et al Langmuir, 2008. 24, 4439-4442). Lids made from glass and other optical quality materials are preferred. Suitable microreactors are also described in US 2010/00361 10 and WO 2010/017487.

Fluorogenic Labels

Any label that becomes able to emit light as a result of incorporation of a nucleotide to a synthesized nucleic acid may be employed in the methods of the invention. Labels can be attached to nucleotides at a variety of locations. Attachment can be made either with or without a bridging linker to the nucleotide. The label may be attached to the base, sugar, or phosphate of the nucleotide. Preferably, the label is attached to the terminal phosphate, so it is cleaved from the nucleotide during replication. Labels may also be attached to non-naturally occurring portions of a nucleotide, e.g., to the delta or epsilon phosphate in a tetra- or pentaphosphate containing nucleotide. Alternatively, labels may be attached to the alpha phosphate and displaced during incorporation of a nucleotide in a synthesized strand. Preferably, when attached to the nucleotide, the label is substantially non-emitting when diffusing free in solution to reduce background that could interfere with real time detection of incorporation.

In certain embodiments, the label is destroyed (or rendered non detectable) once detected. One method to destroy the label is photobleaching. Another method is to employ a catalyst that chemically alters the label after detection. In other embodiments, the label is not destroyed after detection, and the incorporation of nucleotides having the same label is monitored via the incremental increase of signal.

Exemplary labels include phenolic dyes such as fluoresceins (e.g., 6- carboxyfluorescein (6-FAM), 6-carboxyhexachlorofluorescein (6-HEX), 6- carboxytetrachlorofluorescein (6-TET), 6-carboxy-4',5'-dichloro-2',7'- dimethoxy fluorescein (6- JOE), Oregon Green ^1M 488, and Oregon Green™ 514), phenoxazines (such as resorufin), acri dines (such as DDAO), and coumarins (e.g., coumarin 102, 7-hydroxycoumarin, and 6,8- difluoroumbelliferone). The emission and absorption spectra of two exemplary fluorophores, DDAO and resorufin, are shown in Figure IA.

Fluorogenic nucleotide substrates that employ a fluorescein-based fluorophore may have the structure:

where R is a nucleoside base, as described herein, and X is a blocking group that serves to minimize the fluorescence emission of the substrate molecule. This blocking group is, for example, an alkyl group (e.g., such as methyl, ethyl, propyl, isopropyl, butyl), an acyl group (e.g., acetyl), sulfonyl (e.g., SO₂R, where R is C]-C₆ alkyl), an alkyl group interrupted with one or more heteroatoms (e.g., O, N, S, or P), haloalkyl group (e.g., perfluorinated alkyl), cycloalkyl (e.g., with 3-6 ring carbons), carboxy substituted alkyl, sulfonyl substituted alkyl, or any other functional group that prevents the electronic structure of the attached oxygen from imparting significant fluorescence to the substrate molecule (see, e.g., WO 2005/108994). The functional groups R₁-Ri_O are chosen to enhance the properties of the fluorogenic substrate and corresponding fluorophore to satisfy the requirements for single molecule nucleic acid sequencing described above. These groups may be selected from hydrogen, halogen (e.g., F or Cl), sulfonate, carboxy, acyl, alkyl, alkoxy, alkylthio, aryl, heteroaryl (e.g., containing one of O, N, or S), nitro, and hydroxyl (see also U.S. 7,432,372, U.S. 6,162,931, U.S. 6,229,055, and WO 2005/108994 Al).

Another class of fluorogenic substrates has the general formula:

with R, X, and R₁ -R₁₀ as described above. The fluorogenic dyes used in these substrates can be synthesized using methods known in the art (U.S. 6,130,101, U.S. 2005/0026235, Pongev et al., Rus. J. Gen. Chem, 2001).

A third class of fluorogenic compounds has the following structure:

B ase- Sugar-Phosphate- [ S el f-reacting C omponcnt] , where Base is any nucleotide base as described herein, Sugar is any sugar or other such group in a nucleotide as described herein, Phosphate is a polyphosphate, and Self-reacting Component is a moiety that undergoes an intramolecular reaction upon cleavage of the phosphate to which it is connected to form a fluorophore. These compounds are substantially non-fluorescent at the wavelengths where the corresponding fluorophore emits and typically absorb very little at the absorption maximum of the corresponding fluorophore. The Self-reacting Component is of two forms. In one, this component includes a self-immolative linker conjugated to a fluorophore, wherein the conjugation renders the fluorophore substantially non-fluorescent. When the phosphate group is cleaved from the self-immolative linker, it spontaneously reacts, resulting in release of the fluorophore, which is fluorescent again. In another form, this component includes a proto-fluorophore, which is substantially nonfluorescent. Cleavage of the phosphate group from the proto-fluorophore results in an intramolecular reaction, e.g., lactonization, that forms a fluorophore. It will be understood that the compounds depicted above will be linked as is known in the art to produce a nucleotide, as defined herein, having a fluorogenic label.

Self-immolative linkers are known in the art (see, e.g., Zhou et al., ChemBioChem, 2008, 9, 714-718; Levine et al., Molecules, 2008, 13, 204-211 ; Lavis et al., ChemBioChem, 2006, 7, 1151-1154; Richard et al., Bioconjugate Chemistry, 2008, 19, 1707-1718; US2005/0147997 Al; and US2006/0003383 Al). An example of a self-immolative linker is the trimethyl lock linker (Levine et al., Molecules, 2008, 13, 204-211 ; Lavis et al., ChemBioChem, 2006, 7, 1 151-1 154). One class of a nucleotide substrate including a trimethyl lock linker has the general structure:

where R is a nucleotide base, n is an integer ranging from 0 to 4, and X is a blocking group (as discussed above) that serves to minimize the fluorescence emission of the chromophore when it is conjugated to the substrate. The groups R]-R]₁ are all hydrogen atoms in the case of rhodamine but can be modified to fonn derivatives with different chemical, spectral, and photophysical properties. R]-R] i can be hydrogen, halogen (e.g., F), sulfonate, carboxy, acyl, alkyl, alkoxy, alkylthio, aryl, heteroaryl (e.g., containing one of O, N, or S), nitro, or hydroxyl. Exemplary rhodamine dyes include rhodamine B, rhodamine 19, rhodamine 110, rhodamine 116, sulforhodamine B, and carboxyrhodamine.

Derivatives of oxazine dyes can also be employed in a similar fashion:

where R is a nucleoside base, n is an integer between 0 and 4, X is a blocking group (as discussed above) that serves to minimize the fluorescence emission of the chromophore when it is conjugated to the substrate, and R₁-R₇ represent functional groups as discussed for rhodamine.

Benzophenoxazine dyes, such as cresyl violet and its derivates, can also be employed:

where R is a nucleoside base, n is an integer between O and 4, X is a blocking group (as discussed above) that serves to minimize the fluorescence emission of the chromophore when it is conjugated to the substrate, and RpR₈ represent the functional groups as discussed for rhodamine. Examples of benzophenoxazine dyes include 3-imino-3H-phenoxazin-7-amine (oxazine) and 9-imino-9H-benzo[a]phenoxazine-5-amine. The Self-reacting Component may also result in spontaneous generation of a fluorophore, e.g., through cyclization reactions in response to enzymatic digestion. An example of these compounds results in generation of a coumarin fluorophore (see, e.g., Wang et al., Methods in Molecular Medicine, 1998, 23, 71; Wang et al., Bioorganic and Medicinal Chemistry Letters, 1996, 6, 945- 950; and US2001/6,214,330 Bl):

where R represents any suitable substituent for the amine leaving group.

Additional labels are known in the art, e.g., in U.S. Patent Nos. 7,041,812, 7,052,839, 7,125,671, 7,223,541, and 7,244,566; US 2004/015119 and 2010/0036110; and WO 2010/017487.

It will also be understood that the sugar moiety depicted in any of the above structures, i.e., 2'-deoxyribose, may be replaced with any other appropriate group, as described herein (for example, the nucleotide may be a ribonucleotide). Specific fluorogenic labels will be selected based on the excitation and emission wavelengths and compatibility with the reagents employed in the sequencing reactions. Chemical modification can be rationally employed on the fluorogenic labels/fluorophores to impart resistance to effects of reaction components (see, e.g., U.S. 7,432,372, U.S. 6,162,931, U.S. 6,229,055, and WO 2005/108994 Al). Preferred labels are also resistant to photodamage (prior to detection). When detected, labels preferably produce a high photon flux at visible wavelengths with minimal blinking and bleach on a reasonable timescale. Molecular oxygen affects bleaching of fluorophores. Reactions may employ a variety of methods for eliminating molecular oxygen from a reaction sample (including enzymatic systems of catalase and glucose oxidase or protocatechuate 3,4-dioxygenase) are known in the art (see, e.g., US 2007/0161017 Al). Alternately, molecular oxygen concentration can be used to control the average bleaching time of the fluorophore such that a detectable number of photons are emitted prior to bleaching, but only modest laser powers are necessary to bleach the fluorophore. Other molecules are also known to affect the photostability of different fluorophores, such as peroxide and reducing agents (such as DTT, TCEP, and BME). The time or excitation power required to eliminate the fluorophore once detection has occurred may be controlled using these compounds that have either excited-state or ground- state reactivity with the generated fluorophore.

Activating Catalyst

Any catalyst that is capable of acting on a label to render it fluorescent after a nucleotide incorporation event may be used in the invention. Preferably, the activating catalyst does not act on the label prior to incorporation. Preferred catalysts include enzymes such as alkaline phosphatases (e.g., bacterial alkaline phosphatase, shrimp alkaline phosphatase, calf intestinal phosphatase, and antarctic phosphatase), acid phosphatases, galactosidases, horseradish peroxidase, phosphodiesterase, phosphodiesterase, pyruvate kinase, lactic dehydrogenase, lipase, or combinations of enzymes and substrates in a coupled enzyme system such as maltose, maltose phosphorylase, glucose oxidase, horseradish peroxidase, and amplex red (PIPER™ phosphate detection kit, Invitrogen). The activating catalyst may also be an ion in solution, e.g., iodide, hydroxide, or hydronium, a zeolite or other porous catalytic surface, or a metal surface, e.g., platinum, palladium, or molybdenate. Other biological and synthetic catalysts may also be employed. Multiple copies of a particular catalyst may be present to reduce the time required for interaction with the label. The catalyst may be immobilized to a surface of the microreactor or a bead to increase the effective concentration within the reactor.

A nucleic acid replicating catalyst preferably incorporates a fluorogenic label at a rate much slower than the rate at which an activating catalyst converts the fluorogenic-label to a fluorophore, so that sequentially released fluorogenic-labels are sequentially catalyzed by the activating catalyst, thereby reproducing the temporal order of nucleotide addition. Example 1

Figure 4 shows bulk measurements for fluorogenic reverse transcription of primed, homopolymcric RNA molecules by Superscript® II (MMLV-RT RNaseH- mutant). dATP conjugated with DDAO was used as a substrate, and the increase in fluorescence corresponding to nucleotide incorporation was monitored in real-time using a standard fluorometer. These data show that MMLV RT incorporated fluorogenic nucleotides onto an mRNA template, forming an mRNA/DNA hybrid. RT synthesized cDNA on an mRNA template with fluorogenic substrates at a rate of a few seconds per base, even at relatively low substrate concentrations (3 μM).

Example 2

Figure 5 demonstrates reverse transcriptase activity in microreactors as monitored on a fluorescence microscope. Streptavidin-coated polystyrene beads were coated with RNA molecules (UC heteropolymer) that were primed with biotinylated DNA, and these beads were trapped in PDMS microreactors. In the presence of dGTP-resorufin, dATP-DDAO, shrimp alkaline phosphatase, and Superscript III, fluorescent products were built up in a few minutes in microreactors that had mRNA containing beads, demonstrating robust enzyme activity.

OTHER EMBODIMENTS

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the appended claims.

Other embodiments are in the claims. What is claimed is:

Claims

1. A method for profiling RNA expression in a sample, said method comprising the steps of: a) in each of a plurality of optionally sealed microreactors, disposing a mixture in solution phase comprising a single copy of a target RNA from a sample or a DNA copy thereof, a nucleic acid replicating catalyst, and a mixture of nucleotides, wherein said mixture of nucleotides comprises a first nucleotide comprising a first label that is substantially non-fluorescent until after incorporation of said first nucleotide into a nucleic acid based on complementarity to said target RNA or DNA copy thereof; b) allowing continuous template-dependent replication of said target RNAs or DNA copies thereof in said plurality of microreactors; c) sequencing said target RNAs or DNA copies thereof in said plurality of microreactors by detecting in real time the individual incorporation of said first nucleotide during template-dependent replication by monitoring fluorescence emission resulting from said first label; and d) determining from said sequencing the level of expression of RNA in said sample.

2. The method of claim 1, wherein in step (a) said single copy is not an amplification product.

3. The method of claim 1, wherein in step (a), said single copy is said target RNA.

4. The method of claim 1, wherein in step (a), said single copy is said DNA copy of said target RNA.

5. The method of claim 4, further comprising the step of producing said DNA copy from said target RNA.

6. The method of claim 1, wherein said sample is a single cell.

7. The method of claim 1, further comprising the step of Iy sing said sample and separating said RNA from the lysate prior to step (b).

8. The method of claim 6, wherein said single cell is isolated from a sample by laser microdissection.

9. The method of claim 1, wherein said nucleic acid replicating catalyst is a reverse transcriptase.

10. The method of claim 1, further comprising identifying the RNA from a database using a partial sequence.

1 1. The method of claim 1, wherein said sequencing only identifies between 10 and 50 nucleotides of at least one RNA or DNA copy in said plurality of microreactors.

12. The method of claim 1, wherein step (d) comprises determining transcriptional variation of at least one RNA or DNA copy in said plurality of microreactors.

13. The method of claim 1, wherein said RNA is eukaryotic.

14. The method of claim 1, wherein said RNA is prokaryotic.

15. The method of claim 1, wherein said RNA is viral.

16. The method of claim 1, wherein, in each of said plurality of microreactors, said mixture in solution phase further comprises an activating enzyme that renders said first label fluorescent.

17. The method of claim 1, wherein said first label is photobleached after step (c).

18. The method of claim 1, wherein said first label is a phosphate label that is cleaved from said first nucleotide during replication.

19. The method of claim 1, wherein, in each of said plurality of microreactors, said mixture of nucleotides further comprises a second nucleotide comprising a second label that is substantially non-fluorescent until incorporation of said second nucleotide into said nucleic acid based on complementarity to said target nucleic acid, a third nucleotide comprising a third label that is substantially non- fluorescent until incorporation of said third nucleotide into said nucleic acid based on complementarity to said target nucleic acid, and a fourth nucleotide comprising a fourth label that is substantially non-fluorescent until incorporation of said fourth nucleotide into said nucleic acid based on complementarity to said target nucleic acid.

20. The method of claim 1, further comprising repeating steps (b)-(c) at least once.

21. The method of claim 1 , wherein said RNA or DNA copy is immobilized on a surface of said microreactor or a bead disposed in said microreactor.

22. A system for profiling RNA expression in a sample comprising: a plurality of microreactors that are each capable of holding a mixture in solution phase of a single copy of a target RNA or a DNA copy thereof, a nucleic acid replicating catalyst, and a mixture of nucleotides, at least one of which comprises a label that is substantially non- fluorescent until after incorporation of at least one nucleotide into a nucleic acid based on complementarity to said target nucleic acid; a fluorescent microscope for imaging said plurality of microreactors to sequence target RNAs or DNA copies in said microreactors by detecting in real time in each microreactor the incorporation of at least one nucleotide during template-dependent replication of said single copy of said target RNA or DNA copy by monitoring fluorescence from said label resulting from incorporation of said at least one nucleotide; a fluidic delivery system connected to said plurality of microreactors and comprising an inlet for a single cell, a reservoir comprising a lysing reagent, a reservoir comprising a reagent for purifying RNA from a cellular lysate, a chamber for contacting said single cell with said lysing reagent and/or said reagent for purifying RNA, and a pump for delivering single nucleic acid molecules to each of said plurality of microreactors.

23. The system of claim 22, further comprising a reservoir comprising reagents for producing a DNA copy of said RNA.

24. The system of claim 22, further comprising a laser microdissector capable of isolating said single cell from a sample, wherein said dissector is coupled to said inlet for said single cell in said fluidic delivery system.

25. The system of claim 22, wherein said plurality of microreactors comprises between 100,000 and 1 ,000,000 microreactors.

26. The system of claim 22, further comprising a light source capable of photobl each ing said label after detection.

27. The system of claim 22, wherein the excitation source of said fluorescent microscope is capable of photobleaching said label.

28. The system of claim 22, wherein the microreactors comprise poly(dimethyl siloxane) (PDMS).

29. The system of claim 22, wherein said plurality of microreactors comprises a control layer, pressurization of which conformally seals said microreactors against a flat surface, wherein said system further comprises a pressure source.

30. The system of claim 22, further comprising a temperature controller for regulating the temperature of said plurality of microreactors.