Method of generating biocompatible dendrimers for analyte detection with multimodal labeling and signal amplification
阅读说明：本技术 生成用于具有多模式标记和信号放大的分析物检测的生物相容的树枝状聚合物的方法 (Method of generating biocompatible dendrimers for analyte detection with multimodal labeling and signal amplification ) 是由 斯科特·E·弗雷泽 西蒙·雷斯特雷波 约瑟夫·P·邓纳姆 于 2018-02-28 设计创作，主要内容包括：本文描述了使用聚合触发剂以受控的方式由成对的互补的树枝状核酸单体产生树枝状生物相容的聚合物的方法。树枝状单体由能够自组装的有机聚合物和核酸构成。各聚合物包含约200个分枝,该分枝可用于附着标记并构成生物学上相容的信号放大技术。根据上下文,该技术可用于揭示多种分析物(例如特定的核酸分子、小分子、蛋白质和肽)的存在。(Described herein are methods of producing dendritic biocompatible polymers from pairs of complementary dendritic nucleic acid monomers in a controlled manner using a polymerization trigger. Dendritic monomers are composed of organic polymers and nucleic acids that are capable of self-assembly. Each polymer contains about 200 branches that can be used to attach labels and constitute a biologically compatible signal amplification technique. Depending on the context, this technique can be used to reveal the presence of a variety of analytes (e.g., specific nucleic acid molecules, small molecules, proteins, and peptides).)
1. An assembly, comprising:
at least two molecules, wherein each molecule comprises:
a) a nucleic acid hairpin,
b) a nucleic acid stem, wherein the nucleic acid stem,
c) a nucleic acid branch comprising a binding branch and an extension branch, an
d) An organic polymer, and further wherein the nucleic acid hairpin sequence of at least one first molecule is complementary to the nucleic acid-binding branch sequence of at least one second molecule, and wherein the nucleic acid hairpin sequence of the at least one second molecule is complementary to the nucleic acid-binding branch sequence of the at least one first molecule; and
at least one nucleic acid trigger coupled to the analyte binding agent, wherein the nucleic acid trigger is complementary to the nucleic acid stem and the binding branch.
2. The assembly of claim 1, wherein the hairpin sequence and binding branch sequence are about 6-10 nucleotides.
3. The assembly of claim 1, wherein the hairpin sequence and binding branch sequence are about 11-13 nucleotides.
4. The assembly of claim 1, wherein the extension branch comprises about 13-16 nucleotides.
5. The assembly of claim 1, wherein the extension branch comprises about 10-25 nucleotides.
6. The assembly of claim 1, wherein the nucleic acid trigger comprises about 12-48 nucleotides.
7. The assembly of claim 1, wherein the nucleic acid trigger comprises about 34-38 nucleotides.
8. The assembly of claim 1, wherein the nucleic acid stem comprises about 6-15 nucleotides.
9. The assembly of claim 1, wherein the nucleic acid stem comprises about 22-26 nucleotides.
10. The assembly of claim 1, wherein the organic polymer comprises polyethylene glycol.
11. The assembly of claim 10, wherein the polyethylene glycol comprises a length of about 16-20 carbons.
12. The assembly of claim 1, wherein the analyte binding agent comprises a polynucleotide.
13. The assembly of claim 1, wherein the analyte binding agent comprises a peptide or protein.
14. The assembly of claim 1, wherein the analyte binding agent comprises an antibody.
15. The assembly of claim 1, further comprising a marker polynucleotide complementary to the extension branch.
16. The assembly of claim 15, wherein the labeled polynucleotide comprises a fluorophore, a chromophore, a chromogen, a quantum dot, a fluorescent microsphere, a nanoparticle, an elemental label, a metal chelating polymer, a barcode, and/or a sequence barcode.
17. The assembly of claim 1, wherein the assembly further comprises at least two additional molecules, wherein the additional molecules each comprise:
a) a nucleic acid hairpin,
b) a nucleic acid stem, wherein the nucleic acid stem,
c) a nucleic acid branch comprising a binding branch and an extension branch, an
d) An organic polymer, and further wherein the nucleic acid hairpin sequence of at least one additional first molecule is complementary to the nucleic acid binding branch sequence of at least one additional second molecule, and wherein the nucleic acid hairpin sequence of the at least one additional second molecule is complementary to the nucleic acid binding branch sequence of the at least two additional first molecules, and
a linker comprising a nucleic acid address complementary to the extended branches of the at least two molecules and a second trigger complementary to the nucleic acid stems and binding branches of at least two additional molecules.
18. The assembly of claim 17, further comprising a marker polynucleotide complementary to the extended branches of the at least two additional molecules.
19. The assembly of claim 17, wherein the labeled polynucleotide comprises a fluorophore, a chromophore, a chromogen, a quantum dot, a fluorescent microsphere, a nanoparticle, an elemental label, a metal chelating polymer, a barcode, and/or a sequence barcode.
20. A polymerization process, the process comprising:
adding at least two molecules, each comprising a nucleic acid and an organic polymer;
further adding a trigger molecule comprising a nucleic acid; and
triggering self-assembly polymerization, wherein each molecule comprises one or more sequences complementary to another molecule.
21. The method of claim 20, wherein the at least two molecules each comprise a nucleic acid hairpin, a nucleic acid stem, a binding branch, an extension branch.
22. The method of claim 20, wherein the nucleic acid trigger comprises an analyte binding agent.
23. The method of claim 20, comprising generating a detectable signal by binding a labeled polynucleotide complementary to another molecule, wherein the labeled polynucleotide comprises a labeling agent.
24. The method of claim 20, wherein the at least two molecules each comprise:
a) a nucleic acid hairpin,
b) a nucleic acid stem, wherein the nucleic acid stem,
c) a nucleic acid branch comprising a binding branch and an extension branch, an
d) An organic polymer, and further wherein the nucleic acid hairpin sequence of at least one first molecule is complementary to the nucleic acid-binding branch sequence of at least one second molecule, and wherein the nucleic acid hairpin sequence of the at least one second molecule is complementary to the nucleic acid-binding branch sequence of the at least one first molecule; and is
Coupling at least one nucleic acid trigger to the analyte binding agent, wherein the nucleic acid trigger is complementary to the nucleic acid stem and binding branches.
25. The method of claim 24, comprising generating a detectable signal by binding a labeled polynucleotide to the extension branch, wherein the labeled polynucleotide comprises a labeling agent.
26. The method of claim 24, further comprising at least two additional molecules, wherein the additional molecules each comprise:
a) a nucleic acid hairpin,
b) a nucleic acid stem, wherein the nucleic acid stem,
c) a nucleic acid branch comprising a binding branch and an extension branch, an
d) An organic polymer, and further wherein the nucleic acid hairpin sequence of at least one additional first molecule is complementary to the nucleic acid binding branch sequence of at least one additional second molecule, and wherein the nucleic acid hairpin sequence of the at least one additional second molecule is complementary to the nucleic acid binding branch sequence of the at least two additional first molecules, and
e) a linker comprising a nucleic acid address complementary to the extended branches of the at least two molecules and a second trigger complementary to the nucleic acid stems and binding branches of at least two additional molecules.
Technical Field
Described herein are methods and compositions related to dendritic monomers for labeling and detecting analytes.
Background
Innovative protocols are needed to detect analytes of interest in complex mixtures. Most analytes have no intrinsic signal to be used as a detection marker. Therefore, new techniques are urgently needed to label analytes with readily detectable labels (e.g., chromogens or fluorophores). Furthermore, techniques that enable signal amplification are generally preferred over direct labeling methods because they enhance the signal-to-noise ratio, thereby improving the ease and accuracy of detection. Furthermore, the most suitable label employed may vary in particular circumstances, e.g., depending on the sample, the nature of the analyte, or on the context of the assay. Thus, the flexibility of the type of label that can be utilized and whose signal is to be amplified constitutes another desirable feature. An ideal analyte detection method would combine a simple means of detecting analytes while providing labeling flexibility and signal amplification capabilities. Therefore, there is a great need in the art for labeling reagents that are capable of binding to different biological moieties and imparting signal amplification to produce a high signal-to-noise ratio, thereby benefiting the sensitivity, accuracy and reliability of the detection.
Methods and compositions that meet these criteria are described herein. In particular, dendritic biocompatible polymers are generated from pairs of complementary dendritic nucleic acid monomers in a controlled manner, as initiated by the presence of a polymerization trigger. Dendrimers are composed of nucleic acids and organic polymers. Each polymer contains about 200 branches (dendrimers) that can be used to attach labels and constitute a biologically compatible signal amplification technique.
Disclosure of Invention
Described herein is an assembly comprising at least two molecules and at least one nucleic acid trigger coupled to an analyte binding agent, wherein each molecule comprises a nucleic acid hairpin, a nucleic acid stem, nucleic acid branches (including binding branches and extension branches), and an organic polymer, and further wherein the nucleic acid hairpin sequence of at least one first molecule is complementary to the nucleic acid binding branch sequence of at least one second molecule, and wherein the nucleic acid hairpin sequence of at least one second molecule is complementary to the nucleic acid binding branch sequence of at least one first molecule, wherein the nucleic acid trigger is complementary to the nucleic acid stem and binding branches of at least one first molecule. In other embodiments, the hairpin sequence and the binding branch sequence are about 10-24 nucleotides. In other embodiments, the hairpin sequence and the binding branch sequence are about 6-10 nucleotides. In other embodiments, the hairpin sequence and the binding branch sequence are about 11-13 nucleotides. In other embodiments, the extended branch comprises about 10-20 nucleotides. In other embodiments, the extended branch comprises about 13-16 nucleotides. In other embodiments, the extended branch comprises about 10-25 nucleotides. In other embodiments, the nucleic acid trigger comprises about 12-48 nucleotides. In other embodiments, the nucleic acid trigger comprises about 34-38 nucleotides. In other embodiments, the nucleic acid stem comprises about 12-30 nucleotides. In other embodiments, the nucleic acid stem comprises about 6-15 nucleotides. In other embodiments, the nucleic acid stem comprises about 22-26 nucleotides. In other embodiments, the organic polymer comprises polyethylene glycol. In other embodiments, the polyethylene glycol comprises a length of about 16 to 20 carbons. In other embodiments, the analyte binding agent comprises a polynucleotide. In other embodiments, the analyte binding agent comprises a peptide or protein. In other embodiments, the analyte binding agent comprises an antibody. In other embodiments, the method further comprises a labeled polynucleotide complementary to the extended branch. In other embodiments, the tagged polynucleotide comprises a fluorophore, a chromophore, a chromogen, a quantum dot, a fluorescent microsphere, a nanoparticle, an elemental tag, a metal chelating polymer, a barcode, and/or a sequence barcode (sequential barcode).
Further described herein is a polymerization process comprising: adding at least two molecules, each comprising a nucleic acid and an organic polymer; further adding trigger molecules comprising nucleic acids, wherein each molecule comprises one or more sequences complementary to another molecule, and triggering self-assembly polymerization. In other embodiments, the at least two molecules each comprise a nucleic acid hairpin, a nucleic acid stem, a binding branch, an extension branch. In other embodiments, the nucleic acid trigger comprises an analyte binding agent. In other embodiments, the method comprises generating a detectable signal by binding a labeled polynucleotide complementary to another molecule, wherein the labeled polynucleotide comprises a labeling agent. In other embodiments, the at least two molecules each comprise a nucleic acid hairpin, a nucleic acid stem, a nucleic acid branch (including binding and extension branches), and an organic polymer, and further wherein the nucleic acid hairpin sequence of at least one first molecule is complementary to the nucleic acid binding branch sequence of at least one second molecule, and wherein the nucleic acid hairpin sequence of at least one second molecule is complementary to the nucleic acid binding branch sequence of at least one first molecule, and at least one nucleic acid trigger is conjugated to the analyte binding agent, wherein the nucleic acid trigger is complementary to the nucleic acid stem and binding branch of at least one first molecule. In other embodiments, the method comprises generating a detectable signal by binding a labeled polynucleotide to the extension branch, wherein the labeled polynucleotide comprises a labeling agent.
Drawings
FIG. 1 illustrates the components of the system. Two complementary dendrimers are shown, each comprising a three-part structure of hairpin loops, stems and nucleic acid branches (binding and extension branches) and an organic polymer spacer (fig. 1A and 1B). Nucleic acid triggers are shown (FIG. 1C). The nucleic acid domain binding branch 1-hairpin loop 1', stem 2-stem 2' and binding branch 3-hairpin loop 3 'are complementary (i.e., 1-1', 2-2 'and 3-3' are complementary and can hybridize). Domains 5-6 are extended branches. Domain 4 (red) is a spacer element composed of an organic polymer that is critical to maintaining monomer stability and promoting branching function. A trigger (fig. 1C) can bind to
Fig. 2 assembly of the components when assembled. A dendrimer (4) produced by chemical interaction between a trigger (1) and a complementary dendrimer (2). Note the extended branches extending from the polymer (3).
FIG. 3 is a self-assembly mechanism. The nucleic acid trigger may be used directly (fig. 3A), or attached to/extended by another nucleic acid oligonucleotide (fig. 3B), or attached to a solid substrate (e.g., beads or proteins/peptides) (fig. 3C). The trigger may comprise an analyte binding agent, which may be specific for a polynucleotide, peptide, protein, antibody, thereby making the scale-up, polymerization process of figures 1 and 2 an unconnected component step separate from the underlying "detection" technique in which the analyte binds to the analyte binding agent.
FIG. 4 multimodal branch attachments. Examples of direct attachment of labels (5) to dendrimers (4) by hybridization with extended branches (3). It is noted that each branch will eventually be labeled, but only one is shown here for clarity. Labels may include fluorophores, quantum dots, chromogens, oligonucleotides, and the like. It is again emphasized that the "labeling" step herein is separate from the amplification, polymerization process of fig. 1 and 2.
FIG. 5 is a generalized tagging strategy for reducing costs. In a variant of the labeling method, the adaptor oligonucleotide (5) consists of an "address" (the complementary sequence of the branch) and a label-binding sequence (green). This strategy results in significant cost savings since it requires only one labeled oligonucleotide (6) for any number of systems.
Fig. 6 shows an example of a quadratic magnification strategy. Adaptor oligonucleotide (5) containing "address" and secondary trigger sequence (blue in 5) was used to prime the second polymerisation event and thus another round of amplification. This causes a second amplification (i.e., a squared multiplier of n analyte molecules).
FIG. 7 agarose gel electrophoresis. From the left: a dna gradient (ladder) with a monomer only, monomer to initiator (i.e. trigger) ratio of 1/10, a monomer to initiator ratio of 1/50, a monomer to initiator ratio of 1/100, a dna gradient. Note that there are a large number of high molecular weight molecules in lanes 4-5 at the top of the gel, and no monomer at the bottom (all monomers have now been incorporated into the polymer). In contrast, in lane 2 (monomer only), only background signal amplification occurred, and most of the monomer was located at the bottom of the gel.
FIG. 8 in vivo labeling. FIG. 8A Drosophila embryos labeled with dendrimer (containing alexa-488 fluorophore as secondary label) and revealing the expression domain of the segmented gene even-skipeped. FIG. 8B Drosophila embryos (containing alexa-488 fluorophore) labeled with fluorescent beacons generated by double amplification and revealing the expression domain of the segmented gene even-skipeped.
FIG. 9 in vivo labeling. Drosophila embryos labelled with dendrimer (with secondary label containing europium 151) and revealed the expression domain of the segmented gene even-skipeped.
Figure 10 antibody-based detection. In another embodiment, it is demonstrated herein that dendrimers derived from antibodies conjugated with polymerization triggers/initiators can be produced. The amplification is specific, since no polymerization (high molecular weight band) is observed on the unconjugated antibody lane, while only low molecular weight monomers are observed.
FIG. 11 Bar code detection scheme. FIG. 11A the tag-erase-tag strategy works by alternating tagged oligonucleotides (6) that are complementary to the MUSE branches (3) and contain an additional 6-10 nucleotide long overhang. An erasers oligonucleotide (6') that is fully complementary to the sequence of the labelled oligonucleotide may be used to remove the labelled oligonucleotide by branch migration. This enables the addition of new labelled oligonucleotides (7) with different fluorophores. FIG. 11B in the label-quench-label technique, a first oligonucleotide for labeling comprising an additional 15 nucleotide "overhang" sequence, complementary to the branching sequence on the dendrimer, constitutes a first barcode label. "overhangs" are used to anchor dsDNA "quencher" labels comprising two overhangs, one overhang being complementary to the overhang of the first label and the second overhang being used to anchor the next label. "quencher" labeled oligonucleotides contain short-range quenchers, such as dabcyl and a fluorophore. Hybridization of a quencher label to the overhang of a prior label brings the quencher and the prior fluorophore into close proximity, so that the fluorescence of the first fluorophore is quenched and only the fluorophore contained on the "quencher" label can emit a signal. In this way, subsequent "quencher" labels can hybridize to each other n times to generate a barcode.
FIG. 12 marker-quench-marker example. The control shows that 3% laser power is required to detect transcripts labeled with dendrimers herein in the absence of "quencher" labeling. The results of quenching show that the laser power must be increased to 30% to detect the quenched signal. The
FIG. 13 Immunomase. Figure 13A GFP fusion proteins detected with anti-GFP antibody conjugated to MUSE trigger. FIG. 13B Drosophila embryos labeled with dendrimers (containing alexa-488 fluorophore as secondary label) and showing even-skipped exon.
Fig. 14 quantum dot labeling. Drosophila embryos labelled with dendrimers (containing QDot 655 labels) and showing the expression domain of the segmented gene even-skipeped.
Figure 15 immunization of MUSEs. FIG. 15A Drosophila embryos expressing GFP fusion protein (shown in green). FIG. 15B immunofluorescence assay (shown in red) using antibodies against GFP and MUSE amplified with alexa-594 fluorophore.
Steric hindrance of hairpin stability.
FIG. 16 steric hindrance. MCP conjugated to hairpins. (lane 1) amplification without initiator, while the unconjugated hairpin remained stable (lane 3). This indicates that MCP strongly interferes with the meta-stability of the hairpin. Furthermore, amplification was ineffective compared to the unconjugated hairpin (lane 4), whether in the absence (lane 1) or in the presence (lane 2) of the initiator.
Figure 17 evidence of the excellent stability of the very short MUSE hairpin. Very short MUSE hairpins (here 6nt footholds, 10nt stems, 16nt branches) retain their hairpin conformation under storage conditions so that they do not amplify without initiator (lane 1) but are fully amplified with initiator (lane 2) in the absence of rapid cooling. In contrast, HCR hairpins are not actually stored hairpins, so that they are non-specifically amplified (lane 3) and poorly amplified (lane 4) without rapid cooling to a hairpin conformation just prior to the experiment.
Figure 18 rapid amplification of the MUSE hairpin. The short MUSE hairpin (here 10nt foothold, 15nt stem, 12nt branch) was fully amplified within 45 minutes (lane 2) but not without initiator (lane 1).
Figure 19 very rapid amplification of the MUSE hairpin. The very short MUSE hairpin (here 6nt toehold, 8nt stem, 10nt branch) was fully amplified within 4 minutes (lane 2) but not without initiator (lane 1).
Detailed Description
As mentioned above, MUSE (multimode universal signal enhancement) is a nanotechnology that enables signal amplification upon detection of an analyte of interest. MUSEs are highly versatile compared to other detection and labeling techniques in the prior art. First, MUSEs can detect a variety of analytes and are almost completely independent of the detection scheme. Detection is routinely performed for the analyte in question (e.g., in situ hybridization of DNA and RNA analytes, immunohistochemistry of proteins and peptides). Second, MUSEs are widely compatible with different labels (e.g., fluorophores, quantum dots, and elemental labels). These two features allow detection of nearly all types of biological macromolecules, the signals of which are output by any number of selected labels. MUSE achieves this versatility through three component steps: detection, amplification and labeling. Traditional analyte detection schemes involve an unfavorable overlap of these compositional steps. For example, RNA in situ hybridization involves detection directly linked to the label output, usually in a linear fashion. PCR involves overlap between hybridizations for detection, repeated hybridization-conferring amplification, and overlap between amplification directly correlated with output signal via repeated hybridization steps. In contrast, MUSEs are separated into the constituent steps of detection, amplification and labeling by exploiting the properties of self-assembling nucleic acid polymers. Scale-up is achieved by the designed ability to self-assemble monomers into dendrimers. As a result, the generation and propagation of the output signal is separated from amplification in a manner not possible with conventional analyte detection schemes.
The dendrimer-dependent compositions and methods described can be used to reveal the presence of a variety of analytes, including specific nucleic acid molecules, small molecules, proteins, and peptides, thereby providing flexibility in detecting different biological moieties. The compositions and methods comprise: i) a trimolecular molecule consisting of a nucleic acid hairpin loop, stem and nucleic acid branches, further comprising an organic polymer "spacer" as shown in figure 1; ii) a polymerization trigger comprising a single-stranded nucleic acid oligonucleotide; iii) affinity ligands for analyte detection (i.e., analyte binding agents), the composition of which can vary between nucleic acid oligonucleotides, proteins, peptides, etc.
Trimonomers are a key innovation of this technology, enabling flexibility in labeling while maintaining monomer functionality. The generation of nucleic acid polymers from monomers has previously been achieved by hybrid strand reaction (HCR). However, while branched monomers are contemplated as a means to achieve secondary amplification (i.e., a squared multiplier of n analyte molecules), existing monomer detection systems consist only of nucleic acids. Nucleic acid hairpins may be unstable or locked (locked-in) based on a podite-branch interaction. Thus, the HCR method is severely limited by the underlying nucleic acid chemistry to limit the foothold-branch interactions.
To the inventors' knowledge, the second amplification with nucleic acid branched hairpins has not been successful. In addition, there is no existing format that uses two dendrimers for secondary label attachment and thus does not facilitate multimodal detection. In developing the compositions and methods, the inventors have also discovered that the rigidity of the nucleic acid backbone reduces the efficiency of secondary label hybridization, highlighting another limitation of nucleic acid-branched polymers.
To correct for the limitations of branched nucleic acid polymers, an important innovation was the development of an organic polymer "spacer" between the stem and the nucleic acid branches of the nucleic acid hairpin. The spacer minimizes interactions between the foothold and the branches, optimizes hairpin stability, minimizes steric hindrance during hybridization, alters the chemistry of the monomers, and can be further functionalized (e.g., by selecting a photolyzable spacer or a hydrophilic spacer). In addition, the spacer separates the stem and branch of the dendrimer by providing greater flexibility and freedom of movement to the branch, thereby limiting steric hindrance and other potential interactions between the stem of the polymer and the label. The design advantages of MUSE compared to HCR provide a very superior approach.
Robust tolerance for variable reagent purity (robust tolerance)
For example, branched nucleic acid strategies (e.g., HCR) typically require DNA oligonucleotides of nearly 100% purity because truncated monomers may terminate the reaction. If the monomer of 1/10 is truncated, an average of 10 units will result in the polymer ceasing to grow. Only very detailed denaturing PAGE electrophoresis allows purity levels approaching 100%. However, in electrophoretic purification, molecules may be attached to the monomeric oligomer. The attachment chemistry (amino, thiol) of certain alexa fluorophores is strongly influenced by the reagents (urea, ammonium persulfate) used during PAGE denaturation, making it impossible to have both 100% pure oligonucleotides and 100% conjugated (labeled) oligonucleotides. This is a disadvantage of the existing branched nucleic acid techniques and is a direct result of the attachment of the label to the oligonucleotide not being separated from the amplification step.
MUSE provides a solution by separating magnification and labeling. By denaturing the PAGE, the dendrimer can be easily purified. The oligonucleotides we used as labels can be provided by HPLC with a purity of about 80%. In most cases, the truncated label is aligned with the full-length label, thereby enabling robust tolerance to variable purities of reagents. In addition, since the MUSE-labeled oligonucleotide already has a minimum length, the truncated oligonucleotide may not hybridize at all, thereby preventing premature termination of the branching reaction. Thus, truncated labeled oligonucleotides do not affect signal amplification as significantly as truncated monomers.
Reduction of marking costs
Another advantage of MUSE is that it separates monomer costs from labeling costs. Traditional branched nucleic acid techniques (e.g., HCR) involve long and expensive monomers. The necessity of PAGE purification further affects yield. A serious disadvantage of this method is that the required attachment of chemical modifications and labeling molecules is still not achieved due to the subsequent harsh purification steps required. Separating the monomer and tag syntheses also helps to increase yield, which also reduces overall cost.
The disadvantages of conventional branched DNA techniques are exacerbated when groups of labels or multiple labels need to be studied. Traditional branched DNA techniques (e.g. HCR) require a complete detection-amplification-labeling system for any alexa-fluor that is intended to be used. In contrast, MUSE controls costs by separating the cost of attaching chemical modifications and labels from the cost of hairpins. By keeping the magnification system constant, grouped labeling or multiple labeling can be achieved by label exchange. This is particularly advantageous in view of the additional costs associated with the initiator presenting molecules. For MUSE, the initiator presenting molecule is associated with an amplification system. Changing the color of alexa fluor (or ultimately changing the chemistry of the label used) using traditional branched DNA techniques (e.g. HCR) requires a completely new detection-amplification-labeling system.
Advantages of PEG spacer
The polymeric PEG spacer further serves to separate the magnification and labeling by disrupting the continuity of the DNA phosphate backbone. This increases flexibility at the branch-hairpin bend point and minimizes potential base-pairing interactions between the foothold and the branch. Instead of attempting to engineer an engineered dendron (limited by the available nucleic acid design space) consisting of only DNA, the MUSE branching can be altered without modifying the hairpin sequence (and vice versa). This greatly simplifies the design of the MUSE architecture, making it possible to imagine an architecture with hundreds of runs in parallel. Furthermore, it allows the optimization of hairpin and branch sequences independently of each other, thereby allowing the design of optimal hairpins and optimal branches. This is not always possible without spacers.
Label switching
The possibility of post-amplification label exchange is an important advantage of MUSEs compared to traditional branched DNA techniques. Sequence barcode schemes (e.g., seqfash or merish) are becoming increasingly popular in view of their ability to simultaneously analyze thousands of targets. In seqFISH, HCR is used to provide signal amplification. Between barcode rounds, each HCR polymer must be digested with dnase. Thus, each round takes about 24 hours.
In contrast, for example with our label-rub-label approach, the MUSE label can also be removed from the branches by reducing the salt concentration in the buffer or increasing the temperature, or using reagents that reduce the hybridization energy (e.g. formamide). The MUSE labels can be removed and swapped for approximately 2 hours per cycle. This causes a significant shortening of the procedure, since the barcode scheme requires many rounds (up to 30 rounds). Another example of label switching would involve a multimodal label switching example. Here, the user can start the experiment with the fluorescent marker to obtain a high resolution image of his sample, and then rapidly exchange the fluorescent marker with the MCP to obtain highly multiplexed data for the same sample.
Metastability of
Compared to other branched DNA technologies including HCR, MUSE hairpins have significant thermal stability advantages. HCR monomers are described as "metastable" (i.e., their hairpin configuration can be maintained in solution at their use concentration for a considerable period of time). However, over time, particularly during storage and transportation, they give up hairpin secondary structures and adopt a more stable and thermodynamically favored homodimer configuration.
The inventors found that short MUSE hairpins, including very short MSUE hairpins (6-10 nucleotide footholds, less than 15 nucleotide stems), exhibit different and superior chemical characteristics. These MUSE hairpins exceed the metastable state because the only conformation they adopt is the hairpin structure. Some branched DNA techniques (e.g. HCR) require rapid cooling of the hairpin prior to the experiment to ensure formation. This is achieved by: the homodimers were denatured at 95 ℃ for 2-5 minutes and then cooled to room temperature for 30 minutes. The latest MUSE hairpin can be used directly from storage. This change saves more than 18 hours over the HCR reaction in combination with the increase in amplification rate described below. This is also important to the user as it makes MUSE a technology that can be used within a work day.
Speed of amplification
The HCR polymer takes about 12 hours to reach its final size. Current MUSE systems take 1 hour to 15 minutes.
Described herein are assemblies comprising at least two molecules (each comprising a nucleic acid and an organic polymer) and a trigger molecule comprising a nucleic acid, wherein the at least two molecules and the trigger molecule are configured for self-assembly polymerization, further wherein each molecule comprises one or more sequences complementary to another molecule. In other embodiments, each of the at least two molecules comprises a nucleic acid hairpin, a nucleic acid stem, a binding branch, an extension branch. In other embodiments, the nucleic acid trigger comprises an analyte binding agent.
Further described herein are assemblies comprising at least two molecules and at least one nucleic acid trigger coupled to an analyte binding agent, wherein each molecule comprises a nucleic acid hairpin, a nucleic acid stem, a nucleic acid branch (including binding and extension branches), and an organic polymer, and further wherein the nucleic acid hairpin sequence of at least one first molecule is complementary to the nucleic acid binding branch sequence of at least one second molecule, and wherein the nucleic acid hairpin sequence of at least one second molecule is complementary to the nucleic acid binding branch sequence of at least one first molecule, wherein the nucleic acid trigger is complementary to the nucleic acid stem and binding branch of at least one first molecule. In other embodiments, the hairpin sequence and the binding branch sequence are about 6-10 nucleotides. In other embodiments, the hairpin sequence and the binding branch sequence are about 10-24 nucleotides. For example, hairpin sequences and binding branches comprise 6, 7, 8, 9 or 10 nucleotides. In other embodiments, the hairpin sequence and the binding branch sequence are about 11-13 nucleotides. In other embodiments, the extended branch comprises 10-25 nucleotides, including 16-25 nucleotides. In other embodiments, the extended branch comprises about 10-20 nucleotides. For example, the extension branch comprises 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. In other embodiments, the extended branch comprises about 13-16 nucleotides. In other embodiments, the nucleic acid trigger comprises about 12-48 nucleotides. In other embodiments, the nucleic acid trigger comprises about 34-38 nucleotides. In other embodiments, the stem is about 6-15 nucleotides. In other embodiments, the nucleic acid stem comprises about 12-30 nucleotides. For example, the nucleic acid stem comprises 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, the nucleic acid stem comprises about 24 nucleotides. In other embodiments, the organic polymer comprises polyethylene glycol. An example of an assembly of the above sequences configured for self-assembly polymerization comprises at least two molecules, each molecule comprising a 6 nucleotide hairpin sequence and a binding branch, a 10 nucleotide stem of nucleic acid, and a 16 nucleotide extension branch, wherein each molecule comprises one or more sequences complementary to another molecule. Another example of an assembly of the above sequences configured for self-assembly polymerization comprises at least two molecules, each molecule comprising a hairpin sequence of 8 nucleotides and a binding branch, a nucleic acid stem of 10 nucleotides, and an extension branch of 18 nucleotides, wherein each molecule comprises one or more sequences complementary to another molecule. Additional examples of the above sequence assemblies configured for self-assembly polymerization comprise at least two molecules, each molecule comprising a hairpin sequence of 10 nucleotides and a binding branch, a nucleic acid stem of 15 nucleotides, and an extension branch of 25 nucleotides, wherein each molecule comprises one or more sequences complementary to another molecule.
In other embodiments, the polyethylene glycol comprises a length of about 16 to 20 carbons. In other embodiments, the polyethylene glycol comprises a length of about 2 nm. In other embodiments, the polyethylene glycol comprises a length of about 3-8 base pairs. In other embodiments, the polyethylene glycol comprises a length of about 4 base pairs. In various embodiments, the polymer links the nucleic acid stem to the branch. In various embodiments, the at least two molecules are each a monomer comprising: a hairpin sequence of about 6-10 nucleotides, a nucleic acid stem of about 6-15 nucleotides, a binding branch of about 6-10 nucleotides, an extension branch of about 10-25, including 16-25 nucleotides, and a polymer of about 16-20 carbons in length. In various embodiments, the at least two molecules are each a monomer comprising a hairpin sequence of about 11-13 nucleotides, a nucleic acid stem of about 22-26 nucleotides, a binding branch of about 11-13 nucleotides, an extension branch of about 13-16 nucleotides, and a polymer of about 16-20 carbons in length. In other embodiments, the analyte of interest comprises a nucleic acid. In other embodiments, the analyte of interest comprises a small molecule. In other embodiments, the analyte of interest comprises a polymer. In other embodiments, the analyte of interest comprises a peptide or protein.
In other embodiments, the analyte binding agent comprises a polynucleotide. In other embodiments, the analyte binding agent comprises a peptide or protein. In other embodiments, the analyte binding agent comprises an antibody. In other embodiments, the analyte binding agent comprises a peptide or protein. In other embodiments, the analyte binding agent comprises a peptide nucleic acid. In other embodiments, the analyte binding agent comprises a locked nucleic acid.
In other embodiments, the assembly comprises a labeled polynucleotide complementary to the extended branch. In other embodiments, the labeled polynucleotide comprises a fluorophore, chromophore, chromogen, quantum dot, fluorescent microsphere, nanoparticle, elemental label, metal chelating polymer, barcode, and/or sequence barcode, including any number of other labeling reagents known to one of ordinary skill in the art.
In various embodiments, the fluorophore comprises fluorescein, rhodamine, Alexa Fluors, DyLight Fluors, ATTO dyes, or any analog or derivative thereof. In some embodiments, the markers of the present invention include, but are not limited to, fluorescein and chemical derivatives of fluorescein; eosin; a carboxyfluorescein; fluorescein Isothiocyanate (FITC); fluoroesceinamidite (fam); erythrosine; rose bengal; fluorescein secreted by pseudomonas aeruginosa bacteria; methylene blue; a laser dye; rhodamine dyes (e.g., rhodamine 6G, rhodamine B, rhodamine 123, auramine O, Sulforhodmine 101, acid pink and Texas Red). In various embodiments, markers of the invention include Alexa Fluor family of fluorescent dyes, including Alexa-350, Alexa-405, Alexa-430, Alexa-488, Alexa-500, Alexa-514, Alexa-532, Alexa-546, Alexa-555, Alexa-568, Alexa-594, Alexa-610, Alexa-633, Alexa-647, Alexa-660, Alexa-680, Alexa-700, or Alexa-750.
In various embodiments, the quantum dots comprise semiconductor nanocrystals. In various embodiments, the semiconductor is composed of elements from groups II-VI, III-V, and IV of the periodic Table. In various embodiments, the quantum dots include ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, GaN, GaP, GaAs, GaSb, InP, InAs, InSb, AlS, AlP, AlAs, AlSb, PbS, PbSe, Ge, and Si, as well as ternary and quaternary mixtures thereof. In various embodiments, the quantum dots include an overcoat of a semiconductor having a larger band gap. In various embodiments, the semiconductor nanocrystals are characterized by their uniformityIs of nanometer size. By "nano" size is meant less than about
In various embodiments, the assembly further comprises at least two additional molecules and a linker molecule comprising a nucleic acid sequence address (address) complementary to the one or more extended branches of the at least two initial molecules and a secondary trigger for the at least two additional molecules, wherein the at least two additional molecules and the linker molecule are configured for self-assembly polymerization. In various embodiments, the initial at least two molecules and trigger are a first self-assembling polymerization, and the additional at least two molecules and linker molecules are a second self-assembling polymerization. In various embodiments, the first and second self-assembly polymerizations are second order amplifications. In other embodiments, the assembly comprises a labeled polynucleotide complementary to the extended branches of the at least two additional molecules.
Further described herein are kits having an assembly comprising at least two molecules, wherein each molecule comprises a nucleic acid hairpin, a nucleic acid stem, nucleic acid branches (including binding and extension branches), and an organic polymer, and further wherein the nucleic acid hairpin sequence of at least one first molecule is complementary to the nucleic acid binding branch sequence of at least one second molecule, and wherein the nucleic acid hairpin sequence of at least one second molecule is complementary to the nucleic acid binding branch sequence of at least one first molecule, and instructions for using the kit, wherein the nucleic acid trigger is complementary to the nucleic acid stem and binding branches of at least one first molecule. In various embodiments, at least two molecules and a trigger are configured for self-assembly polymerization. In various embodiments, the assembly is capable of producing a polymer comprising 25-50 units of the first molecule, the second molecule, and the nucleic acid trigger sub-assembly, a polymer comprising about 50-100 units of the first molecule, the second molecule, and the nucleic acid trigger sub-assembly, a polymer comprising about 100-150 units of the first molecule, the second molecule, and the nucleic acid trigger sub-assembly, a polymer comprising about 150-200 units of the first molecule, the second molecule, and the nucleic acid trigger sub-assembly, or a polymer comprising more than 200 units of the first molecule, the second molecule, and the nucleic acid trigger sub-assembly.
In various embodiments, the kit further comprises introducing the additional at least two molecules and a linker molecule comprising a nucleic acid sequence address complementary to the one or more extended branches of the initial at least two molecules and a secondary trigger for the additional at least two molecules, wherein the additional at least two molecules and the linker molecule are configured for self-assembly polymerization. In various embodiments, the assembly can be capable of producing a polymer comprising 25-50 units of additional first molecule, second molecule, and linker assembly, a polymer comprising about 50-100 units of additional first molecule, second molecule, and linker assembly, a polymer comprising about 100-150 units of additional first molecule, second molecule, and linker assembly, a polymer comprising about 150-200 units of additional first molecule, second molecule, and linker assembly, or a polymer comprising more than 200 units of additional first molecule, second molecule, and linker assembly.
In other embodiments, the kit comprises labeled polynucleotides complementary to the extended branches of the initial at least two molecules and/or the additional at least two molecules. In various embodiments, the kit comprises two or more marker polynucleotides, each marker polynucleotide being complementary to one or more extended branches of the initial at least two molecules and/or the additional at least two molecules.
Described herein is a method of polymerization, the method comprising: adding at least two molecules, each comprising a nucleic acid and an organic polymer; further adding trigger molecules comprising nucleic acids, wherein each molecule comprises one or more sequences complementary to another molecule, and triggering self-assembly polymerization. In other embodiments, the at least two molecules each comprise a nucleic acid hairpin, a nucleic acid stem, a binding branch, an extension branch. In other embodiments, the nucleic acid trigger comprises an analyte binding agent. In other embodiments, the detectable signal is generated by binding a labeled polynucleotide complementary to another molecule, wherein the labeled polynucleotide comprises a labeling agent. In other embodiments, the at least two molecules each comprise a nucleic acid hairpin, a nucleic acid stem, a nucleic acid branch (including binding and extension branches), and an organic polymer, and further wherein the nucleic acid hairpin sequence of at least one first molecule is complementary to the nucleic acid binding branch sequence of at least one second molecule, and wherein the nucleic acid hairpin sequence of at least one second molecule is complementary to the nucleic acid binding branch sequence of at least one first molecule, and the at least one nucleic acid trigger is coupled to the analyte binding agent, wherein the nucleic acid trigger is complementary to the binding branch and the nucleic acid stem of at least one first molecule. In other embodiments, generating a detectable signal comprises binding a labeled polynucleotide to the extension branch, wherein the labeled polynucleotide comprises a labeling agent. In other embodiments, the labeling reagents include fluorophores, chromophores, chromogens, quantum dots, fluorescent microspheres, nanoparticles, elemental labels, metal chelating polymers, barcodes, and/or sequence barcodes, including any number of other labeling reagents known to those of ordinary skill in the art. In various embodiments, the polymer links the nucleic acid stem to the branch. In various embodiments, the at least two molecules are each a monomer comprising: a hairpin sequence of about 6-10 nucleotides, a nucleic acid stem of about 6-15 nucleotides, a binding branch of about 6-10 nucleotides, an extension branch of about 10-25, including 16-25 nucleotides, and a polymer of about 16-20 carbons in length. In various embodiments, each of the at least two molecules is a monomer comprising a hairpin sequence of about 11-13 nucleotides, a nucleic acid stem of about 12-30 nucleotides, a binding branch of about 11-13 nucleotides, an extension branch of about 13-16 nucleotides, and a polymer of about 16-20 carbons in length. In other embodiments, the nucleic acid trigger comprises about 12-48 nucleotides. In various embodiments, the polymer links the nucleic acid stem to the branch. In various embodiments, the at least two molecules are added in a ratio to the nucleic acid trigger of about 1:25, 1:50, 1:100, 1:200, and all ranges therebetween.
For example, as shown in FIG. 1, the
In various embodiments, the method further comprises introducing at least two additional molecules, and a linker molecule comprising a nucleic acid sequence address complementary to the one or more extended branches of the at least two initial molecules and a secondary trigger for the at least two additional molecules, wherein the at least two additional molecules and the linker molecule are configured for self-assembly polymerization. In various embodiments, the initial at least two molecules and trigger are a first self-assembling polymerization and the introduction of the additional at least two molecules and linker molecules is a second self-assembling polymerization. In various embodiments, the first and second self-assembly polymerizations are second order amplifications. In other embodiments, the assembly comprises a labeled polynucleotide complementary to the extended branches of the at least two additional molecules. For example, the use of
In various embodiments, the self-assembling polymerization comprises incubating the at least two molecules and the trigger molecule for 1 minute to 60 minutes, 1 hour to 12 hours, 12 hours to 24 hours, 24 hours or more. In various embodiments, this includes incubation for 1 minute, 2 minutes, 3 minutes, 4 minutes, 5-10 minutes, 10-30 minutes, 30-60 minutes, 1-2 hours, or more than 2 hours. In various embodiments, the incubation is for 2 hours to 24 hours. In various embodiments, wherein the additional linker molecule comprises an address complementary to the one or more extension branches and a trigger for the additional addition of the at least two molecules, the secondary incubation of the linker molecule and the additional addition of the at least two molecules is from 1 minute to 60 minutes, from 1 hour to 12 hours, from 12 hours to 24 hours, 24 hours or more. In various embodiments, this includes incubation for 1 minute, 2 minutes, 3 minutes, 4 minutes, 5-10 minutes, 10-30 minutes, 30-60 minutes, 1-2 hours, or more than 2 hours. In various embodiments, the incubation is for 2 hours to 24 hours.
Described herein are methods of polymerization comprising adding at least two molecules (each comprising a nucleic acid and an organic polymer) to a material comprising at least one trigger molecule (comprising a nucleic acid), and triggering self-assembly polymerization, wherein each molecule comprises one or more sequences complementary to another molecule. In other embodiments, the at least two molecules each comprise a nucleic acid hairpin, a nucleic acid stem, a binding branch, an extension branch. In other embodiments, the at least one nucleic acid trigger molecule comprises an analyte binding agent. In other embodiments, the at least two molecules each comprise a nucleic acid hairpin, a nucleic acid stem, a nucleic acid branch (including binding and extension branches), and an organic polymer, and further wherein the nucleic acid hairpin sequence of at least one first molecule is complementary to the nucleic acid binding branch sequence of at least one second molecule, and wherein the nucleic acid hairpin sequence of at least one second molecule is complementary to the nucleic acid binding branch sequence of at least one first molecule, and the at least one nucleic acid trigger is coupled to the analyte binding agent, wherein the nucleic acid trigger is complementary to the binding branch and the nucleic acid stem of at least one first molecule.
In various embodiments, the material comprises a substrate, such as a solid substrate or a liquid substrate. In various embodiments, the solid substrate comprises glass, a tissue culture surface, or any similar substrate known to those skilled in the art. In various embodiments, at least one trigger molecule is attached to a solid surface, e.g., a plurality of one or more trigger molecules are deposited on the surface (e.g., an array). In various embodiments, at least one trigger molecule is dispersed within the liquid base.
In various embodiments, the material includes an analyte of interest. In various embodiments, the material is a biological sample, including a whole mount, a tissue slice, one or more tissues and cells, and the like. In various embodiments, the analyte of interest is bound to an analyte binding agent of a trigger molecule. In various embodiments, the biological sample is deposited on a surface of a solid substrate. In various embodiments, the biological sample is dispersed within the liquid substrate.
In other embodiments, the method comprises generating a detectable signal by binding a labeled polynucleotide complementary to another molecule, wherein the labeled polynucleotide comprises a labeling agent. In other embodiments, generating a detectable signal comprises binding a labeled polynucleotide to the extension branch, wherein the labeled polynucleotide comprises a labeling agent. In other embodiments, the method comprises tagging polynucleotides complementary to the extended branches of the initial at least two molecules and/or the additional at least two molecules. In various embodiments, the method comprises more than two marker polynucleotides, each marker polynucleotide being complementary to one or more extended branches of the initial at least two molecules and/or the additional at least two molecules.
In various embodiments, the method is used in combination with detection and/or signal amplification or both of nucleic acid sequences in solution. In various embodiments, the method is used in combination with detection and/or signal amplification or both of nucleic acid sequences in a solid phase (ISH). In various embodiments, the method is used in combination with the detection and/or signal amplification or both of small molecules in solution. In various embodiments, the method is used in combination with detection and/or signal amplification or both of small molecules in a solid phase. In various embodiments, the method is used in combination with detection and/or signal amplification or both of peptides and proteins in solution. In various embodiments, the methods are used in combination with detection and/or signal amplification or both of peptides and proteins in the solid phase. In various embodiments, the method is used in combination with signal amplification (e.g., ELISA and immunofluorescence) from the first antibody. In various embodiments, the method is used in combination with signal amplification (e.g., ELISA and immunofluorescence) from a second antibody.
Also described herein are methods that include providing a sample comprising an analyte of interest. In various embodiments, the method comprises: adding at least two molecules, each molecule comprising a nucleic acid and an organic polymer; further adding trigger molecules comprising nucleic acids, wherein each molecule comprises one or more sequences complementary to another molecule, and triggering polymerization. In other embodiments, the method comprises combining the sample with a trigger and adding at least two molecules, each molecule comprising a nucleic acid and an organic polymer, further adding a trigger molecule comprising a nucleic acid, and triggering polymerization, wherein each molecule comprises one or more sequences complementary to another molecule.
In other embodiments, the at least two molecules each comprise a nucleic acid hairpin, a nucleic acid stem, a binding branch, an extension branch. In various embodiments, the polymer links the nucleic acid stem to the branch. In other embodiments, the nucleic acid trigger comprises an analyte binding agent. In other embodiments, the method comprises generating a detectable signal by binding a labeled polynucleotide complementary to another molecule, wherein the labeled polynucleotide comprises a labeling agent.
In various embodiments, the method further comprises introducing at least two additional molecules, and a linker molecule comprising a nucleic acid sequence address complementary to the one or more extended branches of the at least two initial molecules and a secondary trigger for the at least two additional molecules, wherein the at least two additional molecules and the linker molecule are configured for self-assembly polymerization. In various embodiments, the initial at least two molecules and trigger are a first self-assembling polymerization and the introduction of the additional at least two molecules and linker molecules is a second self-assembling polymerization. In various embodiments, the first and second self-assembly polymerizations are second order amplifications. In other embodiments, the assembly comprises a labeled polynucleotide complementary to the extended branches of the at least two additional molecules.
In other embodiments, the method comprises generating a detectable signal by binding a labeled polynucleotide complementary to another molecule, wherein the labeled polynucleotide comprises a labeling agent. In other embodiments, generating a detectable signal comprises binding a labeled polynucleotide to the extension branch, wherein the labeled polynucleotide comprises a labeling agent. In other embodiments, the method comprises tagging polynucleotides complementary to the extended branches of the initial at least two molecules and/or the additional at least two molecules. In various embodiments, the method comprises more than two marker polynucleotides, each marker polynucleotide being complementary to one or more extended branches of the initial at least two molecules and/or the additional at least two molecules.
In various embodiments, the method includes generation of barcode sequences. In various embodiments, the method comprises adding a first oligonucleotide comprising an overhang sequence, a signal label, and a sequence complementary to an extension branch, and introducing a dsDNA oligonucleotide comprising a quencher label comprising two overhangs, a first dsDNA overhang complementary to the overhang of the first oligonucleotide, and a second dsDNA overhang. In various embodiments, the dsDNA oligonucleotide quencher labels include short-range quenchers, such as dabcyl and a fluorophore. In various embodiments, one or more dsDNA quencher labels can be hybridized n times to each other to generate a barcode.
In various embodiments, the method comprises at least two oligonucleotides, including a tag oligonucleotide and an eraser oligonucleotide. In various embodiments, the labeled oligonucleotide comprises a first overhang sequence and a second overhang sequence that are complementary to the extension branch. In various embodiments, the eraser oligonucleotide is a sequence complementary to the tag oligonucleotide. In various embodiments, the method comprises performing the erasing by introducing an eraser oligonucleotide to the analyte labeled with a labeled oligonucleotide, clearing the eraser-labeled dsDNA oligonucleotide dimers, and adding additional labeled oligonucleotides. In various embodiments, the mark-erase-mark cycle is repeated n times to produce a barcode.
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