Patent Application
20170002405
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 29, 2016, is named NATE-027_ST25.txt and is 170,565 bytes in size.
Current methods for detecting nucleic acid or protein targets in a plurality of samples, in which the identity and quantity of each target for each sample is determined, are time consuming and costly. There exists a need for a new probes, compositions, methods, and kits for simultaneously detecting nucleic acid or protein targets in a plurality of samples.
The present invention relates to a new probes, compositions, methods, and kits for simultaneously detecting nucleic acid or protein targets in a plurality of samples.
A first aspect of the present invention relates to a single-stranded nucleic acid probe including at least three regions: at least a first region capable of binding to a target nucleic acid in a sample, at least a second region capable of binding to at least a first plurality of labeled single-stranded oligonucleotides, in which the first plurality of labeled single-stranded oligonucleotides identifies the target nucleic acid in the sample, and at least a third region capable of binding to at least a second plurality of labeled single-stranded oligonucleotides, in which the second plurality of labeled single-stranded oligonucleotides identifies the sample.
In embodiments of this aspect or any other aspect or embodiment disclosed herein, a target nucleic acid is a synthetic oligonucleotide or is obtained from a biological sample. The second region may include at least one position (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) for binding to the first pluralities of labeled single-stranded oligonucleotides; the first plurality of labeled single-stranded oligonucleotides may include or be complementary to one or more of SEQ ID NO: 1 to SEQ ID NO: 808. The third region may include at least one position (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) for binding to the second pluralities of labeled single-stranded oligonucleotides; the second plurality of labeled single-stranded oligonucleotides may include or be complementary to one or more of SEQ ID NO: 1 to SEQ ID NO: 808. In embodying single-stranded probes, a first plurality of labeled single-stranded oligonucleotides is not identical to a second plurality of labeled single-stranded oligonucleotides.
In any aspect or embodiment of the present invention, there is no upper limit to the number of positions present in a probe's second region and/or in the probe's third region. Additionally, in any aspect or embodiment of the present invention, there is no limit to the number of positions in a second region that can be combined with the number of positions for a third region. More specifically, a first probe may include a second region having one, two, three, four, five, six, seven, eight, nine ten, or more positions and a third region having one, two, three, four, five, six, seven, eight, nine, ten, or more positions. As non-limiting embodiments, a probe may include a second region having two, three, four, five, six, seven, eight, nine, ten, or more positions and a third region having two positions; a probe may include a second region having two, three, four, five, six, seven, eight, nine, ten, or more positions and a third region having three positions; a probe may include a second region having two, three, four, five, six, seven, eight, nine, ten, or more positions and a third region having four positions; a probe may include a second region having two, three, four, five, six, seven, eight, nine, ten, or more positions and a third region having five positions; or a probe may include a second region having two, three, four, five, six, seven, eight, nine, ten, or more positions and a third region having six positions.
The labeled single-stranded oligonucleotide may include deoxyribonucleotides, embodiments of which may have melting/hybridization temperatures of between about 65° C. and about 85° C., e.g., about 80° C. In embodiments, the label monomer of a labeled single-stranded oligonucleotide may be a fluorochrome, quantum dot, dye, enzyme, nanoparticle, chemiluminescent marker, biotin, or another monomer that can be detected directly or indirectly. In embodiments, a label monomer of one position is spectrally or spatially distinguishable from a label monomer of another position, within a region and/or between regions. In embodiments, a label monomer at a position of the second region that is adjacent to a position of the third region differs from a label monomer at the position of the third region that is adjacent to the position of the second region.
In any embodiment or aspect of the present invention, a single-stranded oligonucleotide may lack a label monomer. Thus, a position of a second region and/or a third position may be hybridized with at least one oligonucleotide lacking a detectable label. In these embodiments, a probe will have a “dark spot” adjacent to a position having a detectable signal. The term “dark spot” refers to a lack of signal from a label attachment site on a probe. Dark spots add more coding permutations and generate greater reporter diversity in a population of probes.
In any embodiment or aspect of the present invention, a single-stranded nucleic acid probe may comprise a single-stranded or double-stranded RNA, DNA, PNA, or other polynucleotide analogue spacer between its first region and its second region and/or between its first region and its third region. The spacer may be double-stranded DNA. The spacer may have similar mechanical properties as the probe's backbone. The spacer may be of any length, e.g., 1 to 100 nucleotides, e.g., 2 to 50 nucleotides. The spacer can be shorter than 20 nucleotides or longer than 40 nucleotides and it can be 20 to 40 nucleotides long. Non-limiting examples of a spacer includes the sequences covered by SEQ ID NO: 809 to SEQ ID NO: 813.
In any embodiment or aspect of the present invention, a probe may comprise at least one affinity moiety. The at least one affinity moiety may be attached to the probe by covalent or non-covalent means. Various affinity moieties appropriate for purification and/or for immobilization are known in the art. Preferably, the affinity moiety is biotin, avidin, or streptavidin. Other affinity tags are recognized by specific binding partners and thus facilitate isolation and immobilization by affinity binding to the binding partner, which can be immobilized onto a solid support.
A second aspect of the present invention relates to a composition including at least two single-stranded nucleic acid probes. The at least a first single-stranded nucleic acid probe includes at least three regions: at least a first region capable of binding to a first sequence of a target nucleic acid in a sample, at least a second region capable of binding to at least a first plurality of labeled single-stranded oligonucleotides, in which the first plurality of labeled single-stranded oligonucleotides identifies the target nucleic acid in the sample, and at least a third region capable of binding to at least a second plurality of labeled single-stranded oligonucleotides, in which the second plurality of labeled single-stranded oligonucleotides identifies the sample. The at least a second single-stranded nucleic acid probe includes at least two regions: at least a first region capable of binding to a second sequence of the target nucleic acid in a sample, in which the first and the second sequences of the target nucleic acid are different or to a second target nucleic acid and at least a second region including at least one affinity moiety (e.g., biotin, avidin, and streptavidin).
A third aspect of the present invention relates to a composition including a plurality of single-stranded nucleic acid probes. Each single-stranded nucleic acid probe includes at least three regions: at least a first region capable of binding to a target nucleic acid in a sample, at least a second region capable of binding to a first plurality of labeled single-stranded oligonucleotides, in which the first plurality of labeled single-stranded oligonucleotides identifies the target nucleic acid in the sample, and at least a third region capable of binding to a second plurality of labeled single-stranded oligonucleotides, in which the second plurality of labeled single-stranded oligonucleotides identifies the sample. In embodiments, the plurality of single-stranded nucleic acid probes are capable of binding to different target nucleic acids obtained from the same sample or the plurality of single-stranded nucleic acid probes are capable of binding to the same target nucleic acid obtained from different samples.
A fourth aspect of the present invention relates to a method for simultaneously detecting a target nucleic acid in at least two sample: The method includes steps of: (1) contacting a first sample with one or more first single-stranded nucleic acid probes capable of identifying a first target nucleic acid and the first sample, (2) contacting the first sample with a first plurality of labeled single-stranded oligonucleotides capable of binding to the first single-stranded nucleic acid probes that can identify the first target nucleic acid, (3) contacting the first sample with a second plurality of labeled single-stranded oligonucleotides capable of binding to the first single-stranded nucleic acid probes that can identify the first sample, (4) contacting at least a second sample with one or more at least second single-stranded nucleic acid probes capable of identifying the first target nucleic acid and the at least second sample, (5) contacting the at least second sample with the first plurality of labeled single-stranded oligonucleotides capable of binding to the at least second single-stranded nucleic acid probes that can identify the first target nucleic acid, (6) contacting the at least second sample with at least a third plurality of labeled single-stranded oligonucleotides capable of binding to the at least second single-stranded nucleic acid probes that can identify the at least second sample, (7) pooling the sample of step (3) and the sample of step (6) to form a combined sample, and (8) detecting the first and the at least second single-stranded nucleic acid probes in the combined sample, thereby simultaneously detecting the target nucleic acid in at least two samples. In embodiments, the first sample and the at least second sample are different. The method may further include embodiments of contacting the first and at least second sample with at least a third single-stranded nucleic acid probe comprising at least two regions: at least a first region capable of binding to a second sequence of the first target nucleic acid in a sample, wherein the first and the second sequences of the first target nucleic acid are different or capable of binding to a second target nucleic acid and at least a second region comprising at least one affinity moiety.
A fifth aspect of the present invention relates to a method for simultaneously detecting a target nucleic acid in at least two samples. The method includes steps of: (1) contacting a first sample with one or more first single-stranded nucleic acid probes capable of identifying a first target nucleic acid and the first sample to form one or more first complexes, in which the first single-stranded nucleic acid probes comprise a first plurality of labeled single-stranded oligonucleotides that can identify the first target nucleic acid, (2) contacting the one or more first complexes with a second plurality of labeled single-stranded oligonucleotides capable of binding to the first single-stranded nucleic acid probes that can identify the first sample, (3) contacting at least a second sample with one or more at least second single-stranded nucleic acid probes capable of identifying the first target nucleic acid and the at least second sample to form one or more second complexes, in which the at least second single-stranded nucleic acid probes comprise the first plurality of labeled single-stranded oligonucleotides that can identify the first target nucleic acid, (4) contacting the one or more second complexes with at least a third plurality of labeled single-stranded oligonucleotides capable of binding to the at least second single-stranded nucleic acid probes that can identify the at least second sample, in which the first sample and the at least second sample are different, (5) pooling the sample of step (2) and the sample of step (4) to form a combined sample, and (6) detecting the first and the at least second single-stranded nucleic acid probes in the combined sample, thereby simultaneously detecting the target nucleic acid in at least two samples.
A sixth aspect of the present invention relates to a method for simultaneously detecting a target nucleic acid in at least two samples. The method includes steps of: (1) contacting a first sample with one or more first single-stranded nucleic acid probes capable of identifying a first target nucleic acid and the first sample, in which the first single-stranded nucleic acid probes comprise a first plurality of labeled single-stranded oligonucleotides that can identify the first target nucleic acid and a second plurality of labeled single-stranded oligonucleotides that can identify the first sample, (2) contacting at least a second sample with one or more at least second single-stranded nucleic acid probes capable of identifying the first target nucleic acid and the at least second sample, in which the at least second single-stranded nucleic acid probes comprise the first plurality of labeled single-stranded oligonucleotides that can identify the first target nucleic acid and at least a third plurality of labeled single-stranded oligonucleotides that can identify the at least second sample, (3) pooling the sample of step (1) and the sample of step (2) to form a combined sample, and (4) detecting the first and the at least second single-stranded nucleic acid probes in the combined sample, thereby simultaneously detecting the target nucleic acid in at least two samples. In embodiments of this aspect, the first sample and the at least second sample are different.
A seventh aspect of the present invention relates to a method for simultaneously detecting a target nucleic acid in at least two samples. The method includes steps of: (1) contacting one or more first single-stranded nucleic acid probes capable of identifying a first target nucleic acid and the first sample with a second plurality of labeled single-stranded oligonucleotides capable of binding to the first single-stranded nucleic acid probes that can identify the first sample to form one or more first complexes, in which the first single-stranded nucleic acid probes comprise a first plurality of labeled single-stranded oligonucleotides that can identify the first target nucleic acid, (2) contacting the one or more first complexes with the first sample, (3) contacting one or more at least second single-stranded nucleic acid probes capable of identifying the first target nucleic acid and the at least second sample with at least a third plurality of labeled single-stranded oligonucleotides capable of binding to the at least second single-stranded nucleic acid probes that can identify the at least second sample to form one or more second complexes, in which the second single-stranded nucleic acid probes comprise the first plurality of labeled single-stranded oligonucleotides that can identify the first target nucleic acid, (4) contacting the one or more second complexes with at least a second sample, (5) pooling the sample of step (2) and the sample of step (4) to form a combined sample, and (6) detecting the first and the at least second single-stranded nucleic acid probes in the combined sample, thereby simultaneously detecting the target nucleic acid in at least two samples. In embodiments of this aspect, the first sample and the at least second sample are different.
An eighth aspect of the present invention relates to a kit including at least three containers. A first container includes a plurality of single-stranded nucleic acid probes, each single-stranded nucleic acid probe comprising at least three regions: at least a first region capable of binding to a target nucleic acid, at least a second region capable of binding to a first plurality of labeled single-stranded oligonucleotides, in which the first plurality of labeled single-stranded oligonucleotides identifies the target nucleic acid, and at least a third region capable of binding to at least a second or third plurality of labeled single-stranded oligonucleotides. In embodiments, the first container further includes the first plurality of labeled single-stranded oligonucleotides. A second container includes the second plurality of labeled single-stranded oligonucleotides that can identify the first sample. The at least third container includes at least the third plurality of labeled single-stranded oligonucleotides that can identify the at least second sample. In embodiments, the kit may further include a second single-stranded nucleic acid probe or a plurality of second single-stranded probes each probe including at least two regions: at least a first region capable of binding to a second sequence of the first target nucleic acid in a sample, wherein the first and the second sequences of the first target nucleic acid are different or capable of binding to a second target nucleic acid and at least a second region comprising at least one affinity moiety.
A ninth aspect of the present invention relates to a kit comprising at least four containers. A first container includes a plurality of single-stranded nucleic acid probes, each single-stranded nucleic acid probe comprising at least three regions: at least a first region capable of binding to a target nucleic acid, at least a second region capable of binding to a first plurality of labeled single-stranded oligonucleotides, in which the first plurality of labeled single-stranded oligonucleotides identifies the target nucleic acid, and at least a third region capable of binding to at least a second or third plurality of labeled single-stranded oligonucleotides. A second container includes the first plurality of labeled single-stranded oligonucleotides. A third container includes the second plurality of labeled single-stranded oligonucleotides that can identify the first sample. The at least fourth container includes at least the third plurality of labeled single-stranded oligonucleotides that can identify the at least second sample.
A tenth aspect of the present invention relates to a kit including at least two containers. A first container includes a plurality of single-stranded nucleic acid probes, each single-stranded nucleic acid probe comprising at least three regions: at least a first region capable of binding to a target nucleic acid, at least a second region capable of binding to a first plurality of labeled single-stranded oligonucleotides, in which the first plurality of labeled single-stranded oligonucleotides identifies the target nucleic acid, and at least a third region capable of binding to at least a second or third plurality of labeled single-stranded oligonucleotides. In embodiments, the first container further includes the first plurality of labeled single-stranded oligonucleotides and the second plurality of labeled single-stranded oligonucleotides that can identify a first sample. In embodiments, the at least second container includes the plurality of single-stranded nucleic acid probes and the first plurality of labeled single-stranded oligonucleotides, and the at least third plurality of labeled single-stranded oligonucleotides that can identify at least a second sample.
An eleventh aspect of the present invention relates to probes, compositions, kits, and methods including a single-stranded nucleic acid probe having at least two regions: at least a first region capable of binding to a target nucleic acid in a sample and at least a second region capable of binding to at least a plurality of labeled single-stranded oligonucleotides, in which the plurality of labeled single-stranded oligonucleotides identifies the sample. In embodiments, the second region may include at least one position (e.g., two, three, four, five, six, seven, eight, nine ten, or more) for binding to the pluralities of labeled single-stranded oligonucleotides; the pluralities of labeled single-stranded oligonucleotides may include or be complementary to one or more of SEQ ID NO: 1 to SEQ ID NO: 808. There is no upper limit to the number of positions present in a probe's second region.
Any of the above aspects or embodiments can be adapted for use in a twelfth aspect of the present invention, which relates to detecting protein targets in a plurality of samples. This twelfth aspect extends the prior aspects by further including at least one first protein probe specific for at least one target protein in a sample. The at least one first protein probe includes a first region capable of binding to target protein in a sample and a second region including a partially double-stranded nucleic acid or including a single-stranded nucleic acid. The second region of the protein probe can include a linker, e.g., a photocleavable linker, which when cleaved, can release a portion of the second region from the first region. In the twelfth aspect, a single-stranded nucleic acid probe including at least three regions has a first region capable of binding to a target nucleic acid in which the target nucleic acid is a portion of the first protein probe's second region. In embodiments, the twelfth aspect may further include at least one second protein probe specific for the at least one target protein in a sample, which includes a first region capable of binding to target protein in a sample and a second region including a capture region or a matrix. In embodiments, a protein probe's first region capable of binding to a target protein in a sample may be an antibody, a peptide, an aptamer, or a peptoid. An antibody can be obtained from a variety of sources, including but not limited to polyclonal antibody, monoclonal antibody, monospecific antibody, recombinantly expressed antibody, humanized antibody, plantibodies, and the like. Thus, in any embodiment or aspect of the present invention, a target nucleic acid in a sample may be a portion of a first protein probe that is released from or present in the first protein probe. Such first protein probes include a first region capable of binding to first target protein in a sample and a second region including a partially double-stranded nucleic acid or including a single-stranded nucleic acid. The partially double-stranded nucleic acid or the single-stranded nucleic acid is released from a first protein probe.
Any aspect or embodiment described herein can be combined with any other aspect or embodiment as disclosed herein.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.
The present invention is based in part on new probes, compositions, methods, and kits for simultaneously detecting nucleic acid or protein targets in a plurality of samples.
Unlike previously-described probes, the present invention relates to a probe having a backbone that includes at least one region capable of identifying a target nucleic acid or protein in a sample and at least one region capable of identifying the sample. Two exemplary probes are illustrated in
The region capable of binding to a target nucleic acid is preferably at least 15 nucleotides in length, and more preferably is at least 20 nucleotides in length. In specific embodiments, the target-specific sequence is approximately 10 to 500, 20 to 400, 25, 30 to 300, 35, 40 to 200, or 50 to 100 nucleotides in length.
The probes illustrated in
For a “Target-ID” region, the linear order of labels provides a signal identifying the target nucleic acid. For a “Sample-ID” region, the linear order of labels provides a signal identifying the sample.
Each labeled oligonucleotide may be labeled with one or more detectable label monomers. The label may be at a terminus of an oligonucleotide, at a point within an oligonucleotide, or a combinations thereof. Oligonucleotides may comprise nucleotides with amine-modifications, which allow coupling of a detectable label to the nucleotide.
Labeled oligonucleotides of the present invention can be labeled with any of a variety of label monomers, such as a fluorochrome, quantum dot, dye, enzyme, nanoparticle, chemiluminescent marker, biotin, or other monomer known in the art that can be detected directly (e.g., by light emission) or indirectly (e.g., by binding of a fluorescently-labeled antibody). Preferred examples of a label that can be utilized by the invention are fluorophores. Several fluorophores can be used as label monomers for labeling nucleotides including, but not limited to GFP-related proteins, cyanine dyes, fluorescein, rhodamine, ALEXA Flour™, Texas Red, FAM, JOE, TAMRA, and ROX. Several different fluorophores are known, and more continue to be produced, that span the entire spectrum.
Labels associated with each position (via hybridization of a position with a labeled oligonucleotide) are spatially-separable and spectrally-resolvable from the labels of a preceding position or a subsequent position.
Each position in a probe may be hybridized with at least one labeled oligonucleotide. Alternately, a position may be hybridized with at least one oligonucleotide lacking a detectable label. Each position can hybridize to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 to 100 labeled (or unlabeled) oligonucleotides or more. The number of labeled oligonucleotides hybridized to each position depends on the length of the position and the size of the oligonucleotides. A position may be between about 300 to about 1500 nucleotides in length. The length of the labeled oligonucleotides may vary from about 20 to about 55 nucleotides in length. The oligonucleotides are designed to have melting/hybridization temperatures of between about 65 and about 85° C., e.g., about 80° C. For example, a position of about 1100 nucleotides in length may hybridize to between about 25 and about 45 oligonucleotides, each oligonucleotide about 45 to about 25 nucleotides in length. In embodiments, each position is hybridized to about 34 labeled oligonucleotides of about 33 nucleotides in length. The labeled oligonucleotides are preferably single-stranded DNA. Exemplary oligonucleotides are listed in Table 1.
The number of target nucleic acids and samples detectable by a set of probes depends on the number of positions that the probes' backbones include.
The number of positions on a probe's backbone ranges from 1 to 50. In yet other embodiments, the number of positions ranges from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to 15, 20, 30, 40, or 50, or any range in between. Indeed, the number of positions (for detecting a target nucleic acid and/or for detecting a sample) on a backbone is without limit since engineering such a backbone is well-within the ability of a skilled artisan.
A probe may be chemically synthesized or may be produced biologically using a vector into which a nucleic acid encoding the probe has been cloned.
The labeled oligonucleotides hybridize to their positions under a standard hybridization reaction, e.g., 65° C., 5×SSPE; this allows for self-assembling reporter probes. Probes using longer RNA molecules as labeled oligonucleotide (e.g., as described in US2003/0013091) must be pre-assembled at a manufacturing site rather than by an end user and at higher temperatures to avoid cross-linking of multiple backbones via the longer RNA molecules; the pre-assembly steps are followed by purification to remove excess un-hybridized RNA molecules, which increase background. Use of the short single-stranded labeled oligonucleotide greatly simplifies the manufacturing of the probes and reduces the costs associated with their manufacture.
The probes of the present invention can be used to directly hybridize to a target nucleic acid obtained from a biological sample.
The aforementioned US Patent Publications further describe immobilizing, orientating, and extending a probe pair hybridized to a target nucleic acid.
The at least one affinity moiety may be attached to the capture probe by covalent or non-covalent means. Various affinity moieties appropriate for purification and/or for immobilization are known in the art. Preferably, the affinity moiety is biotin, avidin, or streptavidin. Other affinity tags are recognized by specific binding partners and thus facilitate isolation and immobilization by affinity binding to the binding partner, which can be immobilized onto a solid support. A target- and sample-specific reporter probe may also comprise at least one affinity moiety, as described above.
The probes of the present invention can be used to indirectly hybridize to a target nucleic acid obtained from a biological sample.
In the hybridization/detection system, a probe's target binding region hybridizes to a region of a target-specific oligonucleotide. Thus, the probe's target binding region is independent of the ultimate target nucleic acid obtained from a sample. This allows economical and rapid flexibility in an assay design, as the target (obtained from a biological sample)-specific components of the assay are included in inexpensive and widely-available DNA oligonucleotides rather than the more expensive probes. Therefore, a single set of indirectly-binding probes can be used to detect an infinite variety of target nucleic acids in different experiments simply by replacing the target-specific oligonucleotide portion of the assay.
The aforementioned US Patent Publication further describes immobilizing, orientating, and extending a probe pair hybridized to target-specific oligonucleotides that are in turn hybridized to a target nucleic acid obtained from a biological sample.
The single-stranded nucleic acid probes of the present invention can be used for detecting a target protein obtained from a biological sample.
A probe's backbone is preferably single-stranded DNA, RNA or PNA. It may include one or more positions, e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, and twenty or more positions, each capable of binding to at least a plurality of single-stranded oligonucleotides, e.g., labeled oligonucleotides. There is no upper limit to the number of positions that a probe backbone may contain, e.g., twenty or more, fifty or more, and one hundred or more positions. As described above, the backbone may include, at least, a region for binding to a target nucleic acid, a region for identifying a target, and a region for identifying a sample. The backbone shown in
In embodying probes, a first plurality of labeled single-stranded oligonucleotides is not identical to a second plurality of labeled single-stranded oligonucleotides.
In embodiments, a single-stranded oligonucleotide may lack a label monomer. Thus, a position of a second region and/or a third position may be hybridized with at least one oligonucleotide lacking a detectable label. In these embodiments, a probe will have a “dark spot” adjacent to a position having a detectable signal. The term “dark spot” refers to a lack of signal from a label attachment site on a probe. Dark spots add more coding permutations and generate greater reporter diversity in a population of probes.
In embodiments, at least one probe may comprise a single-stranded or double-stranded RNA, DNA, PNA, or other polynucleotide analogue spacer between its first region and its second region and/or between its first region and its third region. The spacer may be double-stranded DNA. The spacer may have similar mechanical properties as the probe's backbone. The spacer may be of any length, e.g., 1 to 100 nucleotides, e.g., 2 to 50 nucleotides. The spacer can be shorter than 20 nucleotides or longer than 40 nucleotides and it can be 20 to 40 nucleotides long. Non-limiting examples of a spacer includes the sequences covered by SEQ ID NO: 809 to SEQ ID NO: 813.
A probe backbone may include only a single position, with the single position identifying the sample (as shown in
Probes can be detected and quantified using commercially-available cartridges, software, systems, e.g., the nCounter® System using the nCounter® Cartridge.
For the herein-described probes, association of label code to target sequence is not fixed. This allows a single set of backbones to be used to generate different codes during hybridization to different samples, by combining it with differently colored pools of oligonucleotides. Following hybridization, the samples are pooled and processed together, as the resulting barcodes will be unique to each sample and can be assigned back to their sample of origin following data collection. An example is the following:
A set of 96 six-position backbones may be used to detect up to 96 different target nucleic acids (either directly or indirectly) or proteins. Oligonucleotide pools (i.e., a plurality of labeled single-stranded oligonucleotides) for positions 1 to 4 of each backbone are associated with fixed colors, such that the four position code for a particular target nucleic acid/protein is always the same, regardless of the hybridization reaction. Positions 5 and 6, although they have a fixed sequence for any given backbone, are given a different color for each sample by coupling the oligonucleotide pool for each position separately to different colored-labels. By producing a differentially-labeled probe for each sample, samples (comprising the target nucleic acid and hybridized probes) can be pooled after the hybridization reaction. The pooled samples can then be processed together and all labeled probes (i.e., barcodes) are imaged together. Then, obtained data is de-convoluted back into the original samples after scanning, thereby tallying the identity of all the barcodes in the image. Such multiplexing greatly increases the throughput of the system.
In a six-position, four color system (i.e., yellow, red, blue, and green fluorophores), the possible combinations of gene-plex and sample-plex are many, depending on how many positions are dedicated to identifying a target nucleic acid or protein and how many positions are dedicated to identifying a sample. When plexing eight samples together (two positions of a probe dedicated for sample identity), each column of a 96-well plate is pooled and each pool is detected on a single lane of a twelve lane cartridge, e.g., an nCounter® Cartridge. When plexing thirty-two samples together (three positions of a probe dedicated for sample identity), a 384-well plate can be detected on a single twelve lane cartridge, e.g., an nCounter® Cartridge.
A kit including six-position probes contains reagents and probes sufficient to detect up to 96 target nucleic acids or proteins in a 96 well format or up to 24 target nucleic acids or proteins in a 384 well format.
An exemplary protocol, using NanoString Technologies®'s nCounter® systems for detecting nucleic acids, is described as follows. Approximately 50 to 100 ng of total RNA per sample and/or a lysate of about 1,000 to about 2500 cells per sample in a total volume of about 5 μl (volume adjusted with RNAse free water, if necessary). Samples are added to a thermocycler-compatible 96-well plate. For a 96-well plate of samples, a kit may include eight tubes (labeled A to H) of TagSet reagents, with each tube containing enough reagents to set up one row of assays (12 samples). A mastermix is made for each of tubes A to H (i.e., Mastermix A to Mastermix H) by adding hybridization buffer and the target-specific first and second probes diluted to the appropriate concentration. 10 μl of Mastermix A is pipetted into each well in row A, 10 μl of Mastermix B is pipetted into each well in row B, and so forth, until Mastermix H has been pipetted. The plate is sealed and heated overnight at about 67° C. in a thermocycler with a heated lid, allowing hybridization of labeled oligonucleotides to appropriate positions of a probe and allowing the probes to hybridize to their target nucleic acids. If the probe indirectly binds to a target nucleic acid obtained from a sample, additional target-specific oligonucleotides (which are bound by a probe and bind to the target nucleic acid obtained from a sample) are include in mastermixes. These target-specific oligonucleotides may not be included in kit as they can be commercially synthesized. The sealing is removed from the plate and assays (samples) for each column are pooled into a twelve-tube strip such that a first pooled sample will contain samples from wells A1 to H1, a second pooled sample will contain samples from wells A2 to H2, and so forth. The twelve-tube strip is placed into a NanoString Technologies® Prep Station and processed using a standard nCounter® protocol, which ultimately scans an nCounter® cartridge and de-convolutes data into individual samples by the ordering of labeled oligonucleotides hybridized to the probes.
A target nucleic acid obtained from a sample may be DNA or RNA and preferably messenger RNA (mRNA).
Probes of the present invention can be used to detect a target nucleic acid or protein obtained from any biological sample. As will be appreciated by those in the art, the sample may comprise any number of things, including, but not limited to: cells (including both primary cells and cultured cell lines), cell lysates or extracts (including but not limited to protein extracts, RNA extracts; purified mRNA), tissues and tissue extracts (including but not limited to protein extracts, RNA extracts; purified mRNA); bodily fluids (including, but not limited to, blood, urine, serum, lymph, bile, cerebrospinal fluid, interstitial fluid, aqueous or vitreous humor, colostrum, sputum, amniotic fluid, saliva, anal and vaginal secretions, perspiration and semen, a transudate, an exudate (e.g., fluid obtained from an abscess or any other site of infection or inflammation) or fluid obtained from a joint (e.g., a normal joint or a joint affected by disease such as rheumatoid arthritis, osteoarthritis, gout or septic arthritis) of virtually any organism, with mammalian samples being preferred and human samples being particularly preferred; environmental samples (including, but not limited to, air, agricultural, water and soil samples); biological warfare agent samples; research samples including extracellular fluids, extracellular supernatants from cell cultures, inclusion bodies in bacteria, cellular compartments, cellular periplasm, and mitochondria compartment.
A probe's region capable of binding to a target protein include molecules or assemblies that are designed to bind with at least one target protein, at least one target protein surrogate, or both; and can, under appropriate conditions, form a molecular complex comprising the protein probe and the target protein. The terms “protein”, “polypeptide”, “peptide”, and “amino acid sequence” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids or synthetic amino acids. The terms also encompass an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. As used herein the term “amino acid” refers to either natural and/or unnatural or synthetic amino acids, including but not limited to glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.
The biological samples may be indirectly derived from biological specimens. For example, where the target nucleic acid is a cellular transcript, e.g., an mRNA, the biological sample of the invention can be a sample containing cDNA produced by a reverse transcription of mRNA. In another example, the biological sample of the invention is generated by subjecting a biological specimen to fractionation, e.g., size fractionation or membrane fractionation.
The biological samples of the invention may be either “native,” i.e., not subject to manipulation or treatment, or “treated,” which can include any number of treatments, including exposure to candidate agents including drugs, genetic engineering (e.g., the addition or deletion of a gene).
In embodiments, a first sample differs from a second sample in an experimental manipulation, e.g., the presence of absence of an applied drug or concentration thereof. This embodiment is particularly significant in cultured cells which may be exposed to a variety of controlled conditions.
In some embodiments, the probes, compositions, methods, and kits described herein are used in the diagnosis of a condition. As used herein the term “diagnose” or “diagnosis” of a condition includes predicting or diagnosing the condition, determining predisposition to the condition, monitoring treatment of the condition, diagnosing a therapeutic response of the disease, and prognosis of the condition, condition progression, and response to particular treatment of the condition. For example, a blood sample can be assayed according to any of the probes, methods, or kits described herein to determine the presence and/or quantity of markers of a disease or malignant cell type in the sample (relative to the non-diseased condition), thereby diagnosing or staging the a disease or a cancer.
A kit of the present invention can include other reagents as well, for example, buffers for performing hybridization reactions, linkers, restriction endonucleases, and DNA ligases. A kit also will include instructions for using the components of the kit, including, but not limited to, information necessary to hybridize labeled oligonucleotides to a probe, to hybridize a probe to a target-specific oligonucleotide, and/or to hybridize a probe or target-specific oligonucleotide to a target nucleic.
As used in this Specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive and covers both “or” and “and”.
Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.”
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although other probes, compositions, methods, and kits similar, or equivalent, to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to practice the present invention, and are not intended to limit the scope of what the inventors regard as the invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts and concentrations) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, temperature is in degrees Centigrade and pressure is at or near atmospheric.
This Example provides data using probes have six positions which include four positions for target identification and two positions for sample identification. Such probes can be detected with the NanoString Technologies® Digital Analyzer post sample processing.
Single-stranded nucleic acid probes used in this assay included a first region of a unique thirty-five deoxynucleotide target binding domain and six consecutive positions for binding labeled oligonucleotides. Each position was 1100 deoxynucleotides in length and had a unique sequence. The first four positions, which were adjacent to the target binding domain, were for identifying the target nucleic acid and the next two positions were for identifying the sample.
Each position of a probe backbone was an approximately 1100 nucleotide sequence. Twenty-four approximate 1100-nucleotide sequences, as described in US2010/0047924 (the contents of which are incorporated herein by reference in its entirety) were used to form backbones. For each position, a set of single-stranded DNA oligonucleotides was designed; together these oligonucleotides were complementary to the entirety of each 1100-nucleotide sequence. Each individual oligonucleotide in the set was designed to have melting temperature (Tm) of approximately 80° C. in 5×SSPE (typically ranging from 78 to 85° C.). Sequences for the single-stranded DNA oligonucleotides are listed in Table 1. All oligonucleotides were synthesized with 5′ amine modifications to attach fluorescent labels. Fluorescent labels coupled to these 5′ amine modifications were Alexa Fluor 488 5-TFP (2,3,5,6-Tetrafluorophenyl Ester) (“Blue”), Alexa Fluor 546 NHS Ester (Succinimidyl Ester) (“Green”), Texas Red-X NHS Ester (Succinimidyl Ester) (“Yellow”), or Alexa Fluor 647 NHS Ester (Succinimidyl Ester) (“Red”) Coupling used standard methods.
Hybridization reactions were performed as described in, e.g., US2014/0371088.
This Example is illustrated in
In 30 μl hybridization reactions, the following reagents were combined (to a final concentration shown): SSPE (5x), Oligo A's (20 pM each), Oligo B's (100 pM each), UCP-3BF2 (5200 pM), 26 Backbones (25 pM each), labeled oligonucleotides (at a 2:1 ratio relative to backbone sequence), and cell lysate from A431 cells (endogenous RNAs from these lysates are the target nucleic acid obtained from a sample). Hybridization reactions were performed in separate PCR tubes in a thermocycler overnight at 67° C. Backbone sequences and labeled oligos used for each sample are listed in Table 2 and Table 3.
After the hybridization reactions, samples were either processed as single samples (a single-plex assay) or pooled into a combined sample with seven other samples (a multi-plex assay). Samples were processed on a NanoString Technologies® Prep Station and codes were counted with a Gen2 Digital Analyzer.
To clarify Tables 2 and 3, DV2 tag-306, as an example, which has an underlying spot sequence of 3-5-10-16-17-22, would comprise (in order) a first position hybridized to a plurality of yellow fluorophore labeled oligonucleotides, a second position hybridized to a plurality of blue fluorophore labeled oligonucleotides, a third position hybridized to a plurality of green fluorophore labeled oligonucleotides, and a fourth position hybridized to a plurality of red fluorophore labeled oligonucleotides; the first through fourth positions are for identifying a target nucleic acid. The DV2 tag-306 would identify the sample as Sample A if it further comprises (in order) a fifth position hybridized to a plurality of a green fluorophore labeled oligonucleotides followed by a sixth position hybridized to a plurality of a blue fluorophore labeled oligonucleotides. However, the DV2 tag-306 would identify the sample as Sample B if it instead further comprises fifth and six positions that are hybridized to a plurality of blue fluorophore labeled oligonucleotides and green fluorophore labeled oligonucleotides.
Spot sequences/Spot IDs 1, 5, 9, 13, 17, and 21 correspond to SEQ ID NO: 1 to SEQ ID NO: 33, SEQ ID NO: 133 to SEQ ID NO: 166, SEQ ID NO: 268 to SEQ ID NO: 302, SEQ ID NO: 405 to SEQ ID NO: 437, SEQ ID NO: 542 to SEQ ID NO: 574, and SEQ ID NO: 675 to SEQ ID NO: 707, respectively.
Spot sequences/Spot IDs 2, 6, 10, 14, 18, and 22 correspond to SEQ ID NO: 34 to SEQ ID NO: 66, SEQ ID NO: 167 to SEQ ID NO: 200, SEQ ID NO: 303 to SEQ ID NO: 336, SEQ ID NO: 438 to SEQ ID NO: 473, SEQ ID NO: 575 to SEQ ID NO: 606, and SEQ ID NO: 708 to SEQ ID NO: 741 respectively.
Spot sequences/Spot IDs 4, 8, 12, 16, 20, and 24 correspond to SEQ ID NO: 101 to SEQ ID NO: 132, SEQ ID NO: 234 to SEQ ID NO: 267, SEQ ID NO: 371 to SEQ ID NO: 404, SEQ ID NO: 508 to SEQ ID NO: 541, SEQ ID NO: 641 to SEQ ID NO: 674, and SEQ ID NO: 775 to SEQ ID NO: 808, respectively.
Spot sequences/Spot IDs 3, 7, 11, 15, 19, and 23 correspond to SEQ ID NO: 67 to SEQ ID NO: 100, SEQ ID NO: 201 to SEQ ID NO: 233, SEQ ID NO: 337 to SEQ ID NO: 370, SEQ ID NO: 474 to SEQ ID NO: 507, SEQ ID NO: 607 to SEQ ID NO: 640, and SEQ ID NO: 742 to SEQ ID NO: 774, respectively.
The steps used in Example 2 are similar to those described in Example 1 with the exception that the six position probe backbone used in this Example had three positions for target identification and three positions for sample identification. Here, the first three positions adjacent to the thirty-five deoxynucleotide target binding domain were for target identification. A schematic of a backbone used in this Example is shown in
After the hybridization reactions, samples were either processed as single samples (a single-plex assay) or pooled into a combined sample with thirty-one other samples (a thirty-two sample multi-plex assay). Samples were processed on a NanoString Technologies® Prep Station and codes were counted with a Gen2 Digital Analyzer.
The contents of Tables 4 and 5 are similar to the contents of Tables 2 and 3, respectively. Thus, DV2 tag-418, as an example, which has an underlying spot sequence of 3-5-10-13-18-24, would comprise (in order) a first position hybridized to a plurality of yellow fluorophore labeled oligonucleotides, a second position hybridized to a plurality of blue fluorophore labeled oligonucleotides, and a third position hybridized to a plurality of green fluorophore labeled oligonucleotides; the first through third positions are for identifying a target nucleic acid. The DV2 tag-418 would identify the sample as Sample A if it further comprises (in order) a fourth position hybridized to a plurality of a blue fluorophore labeled oligonucleotides, a fifth position hybridized to a plurality of a green fluorophore labeled oligonucleotides, and a sixth position hybridized to a plurality of a blue fluorophore labeled oligonucleotides. However, the DV2 tag-418 would identify the sample as Sample B if it instead further comprises fourth, fifth and six positions that are hybridized to a plurality of blue fluorophore labeled oligonucleotides, green fluorophore labeled oligonucleotides, and yellow fluorophore labeled oligonucleotides.
From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
All citations to sequences, patents and publications in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.
This application claims priority to and the benefit of U.S. Provisional Application No. 62/186,818, filed Jun. 30, 2015, the contents of which are incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62186818 | Jun 2015 | US |
