Modular system and method for quantifying target polynucleotides using barcoded oligonucleotides with enhanced flexibility and error recovery

Abstract

The present invention relates to a method for quantifying target polynucleotides, particularly RNA molecules such as 16S rRNA, through a process that converts the abundance of target molecules into the abundance of predefined barcoded oligonucleotides The invention enables reduced nonspecific binding, protocol flexibility with pausing and re-entry points, and versatile, optionally multiplexed data acquisition including flow-cytometry-like or imaging-based measurements.

Description

[0001] Modular System and Method for Quantifying Target Polynucleotides Using Barcoded Oligonucleotides with Enhanced Flexibility and Error Recovery

[0002] The present invention relates to the field of polynucleotide quantification, particularly RIMA quantification. More specifically, the invention provides a method for converting the abundance of target polynucleotides, such as RNA molecules, into the abundance of predefined, barcoded oligonucleotides, enabling efficient storage, detection, and quantification.

[0003] Quantifying RNA molecules is often challenging due to several factors. The secondary structure and steric hindrance of RNA can affect hybridization and probe accessibility, while the chemical instability of RNA makes it prone to degradation. Additionally, the rigidity of workflows, where consecutive steps in RNA selection and labelling cannot be paused or repeated, limits flexibility. Another issue is nonspecific binding, which leads to background noise and reduced sensitivity. Furthermore, the limited detection modalities often restrict quantification to microscopy-based methods.

[0004] Existing approaches, such as those described Islam et al., 2013 address these limitations by introducing unique molecular identifiers (UMIs), which tag each RNA molecule with a distinct barcode. This helps overcome nonspecific binding by providing molecule-specific labeling, reducing background noise and increasing sensitivity.

[0005] However, this RNA quantification method needs an improvement because it relies heavily on sequencing-based detection to quantify the barcoded molecules, which can be resourceintensive and limits real-time or non-sequencing-based applications. Additionally, the workflow does not explicitly address the flexibility to pause or repeat steps, making it less adaptable for scenarios requiring modular processing. Furthermore, while UMIs reduce amplification noise, they do not resolve issues related to the chemical instability of RNA or provide mechanisms to release and quantify barcodes independently of the RNA-probe complex. These gaps highlight the need for a method that integrates modularity, improved barcode handling, and compatibility with diverse detection modalities beyond sequencing.

[0006] The objective of the invention is to address these limitations and provide a method for RNA quantification that is modular, flexible, and efficient. The invention aims to improve workflow adaptability by allowing steps such as barcode attachment, removal of unbound components, and barcode release to be performed independently or iteratively. Additionally, it seeks to reduce the reliance on sequencing-based detection by enabling alternative quantification methods through the release and measurement of barcodemodules. By addressing issues such as nonspecific binding, RIMA instability, and inflexible workflows, the invention provides a robust solution for accurate and sensitive RNA quantification across a wide range of applications.

[0007] This is achieved by claim 1 which discloses a method of quantifying target polynucleotides comprising the steps of: (a) forming a conversion complex by attaching the target polynucleotide (analyte) to a carbon copy compound that includes a barcode module, a linkage module, and hybridization-specific design for the target; (b) removing unbound carbon copy compounds to reduce background noise and improve specificity; (c) releasing the barcode module from the conversion complex, enabling independent handling and measurement of the barcodes; and (d) quantifying the released barcode module to determine the abundance of the target polynucleotide. This modular and sequential workflow allows for increased flexibility, reduced background interference, and compatibility with multiple detection methods beyond sequencing.

[0008] The method, in more specific terms, comprises several steps. First, a conversion complex is formed by attaching a target polynucleotide to a carbon copy compound, which includes a barcode module for identification and quantification, and a linkage module with a handle sequence designed to specifically select the target polynucleotide. Next, the conversion complex is attached to a purification compound that contains a filter module (such as a filter column, or a surface-based filter module which may be a magnetic bead or another substrate) and a capture module (such as streptavidin) that binds to the attachment module of the capture compound (e.g., biotin). The unbound components are then removed through the filter module, isolating the conversion complex. After purification, the barcode module is released by cleaving a cleavage module (for example, a disulfide bond). Finally, the released barcode module is quantified using detection techniques such as fluorescence microscopy, flow cytometry, qPCR, or sequencing. This process enables the accurate quantification of the original target polynucleotide based on the measured abundance of the barcode modules.

[0009] In another aspect of the invention, the process of quantifying target polynucleotides may include a step of forming a conversion complex by attaching an analyte to a carbon copy compound. The formation of the conversion complex may be achieved by annealing in a thermocycler. Alternatively, the formation of the conversion complex may be performed by contacting the analyte and the carbon copy compound at room temperature. In another embodiment, the formation of the conversion complex may involve changing the melting temperature by the introduction or extraction of chaotropic or cosmotropic substances. The extraction of these chaotropic or cosmotropic substances may be performed through a semipermeable membrane.In exemplary implementations, the process may be carried out using defined buffers and preprocessing workflows. Suitable buffers include, for instance: (i) A storage buffer for surface reagents and surface incubation, comprising Tris-HCI, about 5 to 50 mM, at a pH of about 7.4 to 8.0, and NaCI, about 50 to 600 mM, optionally comprising 0.01% to 0.1% Tween-20, in an aqueous solution, wherein all incubations can be carried out using this buffer except for DNA origami incubation, and the buffer may also be used as a washing buffer to remove surface reagents from a substrate or slide. For example, this buffer may be Buffer A+, comprising 10 mM Tris-HCI pH 7.5, 100 mM NaCI and 0.05% Tween-20; (ii) A buffer for DNA origami applications, suitable for use in folding, incubation, selective elution during purification, or as a washing buffer, selected from the following options: 1-A buffer comprising Tris-HCI, about 5 to 50 mM, at a pH of about 7.4 to 8.0, MgCh about 7 to 20 mM, optionally comprising Tween-20 about 0.05% to 1% for folding applications, and comprising Tween-20 about 0.05% to 1% for incubation, elution, and washing applications, optionally comprising NaCI about 5 mM and EDTA about 1 mM, in an aqueous solution. 2- A buffer comprising NaCI, about 75 to 600 mM, and sodium citrate, about 7.5 to 60 mM, optionally comprising Tween-20 about 0.05% to 1% for folding applications, and comprising Tween-20 about 0.05% to 1% for incubation, elution, and washing applications, at a pH of about 7.0, in an aqueous solution. For example, this buffer may be Buffer B+, comprising 10 mM Tris-HCI pH 8.0, 10 mM MgCh and 1 mM EDTA; and (iii) A buffer for RNA applications, suitable for use in annealing, incubation, or selective elution during purification, comprising NaCI, about 75 to 600 mM, sodium citrate, about 7.5 to 60 mM, optionally comprising Tween-20 about 0.05% to 1% for incubation and elution applications, at a pH of about 7.0, in an aqueous solution. For surface incubation, the buffer may optionally comprise Denhardt's solution, about lx to 5x, and dextran, about 1% to 5%. For example, this buffer may be R.X20 buffer, comprising 4x SSC, 5% dextran sulfate, 0.1% Tween-20 and 5x Denhardt's solution. Exemplary preprocessing steps may include sample collection in nucleic-acid stabilizing buffer, mechanical lysis, column-based RNA purification with optional DNase treatment, and filtration through high-molecular-weight cutoff filters. Exemplary in-silico design workflows for selecting linkage modules comprise target sequence alignment, probe generation using DECIPHER DesignProbe algorithms, and analysis of predicted RNA accessibility, yielding 23-nt linkage sequences.

[0010] The types of targets for the process of quantifying target polynucleotides may comprise rRNA. In addition to rRNA, the targets may further comprise mRNA. In another embodiment, the targets may also include RNA in vitro transcribed from genomic DNA. In further embodiments, the target polynucleotide may be DNA, including genomic DNA, complementary DNA (cDNA), or DNA obtained by PCR amplification.The carbon copy compound used in the process of quantifying target polynucleotides may be a DNA oligonucleotide. The carbon copy compound may further comprise a barcode module for identification and quantification. Additionally, the carbon copy compound may include a linkage module. The linkage module may be a handle sequence. The handle sequence may be designed to specifically select RNA of one taxon out of the pool of all RNA molecules.

[0011] In certain embodiments, the invention is implemented using multi-analyte or multiplexed architectures, in which several target polynucleotides in the same sample are each hybridised to distinct carbon copy compounds. Each carbon copy compound may comprise a different cleavage module, enabling orthogonal and sequential release of corresponding barcode modules in separate processing steps. For example, a first subset of target analytes may be linked to carbon copy compounds containing a disulfide-based cleavage module, a second subset to carbon copy compounds containing a uracil-containing cleavage module, and a third subset to carbon copy compounds containing a hybridisationbased cleavage module comprising a TH / II / II* strand-displacement motif. Activation of a given cleavage module does not substantially affect the others.

[0012] In some embodiments, a two-handle capture configuration is used. In such cases, the analyte is simultaneously hybridised to (i) a carbon copy compound comprising a barcode module and a linkage module, and (ii) a capture compound comprising a second linkage module and an attachment module. Capture compounds suitable for this purpose are provided as Seq-ID Nos. 33-36. The attachment module hybridises or binds to a capture module of a purification compound, such as streptavidin-coated magnetic beads or a synthetic oligo-based capture module (e.g., Seq-ID No. 4), allowing physical separation of bound and unbound material.

[0013] Certain assay architectures involve stepwise assembly of the carbon copy compound during the workflow. For example, in the LF assay configuration, the analyte may be initially hybridised to the linkage-module strand and cleavage-module duplex (Seq-ID No. 31 and its associated II / II* elements). The barcode module (Seq-ID No. 30) may then be added in a subsequent hybridisation step performed on the immobilised purification compound. Release of the barcode module may be triggered by addition of a strand-displacement reagent such as Seq-ID No. 32.

[0014] Following cleavage of the barcode module, the released barcode may be immobilised on a solid support for quantification. Immobilisation may be achieved via thiol-maleimide coupling on maleimide-functionalised surfaces, via thiol-gold chemistry on gold beads or gold-coated substrates, or via click-chemistry groups such as DBCO-azide. Quantificationmay then be achieved with barcode-readout extensions (e.g., Seq-ID No. 3) attached to DNA origami metafluorophores, enabling single-molecule readout, DNA-PAINT microscopy, or flow-cytometry-like data acquisition.

[0015] Representative implementations of these multi-cleavage and two-handle capture workflows are disclosed in the in exemplary implementations, including a multiplex assay enabling sequential quantification of Listeria monocytogenes, Staphylococcus aureus, Pseudomonas aeruginosa, and Lactobacillus fermentum 16S rRNA from the same sample.

[0016] In some embodiments, the carbon copy compound comprises a barcode module, a cleavage module and a linkage module arranged on a single oligonucleotide backbone. For example, the carbon copy compound may have the general structure "barcode module - T - S-S - T - linkage module" or "barcode module - C6-S-S-C6 - linkage module", wherein the cleavage module comprises a bifunctional disulfide linker, such as a C6-thio modifier, that replaces a stretch of non-probe nucleotides (for example a " I I I I I " or "AAAAA" spacer). In other embodiments, the arrangement may be inverted, for example "linkage module - C6-S-S-C6 - barcode module" or "linkage module - S-S - barcode module". The barcode module and linkage module may each be a capture or handle sequence as described herein, while the disulfide-containing linker is placed between them to constitute the cleavage module.

[0017] In further embodiments, the barcode module and / or the linkage module additionally comprise an immobilization moiety, such as a biotin, a dibenzocyclooctyne (DBCO) group, an azide, or other click-chemistry compatible group, at the 5' or 3' end and / or internally. For example, the barcode module may be provided as a DBCO-modified oligonucleotide, while the linkage module is provided as a non-modified oligonucleotide, or vice versa.

[0018] The process of quantifying target polynucleotides may further include a step of removing unbound carbon copy compounds. The removal of these unbound compounds may be performed using size purification methods. Examples of size purification methods may include the use of SPRI beads, gel electrophoresis, or PEG purification. In another embodiment, the size purification method may involve high-performance liquid chromatography (HPLC).

[0019] The purification process may also be affinity-based. The affinity-based purification may comprise biotin-streptavidin binding, click chemistry, HIS-tag / Nickel-NTA interactions, or the SpyTag / SpyCatcher system. Alternatively, the affinity-based purification may use sequence-specific hybridization.The purification may also be achieved using physical separation means. In one embodiment, the physical separation means may be magnetic beads. In another embodiment, the physical separation means may be centrifuging beads.

[0020] The process of quantifying target polynucleotides may further comprise a step of releasing at least the barcode module from the conversion complex. The release of the barcode module may be achieved by cleavage of a disulfide bond. The cleavage of the disulfide bond may be performed using a reducing agent. The reducing agent may be TCEP or DTT.

[0021] Alternatively, the barcode module may be released by toehold-mediated strand displacement when the carbon copy compound comprises a hybridization complex of two oligonucleotides, where the barcode module and the linkage module are located on opposite strands in the hybridization complex. In another embodiment, the release of the barcode module may be achieved by disconnecting the linkage module-analyte bond using strand displacement.

[0022] In further embodiments, the cleavage module is not a disulfide bond but comprises a chemically cleavable nucleobase and / or a hybridization-based motif. For example, the cleavage module may comprise at least one uracil base located between the barcode module and the linkage module, which is cleavable by an uracil-specific endonuclease and glycosylase, for example a USER-type enzyme mix. In another example, the cleavage module may comprise at least one 8-oxo-7,8-dihydroguanine (8-oxoG) residue which can be converted into an abasic (AP) site by an N-glycosylase and subsequently cleaved at the AP site by an AP-lyase and / or a restriction endonuclease specifically recognizing the generated cleavage site. In yet another embodiment, the cleavage module comprises a hybridized DNA double helix formed by complementary sequences A and A*, which is flanked on at least one side by a toehold sequence TH or TH* that permits sequencespecific toehold-mediated strand displacement. In such configurations, the barcode module and the linkage module may be located on separate oligonucleotides, and the carbon copy compound may be provided as two hybridized oligonucleotides, for example in the form "linkage module - A" hybridized to "barcode module - TH - A*" or "TH - A - linkage module" hybridized to "barcode module - A*". The hybridized carbon copy compound can be obtained by mixing the respective oligonucleotides, each at about 1 mM in a suitable hybridization buffer comprising Tris-HCI, MgCh and EDTA, incubating the mixture at room temperature for about 2 hours, and purifying the resulting hybrid from non-hybridized oligonucleotides by a size- or affinity-based method such as agarose gel extraction, centrifugal ultrafiltration using a spin column with a molecular weight cut-off of about 3 kDa, or solid-phase reversible immobilization (SPRI) bead purification. Alternatively, the two-strand carbon copy compound can be assembled stepwise during the assay, forexample by first forming a conversion complex comprising the linkage-module strand "linkage module - A" hybridized to the analyte and, at a later stage in a purification compound comprising a filter module, capture module, capture compound, analyte and "linkage module - A*", adding the complementary barcode strand "barcode module - TH - A*" at a concentration of about 1 mM, incubating to allow hybridization, and washing with buffer to obtain a purification compound that comprises the fully assembled carbon copy compound in which the barcode module and the linkage module are connected via the A / A* duplex and the toehold sequence.

[0023] In exemplary workflows, the constituent oligonucleotides may be mixed at approximately 1 mM each in Buffer B+, incubated on a rotator for about 2 hours at room temperature, and purified from non-hybridized material by methods such as SPRI beads, agarose gel extraction or centrifugal ultrafiltration. In some workflows, assembly may occur directly on the purification compound, as illustrated by the Lactobacillus fermentum assay described in the exemplary implementations, wherein the barcode-containing strand is added only after analyte capture.

[0024] The process of quantifying target polynucleotides may further comprise a step of quantifying the released barcode module. The quantification may be performed by immobilizing fluorescent DNA origami barcodes that hybridize to the barcode module. In one embodiment, the quantification may be carried out by a single-molecule readout. The single-molecule readout may be achieved by fluorescent detection, or flow cytometry.

[0025] In another embodiment, the quantification may involve the use of barcoded DNA origami nanoparticles. The process may also use bulk quantification methods to quantify the barcode module. Bulk quantification methods may comprise impedance-based detection, capillary electrophoresis, qPCR, or sequencing. In a specific embodiment, the sequencing may be impedance-based sequencing.

[0026] In one variation, the conversion complex may further comprise a capture compound. The capture compound may include a linkage module and an attachment module. The capture compound may bind to the conversion complex via the linkage module. The attachment module of the conversion complex may attach to a capture module of a purification compound. The purification compound may comprise a filter module and a capture module.

[0027] The process may further include filtering out all the modules and compounds that are not attached to the filter module.The invention is also defined by the following numbered embodiments. These embodiments are abbreviated by the letter "P" followed by a number. When reference is herein made to a process embodiment, those embodiments are meant.

[0028] Pl. A process of quantifying target polynucleotides comprising a step of forming a conversion complex by attaching an analyte to a carbon copy compound.

[0029] P2. The process in accordance with the preceding embodiment, wherein the formation of the conversion complex is done by annealing in a thermocycler.

[0030] P3. The process in accordance with the preceding embodiment, wherein the formation of the conversion complex is done by contacting the analyte and a carbon copy compound at room temperature.

[0031] P4. The process in accordance with the preceding embodiments, wherein the formation of the conversion complex is done by changing the melting temperature by introduction or extraction of chaotropic or cosmotropic substances.

[0032] P5. The process in accordance with the preceding embodiment, wherein the extraction of chaotropic or cosmotropic substances is done through a semipermeable membrane.

[0033] P6. The process in accordance with any of the preceding embodiments, wherein the types of targets comprise rRNA.

[0034] P7. The process in accordance with the preceding embodiments, wherein the types of targets comprise mRNA.

[0035] P8. The process in accordance with the preceding embodiment, wherein the types of targets further comprise RNA in vitro transcribed from genomic DNA.

[0036] P9. The process in accordance with any of the preceding embodiments, wherein the carbon copy compound is a DNA oligonucleotide.

[0037] PIO. The process in accordance with embodiment Pl, wherein the carbon copy compound comprises a barcode module.

[0038] Pll. The process in accordance with the preceding embodiment, wherein the carbon copy compound further comprises a linkage module.P12. The process in accordance with the preceding embodiment, wherein the linkage module is a handle sequence.

[0039] P13. The process in accordance with the preceding embodiment, wherein the handle sequence is designed to specifically select RNA of one taxon out of the pool of all RNA molecules.

[0040] P14. The process in accordance with any of the preceding embodiments, further comprising a step of removing unbound carbon copy compounds.

[0041] P15. The process in accordance with the preceding embodiment, wherein the removal of non-bound carbon copy compounds can be done by size purification methods.

[0042] P16. The process in accordance with the preceding embodiment, wherein the size purification method comprises SPRI beads.

[0043] P17. The process in accordance with the preceding embodiment, wherein the size purification method is gel electrophoresis.

[0044] P18. The process in accordance with the preceding embodiment, wherein the size purification method is PEG purification.

[0045] P19. The process in accordance with the preceding embodiment, wherein the size purification method comprises HPLC.

[0046] P20. The process in accordance with any of the preceding embodiments, wherein the purification method is affinity-based.

[0047] P21. The process in accordance with the preceding embodiment, wherein the affinity-based purification comprises biotin-streptavidin binding.

[0048] P22. The process in accordance with the preceding embodiment, wherein the affinity-based purification comprises click chemistry.

[0049] P23. The process in accordance with the preceding embodiment, wherein the affinity-based purification comprises HIS-tag / Nickel-NTA.

[0050] P24. The process in accordance with the preceding embodiment, wherein the affinity-based purification comprises SpyTag / SpyCatcher.P25. The process in accordance with the preceding embodiment, wherein the affinity-based purification comprises sequence-specific hybridization.

[0051] P26. The process in accordance with the preceding embodiment, wherein the purification is done using physical separation means.

[0052] P27. The process in accordance with the preceding embodiment, wherein the physical separation means comprises magnetic beads.

[0053] P28. The process in accordance with the preceding embodiment, wherein the physical separation means comprises centrifuging beads.

[0054] P29. The process in accordance with any of the preceding embodiments, further comprising a step of releasing at least the barcode module from the conversion complex.

[0055] P30. The process in accordance with the preceding embodiment, wherein the release of the barcode module is achieved by cleavage of a disulfide bond.

[0056] P31. The process in accordance with the preceding embodiment, wherein the cleavage of the disulfide bond is performed using a reducing agent.

[0057] P32. The process in accordance with the preceding embodiment, wherein the reducing agent is TCEP.

[0058] P33. The process in accordance with the preceding embodiment, wherein the reducing agent is DTT.

[0059] P34. The process in accordance with any of the preceding embodiments, wherein the release of the barcode module is achieved by toehold-mediated strand displacement when the carbon copy compound comprises a hybridization complex of two oligonucleotides and wherein the barcode module and linkage module are located on opposite strands in the hybridization complex.

[0060] P35. The process in accordance with any of the preceding embodiments, wherein the release of the barcode module is achieved by disconnecting the linkage module-analyte bond using strand displacement.P36. The process in accordance with any of the preceding embodiments, further comprising a step of quantifying the released barcode module.

[0061] P37. The process in accordance with the preceding embodiment, wherein the quantification of the barcode module is performed by immobilizing fluorescent DNA origami barcodes that hybridize to the barcode module.

[0062] P38. The process in accordance with the preceding embodiment, wherein the quantification of the barcode module is performed by single-molecule readout.

[0063] P39. The process in accordance with the preceding embodiment, wherein single-molecule readout is achieved by fluorescent detection.

[0064] P40. The process in accordance with the preceding embodiment, wherein single-molecule readout is achieved by DNA-PAINT microscopy.

[0065] P41. The process in accordance with the preceding embodiment, wherein single-molecule readout is achieved by flow cytometry.

[0066] P42. The process in accordance with any of the preceding embodiments, wherein the quantification of the barcode module is performed using barcoded DNA origami nanoparticles.

[0067] P43. The process in accordance with any of the preceding embodiments, wherein bulk quantification methods are used for quantifying the barcode module.

[0068] P44. The process in accordance with the preceding embodiment, wherein bulk quantification methods comprise impedance-based detection.

[0069] P45. The process in accordance with the preceding embodiment, wherein bulk quantification methods comprise capillary electrophoresis.

[0070] P46. The process in accordance with the preceding embodiment, wherein bulk quantification methods comprise qPCR.

[0071] P47. The process in accordance with the preceding embodiment, wherein bulk quantification methods comprise sequencing.P48. The process in accordance with the preceding embodiment, wherein sequencing comprises impedance-based sequencing.

[0072] P49. The process in accordance with any of the preceding embodiments, wherein the conversion complex further comprises a capture compound.

[0073] P50. The process in accordance with the preceding embodiment, wherein the capture compound comprises a linkage module and an attachment module.

[0074] P51. The process in accordance with the preceding embodiment, wherein the capture compound binds to the conversion complex via the linkage module.

[0075] P52. The process in accordance with the preceding embodiment, wherein the attachment module of the conversion complex attaches to a capture module of a purification compound.

[0076] P53. The process in accordance with the preceding embodiment, wherein the purification compound comprises a filter module and a capture module.

[0077] P54. The process in accordance with the preceding embodiment, wherein all the modules and compounds that are not attached to a filter module are filtered out.

[0078] P55. The process in accordance with any of the preceding embodiments, wherein the target polynucleotides comprise 16S rRNA of at least one bacterial taxon, in particular at least one of Listeria monocytogenes, Staphylococcus aureus, Pseudomonas aeruginosa, Lactobacillus fermentum, Salmonella enterica, Bacillus subtilis or Enterococcus faecalis.

[0079] P56. The process in accordance with any of the preceding embodiments, wherein the analyte is obtained from a stool sample collected in a tube comprising a nucleic acid stabilization solution, such as a DNA / RNA preservation buffer, and stored at or below -20 °C prior to RNA isolation.

[0080] P57. The process in accordance with embodiment P56, wherein RNA isolation comprises mechanical lysis in a bead-containing lysis tube, followed by column-based RNA purification including DNase treatment and filtration through a high-molecular-weight cutoff filter to provide a purified RNA primary sample.

[0081] P58. The process in accordance with any of the preceding embodiments, wherein the cleavage module of the carbon copy compound comprises a uracil base and the release ofthe barcode module is achieved by treatment with a uracil-specific endonuclease and glycosylase and / or a USER-type enzyme mixture.

[0082] P59. The process in accordance with any of the preceding embodiments, wherein the cleavage module of the carbon copy compound comprises an 8-oxo-7,8-dihydroguanine (8-oxoG) and the release of the barcode module is achieved by converting 8-oxoG into an apurinic / apyrimidinic (AP) site using an N-glycosylase and subsequently cleaving the AP site using an AP-lyase.

[0083] P60. The process in accordance with any of the preceding embodiments, wherein the cleavage module comprises a hybridization construct of two complementary sequences A and A* flanked by at least one toehold sequence TH, and wherein release of the barcode module is achieved by toehold-mediated strand displacement using a cleavage reagent complementary to at least TH and one of A or A*.

[0084] P61. The process in accordance with any of the preceding embodiments, wherein the carbon copy compound comprises a disulfide-containing linker between the barcode module and the linkage module and, after reduction of the disulfide bond, a free thiol group is generated on the barcode module.

[0085] P62. The process in accordance with the preceding embodiment, wherein the barcode module comprising the free thiol group is covalently attached to a maleimide-functionalized solid substrate, in particular to a maleimide-functionalized microscopy cover slip arranged in a flow channel, preferentially a microfluidic flow channel, or a well of a well plate.

[0086] P63. The process in accordance with any of the preceding embodiments, wherein the barcode module further comprises an immobilization module selected from a strained alkyne, such as a DBCO group, and the quantification step comprises covalently attaching the barcode module to an azide-functionalized surface by copper-free click chemistry.

[0087] P64. The process in accordance with any of the preceding embodiments, wherein the quantification step is carried out in a flow chamber formed by a microfluidic slide and a functionalized cover slip, the flow chamber being filled, incubated and washed with defined buffers to immobilize barcode modules and / or DNA origami barcodes on the surface and to remove unbound components.

[0088] P65. The process in accordance with any of the preceding embodiments, wherein the fluorescent DNA origami barcodes comprise DNA nanostructures folded from a scaffold strand and a plurality of staple strands as described for DNA origami metafluorophores,and wherein each DNA origami barcode comprises a capture extension complementary to the barcode module and at least one fluorescent dye.

[0089] P66. The process in accordance with any of the preceding embodiments, wherein a plurality of different analytes present in the same sample are each attached to a distinct carbon copy compound comprising a distinct cleavage module, and the process comprises sequentially cleaving the different cleavage modules in separate steps and quantifying the corresponding barcode modules on separate readout substrates.

[0090] P67. The process in accordance with the preceding embodiment, wherein a first subset of analytes is attached to carbon copy compounds comprising a disulfide cleavage module, a second subset of analytes is attached to carbon copy compounds comprising a uracil-containing cleavage module, and optionally a third subset of analytes is attached to carbon copy compounds comprising a toehold-based hybridization cleavage module.

[0091] P68. The process in accordance with any of the preceding embodiments, wherein at least two different analytes are quantified using carbon copy compounds that share the same barcode module sequence and are detected with the same type of DNA origami barcode, and wherein the different analytes are distinguished by being quantified in different readout experiments and / or on different readout substrates.

[0092] P69. The process in accordance with any of the preceding embodiments, wherein at least one capture compound comprises a handle sequence designed by an in-silico pipeline comprising alignment of target sequences from a reference database, design of candidate probes using a probe-design algorithm, evaluation of secondary structure and accessibility using an RNA folding prediction tool, and selection of a handle sequence based on a combined score for specificity and accessibility.

[0093] P70. The process in accordance with any of the preceding embodiments, wherein buffers used in the formation of the conversion complex, in purification and / or in quantification comprise one or more of:

[0094] - a buffer comprising Tris-HCI, NaCI and a non-ionic detergent,

[0095] - a buffer comprising Tris-HCI, MgCh and EDTA, and

[0096] - a hybridization buffer comprising a saline sodium citrate buffer, dextran sulfate, a nonionic detergent and a blocking reagent such as Denhardt's solution.

[0097] In addition to the embodiments illustrated in the accompanying drawings, the invention further encompasses alternative cleavage architectures disclosed herein, including photon-induced cleavage, redox-based cleavage such as disulfide-based cleavage, Nucleophile-cleavage and pH-cleavage, enzymatic cleavage such as uracil-dependent enzymatic cleavage, 8-oxoG / AP-site cleavage, and hybridisation-based cleavage using toehold-mediated strand displacement. These cleavage mechanisms may be implemented in carbon copy compounds having single-strand or multi-strand architectures, optionally including stepwise assembly, and are exemplified in the Sequence Listing and in exemplary implementations. The order of steps in these alternative architectures does not need to follow the order illustrated for other embodiments.

[0098] The present invention will now be described with reference to the accompanying drawings which illustrate embodiments of the invention, without limiting the scope of the invention.

[0099] Fig. 1 schematically illustrates a target analyte 1 (e.g. rRNA) hybridized to a carbon copy compound 2, the carbon copy compound comprising a linkage module 3 and a barcode module 4.

[0100] Fig. 2 schematically illustrates a conversion complex 21 formed by hybridizing a target analyte 1 with a carbon copy compound 2 comprising a linkage module 3, a barcode module 4 and a cleavage module 5, and by further hybridizing the target analyte 1 to a capture compound 6 comprising a linkage module 7 and an attachment module 8, the capture compound 6 being bound to a purification compound 9 comprising a capture module 10 and a filter module 11.

[0101] Fig. 3 schematically illustrates cleavage of the cleavage module 5 of the conversion complex 21 of Fig. 2 to release the barcode module 4 carrying an immobilisation module 12 from the remainder of the conversion complex 21, which remains attached to the purification compound 9 via the capture module 10 and the filter module 11.

[0102] Fig. 4 schematically illustrates binding of a readout compound 15 to the immobilised barcode module 4 of Fig. 3, the readout compound 15 comprising a barcode readout module 13 hybridized to the barcode module 4 and a detection module 14 (e.g. a DNA origami structure carrying fluorophores).

[0103] Fig. 5 schematically illustrates immobilisation of the readout compound 15 of Fig.

[0104] 4 on a detection substrate 16 (e.g. a glass slide), via the immobilisation module 12 that is bound to the substrate 16, while the detection module 14 provides a detectable signal.Fig. 6 schematically illustrates a variation in which, after cleavage of the cleavage module 5, the barcode module 4 carries thiol groups 17, which are coupled to the readout compound 15 comprising the barcode readout module 13 and the detection module 14, while the remaining part of the conversion complex 21 remains attached to the purification compound 9.

[0105] Fig. 7 schematically illustrates covalent immobilisation of the thiol-bearing barcode module 4 on a gold bead 18. After cleavage of the disulfide-containing cleavage module 5 of the carbon copy compound 3, a free thiol group 17 is generated on the barcode module 4. The thiol group 17 forms a covalent Au-S bond with the gold surface of the bead 18, thereby immobilising the barcode module 4 on the solid substrate. The analyte 1, linkage modules 7 and 8, the capture module 10, and the filter module 11 remain bound as part of the purification compound 9. The detection module 14 of the readout compound 15 hybridises to the immobilised barcode module 4 and remains available for optical readout.

[0106] Fig. 8 schematically illustrates immobilisation of the thiol-bearing barcode module 4 on a maleimide-functionalised solid surface 19 after cleavage of the cleavage module 5 of the carbon copy compound 2. Reduction of the disulfide-containing cleavage module 5 generates a free thiol group 17 on the barcode module 4, which subsequently forms a covalent thioether bond with the maleimide groups on the solid substrate 19. The analyte 1 remains hybridised to the carbon copy compound 2, and the capture compound 6, comprising the linkage module 7 and the attachment module 8, is retained on the purification compound 9 via the capture module 10 and the filter module 11. A readout compound 15, comprising a detection module 14, hybridises by sequence complementarity to the immobilised barcode module 4 and remains available for optical detection.

[0107] Fig. 9 schematically illustrates a conversion complex 21 comprising a target analyte 1 hybridised to both a carbon copy compound 2 and a capture compound 6. The carbon copy compound 2 comprises a linkage module 3, a barcode module 4, and an immobilisation module 12. An orthogonal cleavage module 20 is arranged between the linkage module 3 and the barcode module 4, allowing selective release of the barcode module 4 under cleavage conditions specific for the orthogonal cleavage chemistry. The capture compound 6comprises a linkage module 7 that hybridises to the analyte 1, and an attachment module 8 that binds to the capture module 10 of a purification compound 9, the latter comprising a filter module 11 enabling physical separation of bound and unbound constituents. Together, the analyte 1, the carbon copy compound 2, and the capture compound 6 form the conversion complex 21, which is immobilised on the purification compound 9 to facilitate selective processing and subsequent cleavage of the barcode module 4 via the orthogonal cleavage module 20.

[0108] Fig. 10 schematically illustrates cleavage of the orthogonal cleavage module 20 of the carbon copy compound 3 within the conversion complex, resulting in release of the barcode module 4 carrying the immobilisation module 12. The analyte 1 remains hybridised to the capture compound 6, which comprises a linkage module 7 and an attachment module 8. The capture compound 6 is retained on the purification compound 9 through binding between the attachment module 8 and the capture module 10, the latter being associated with the filter module 11 for physical separation. Cleavage at the orthogonal cleavage module 20 detaches the barcode module 4 and its immobilisation module 12 from the remainder of the carbon copy compound 3, while the analyte 1, the capture compound 6, and the purification compound 9 remain bound for further processing.

[0109] Fig. 11 schematically illustrates a conversion complex 21 in which the target analyte 1 is hybridised to both a carbon copy compound 2 and a capture compound 6. The carbon copy compound 2 comprises a linkage module 3, an orthogonal cleavage module 20, a barcode module 4, and an immobilisation module 12. The orthogonal cleavage module 20 allows selective release of the barcode module 4 under cleavage conditions specific to the orthogonal cleavage chemistry, while leaving other cleavage modules in the system unaffected. The capture compound 6 comprises a linkage module 7 hybridising to the analyte 1, and an attachment module 8 that binds to the capture module 10 of a purification compound 9, the purification compound 9 further comprising a filter module 11 for physical separation of bound and unbound components. Together, the analyte 1, the carbon copy compound 2, and the capture compound 6 form the conversion complex 21, which is immobilised on the purification compound 9 to enable selective processing, orthogonal cleavage at module 20, and subsequent release and capture of the barcode module 4 carrying the immobilisation module 12.Fig. 12 schematically illustrates cleavage of the orthogonal cleavage module 20 of the carbon copy compound 2 within the conversion complex 21, resulting in separation of the barcode module 4 carrying the immobilisation module 12 from the remainder of the complex.

[0110] The analyte 1 remains hybridised to the linkage module 7 of the capture compound 6, which further comprises an attachment module 8 bound to the capture module 10 of the purification compound 9. The purification compound 9 additionally comprises the filter module 11, which enables physical separation of bound species from unbound components. Cleavage at the orthogonal cleavage module 20 detaches the barcode module 4 (together with its immobilisation module 12) from the carbon copy compound 2, while the analyte 1, the capture compound 6, and the purification compound 9 remain bound for further processing or downstream steps.

[0111] In the following, a Sequence Listing is shown which contains the sequences SEQ ID NO:1 to SEQ ID NO:36. To the extent that the description, the claims and / or the drawings refer to sequence identifiers such references relate to the corresponding sequences as set forth in said Sequence Listing.

[0112]

[0113]

Claims

Claims1. A method of quantifying target polynucleotides, the method comprising: (a) forming a conversion complex by attaching an analyte to a carbon copy compound; (b) removing unbound carbon copy compounds from the mixture; (c) releasing at least a barcode module from the conversion complex; and (d) quantifying the released barcode module; wherein the carbon copy compound comprises a barcode module, a linkage module, and is designed to hybridize specifically to the target polynucleotide.

2. The method according to claim 1, wherein the formation of the conversion complex is performed by modifying the melting temperature of the analyte-carbon copy compound hybrid through the introduction or extraction of chaotropic or cosmotropic substances.

3. The method according to claim 2, wherein the extraction of chaotropic or cosmotropic substances is performed using a semipermeable membrane / s.

4. The method according to any of claims 1 to 3, wherein the removal of unbound carbon copy compounds is achieved through a size-based purification method selected from the group consisting of: (a) solid-phase reversible immobilization (SPRI) beads, (b) gel electrophoresis, (c) polyethylene glycol (PEG) precipitation, and (d) high-performance liquid chromatography (HPLC).

5. The method according to any of claims 1 to 3, wherein the removal of unbound carbon copy compounds is achieved through affinity-based purification, comprising at least one of the following: (a) biotin-streptavidin binding, (b) click chemistry, (c) HIS-tag / Nickel-NTA, (d) SpyTag / SpyCatcher, and (e) sequence-specific hybridization.

6. The method according to any of claims 1 to 5, wherein the barcode module is released from the conversion complex through cleavage of a disulfide bond using a reducing agent selected from tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT).

7. The method according to any of claims 1 to 5, wherein the barcode module is released through toehold-mediated strand displacement, wherein the carbon copy compound comprises a hybridization complex of two oligonucleotides, and thebarcode module and linkage module are located on opposite strands in the hybridization complex.

8. The method according to any of claims 1 to 7, wherein the released barcode module is quantified using single-molecule readout techniques selected from the group consisting of: (a) fluorescence detection, and (c) flow cytometry.

9. The method according to any of claims 1 to 7, wherein the released barcode module is quantified using barcoded DNA origami nanoparticles capable of hybridizing to the barcode module.

10. The method according to any of claims 1 to 7, wherein the barcode module is quantified in bulk and the bulk quantification is performed using at least one method selected from: (a) impedance-based detection, (b) capillary electrophoresis, (c) quantitative PCR (qPCR), and (d) sequencing.

11. The method according to claim 10, wherein sequencing comprises impedance-based sequencing.

12. The method according to any of claims 1 to 11, wherein the carbon copy compound further comprises a handle sequence, wherein the handle sequence is specifically designed to selectively hybridize with RNA of a specific taxon from a mixed population of RNA molecules.

13. The method according to any of claims 1 to 12, wherein the conversion complex further comprises a capture compound comprising: (a) a linkage module for binding to the conversion complex; and (b) an attachment module that binds to a capture module of a purification compound.

14. The method according to claim 13, wherein the purification compound comprises:(a) a filter module to remove unbound modules or compounds, and (b) a capture module for retaining the capture compound through specific interactions.

15. The method according to any of claims 1 to 14, wherein the conversion complex is formed under room temperature conditions without the need for thermal cycling.

16. The method according to any of claims 1 to 15, wherein the carbon copy compound comprises a cleavage module containing a uracil nucleotide, and step (c) comprises contacting the conversion complex with a uracil-specific endonuclease andglycosylase and / or a USER-type enzyme mix to cleave the cleavage module and release the barcode module.

17. The method according to any of claims 1 to 16, wherein the carbon copy compound comprises a cleavage module containing an 8-oxo-7,8-dihydroguanine (8-oxoG), and step (c) comprises contacting the conversion complex with an N-glycosylase to generate an abasic site and an AP-lyase to cleave the abasic site and release the barcode module.

18. The method according to any of claims 1 to 17, wherein the cleavage module comprises a hybridisation construct of two complementary oligonucleotide sequences A and A* flanked by at least one toehold sequence (TH), and step (c) comprises adding a cleavage reagent complementary to at least TH and one of A or A* to trigger toehold-mediated strand displacement and release of the barcode module.

19. The method according to any of claims 1 to 18, wherein the barcode module, after release in step (c), comprises a thiol group and is covalently coupled to a maleimide- functionalised solid support, in particular a maleimide-functionalised microscopy cover slip arranged in a microfluidic flow channel.

20. The method according to any of claims 1 to 19, wherein the barcode module comprises a strained alkyne group, in particular a dibenzocyclooctyne (DBCO) group, and is covalently coupled to an azide-functionalised surface by copper-free click chemistry.

21. The method according to any of claims 1 to 20, wherein step (d) comprises performing the quantification in a flow chamber formed between a microfluidic slide and a functionalised cover slip, the flow chamber being filled, incubated and washed with defined buffers to immobilise the barcode modules and / or readout compounds on the surface.

22. The method according to any of claims 1 to 15, wherein the conversion complex further comprises a capture compound that is an oligonucleotide comprising (i) a linkage module that hybridises to the analyte and (ii) an attachment module that hybridises to a capture module on a purification compound, thereby forming a two- handle capture configuration.

23. The method according to claim 22, wherein the purification compound comprises streptavidin-coated magnetic beads as the filter module and a biotinylated capture module, and the capture compound attaches to the purification compound by biotinstreptavidin binding.

24. The method according to any of claims 1 to 23, configured for multiplex detection of at least two different target polynucleotides in the same sample, wherein different carbon copy compounds targeting different polynucleotides comprise different cleavage modules, and wherein step (c) comprises sequentially cleaving the different cleavage modules in separate reactions and quantifying the corresponding barcode modules in separate readout experiments.

25. The method according to claim 24, wherein a first subset of target polynucleotides is linked via carbon copy compounds comprising a disulfide-based cleavage module, and a second subset of target polynucleotides is linked via carbon copy compounds comprising a uracil-containing or toehold-based cleavage module.

26. The method according to any of claims 1 to 25, wherein the target polynucleotides comprise 16S rRNA from a biological sample, in particular a stool sample.

27. The method according to claim 26, wherein the 16S rRNA is derived from at least one bacterial taxon selected from Listeria monocytogenes, Staphylococcus aureus, Pseudomonas aeruginosa, Lactobacillus fermentum, Salmonella enterica, Bacillus subtilis and Enterococcus faecalis.

28. The method according to any of claims 1 to 27, wherein the analyte is obtained from a stool sample collected in a tube comprising a nucleic acid-stabilising solution and processed by bead-beating and column-based RNA extraction including DNase treatment and filtration.

29. The method according to any of claims 1 to 28, wherein the handle sequence of the linkage module is designed by an in silico pipeline comprising: (a) aligning reference sequences of the target region from a sequence database; (b) generating candidate probes by a probe-design algorithm; (c) evaluating secondary structure and nucleotide accessibility using an RNA folding prediction tool; and (d) selecting a handle sequence based on a combined specificity and accessibility score, in particular a handle of about 23 nucleotides in length.

30. The method according to any of claims 8 to 11 and 19 to 21, wherein the singlemolecule readout uses DNA origami metafluorophores comprising a DNA nanostructure carrying a capture extension complementary to the barcode module and at least one fluorescent dye.

31. The method according to claim 30, wherein the DNA origami metafluorophores are immobilised in the flow chamber and detected by single-molecule fluorescence microscopy or DNA-PAINT microscopy.

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