Accelerated multiplexed assay development via sequence transduction and universal detection

Abstract

Embodiment herein provides a method for substituting a molecule of a predetermined sequence for a molecule of a target sequence. The method comprises identifying a target sequence, and substituting the target sequence with a predetermined sequence, such that one or more molecules of the predetermined sequence serve to represent the absence, the presence and the amount of one or more molecules of the target sequence.

Description

ACCELERATED MULTIPLEXED ASSAY DEVELOPMENT VIA SEQUENCE TRANSDUCTION AND UNIVERSAL DETECTIONBACKGROUNDTechnical Field

[0001] The embodiments herein generally relate to molecular diagnostics and assay development, and more particularly relate to a method for accelerating the development of multiplexed assays used in diagnostics, life sciences research, and drug discovery. Specifically, the embodiments herein relate to a method for substituting a molecule of a predetermined sequence for a molecule of a target sequence and the use of sequence transduction and universal detection assay to overcome challenges in sensitivity and specificity.Description of the Related Art

[0002] Multiplexed assays are important for testing samples against multiple targets in diagnostics, life sciences and drug discovery. However, designing such assays has become a bottleneck due to challenges in achieving simultaneous high sensitivity and specificity. Sensitivity requires that even a very small number of copies of each target molecule should trigger detection, while specificity ensures that detection is not triggered in the absence of the specific target.

[0003] In highly multiplexed systems, specificity becomes more challenging due to the increased risk of cross-reactivity from unintended interactions. For example, in a highly multiplexed polymerase chain reaction (PCR) reaction, the number of primer strands is twice the number of targets, which causes the number of potential cross-reactive interactions to grow quadratically as the number of targets increases. This leads to an increased occurrence of unintended interactions, which may result in runaway amplification in a channel that lacks the target. Such occurrences may cause false positives, and the test erroneously reports the presence of a target that was not actually present.

[0004] As the number of targets increases, achieving sensitivity becomes more challenging. This is due to the competition among targets for amplification. In the absence of perfect symmetry, this competitive amplification results in some targets outperformingothers. The targets that lose this competition amplify poorly, which causes the minimum detectable copy number for these targets to be very high.

[0005] Existing assay development processes rely on iterative design-measure-learn (DML) loops to address these issues. In each loop, they evaluate the assay’s shortcomings in terms of targets with inadequate specificity and sensitivity, and then adjust the design to improve performance. As a result, this iterative process may take 3 to 4 years depending on the number of targets. Such prolonged timeline delays the availability of critical assays during emergencies and increases the cost of life sciences R&D, diagnostics, and drug discovery.

[0006] Existing solutions for multiplexed assay development include multiplexed PCR, microarrays, next-generation sequencing (NGS), and high-resolution melting (HRM) curves. However, each of these approaches has significant drawbacks. For example, the multiplexed PCR faces the challenge of competitive amplification, where multiple targets vie for limited resources during amplification. In the absence of perfect symmetry, this competition can result in some targets outperforming others. Targets that lose this competition experience reduced PCR efficiency, leading to a higher limit of detection. As a result, a relatively high copy number of the target is required for successful detection, which can cause analytical false negatives when the assay is tested. This issue necessitates extensive optimization to balance amplification efficiencies across all targets.

[0007] The microarrays require the creation of new arrays for each set of targets, which raises costs due to limited reusability. If the same DNA sequences are used across multiple microarrays, the cost per array may be reduced as large quantities of the same probes may be synthesized and applied to multiple arrays. However, currently, no single array may dynamically adapt to the changing requirements for detecting different targets. As the set of targets of interest evolves, new arrays may be created, which further drives up costs.

[0008] The next-generation sequencing (NGS) machines may misread a base. Even if the probability of misreading any particular base is small over the length of the genome, it may lead to numerous misreads. To mitigate this issue and ensure accuracy, sequencing machines may read sequences from the same region of the genome with high coverage, typically 30X or more. This approach helps correct errors by providing multiple reads of each base, but it also results in a substantial increase in costs and raises the expense of sequencingby 30-fold or more.

[0009] The high-resolution melting (HRM) curves may serve as fingerprints for detecting and quantifying specific DNA molecules. However, for each new target, the corresponding amplicon’s melting curve may be required to be determined experimentally before the pattern is fed into algorithms for detection. Further, previous classification rules need to be re-learned whenever a new target is added. This makes the entire learning process time-consuming and non-modular.

[0010] Thus, there is a need to overcome these drawbacks to enable faster and more efficient development of multiplexed assays.SUMMARY

[0011] In view of the foregoing, according to a first aspect, there is provided a method for substituting a molecule of a predetermined sequence for a molecule of a target sequence. The method comprises identifying a target sequence, and substituting the target sequence with a predetermined sequence, such that one or more molecules of the predetermined sequence serve to represent the absence, the presence and the amount of one or more molecules of the target sequence.

[0012] In some embodiments, the method comprises generating a molecular construct that specifically associates with the target sequence and generating, directly or indirectly, the predetermined sequence that is independent of the target sequence, thereby enabling subsequent molecular operations to be performed using the predetermined sequence rather than the target sequence.

[0013] In some embodiments, the target sequence is transduced / substituted into the predetermined sequence, using a sequence transduction process, by hybridizing a padlock probe having at least two outer arms complementary to the target sequence and at least one inner arm encoding the predetermined sequence, followed by ligation to generate a circularized padlock probe in the presence of the target sequence. The predetermined sequence is detected or quantified to determine the presence or quantity of the corresponding target sequence.

[0014] In some embodiments, the predetermined sequence is detected or quantifiedto determine the presence or quantity of the corresponding target sequence. In some embodiments, the method comprises digesting non-circularized probes and linear nucleic acids using a plurality of exonucleases to selectively retain only the circularised padlock probe, and amplifying the predetermined sequence from the circularized padlock probe using a plurality of universal primers shared across multiple predetermined sequences before detecting or quantifying the predetermined sequence.

[0015] In some embodiments, the predetermined sequence is detected by performing a quantitative real-time PCR using a shared primer set and hydrolysis probe, wherein the predetermined sequence is selected to generate distinct melting curves to enable high-resolution melting analysis. In some embodiments, the plurality of exonucleases degrades linear single-stranded and double-stranded DNA, other than the circularized padlock probe. In some embodiments, the predetermined sequences are configured to have a large pairwise Hamming distance to enable error correction during Next-Generation Sequencing.

[0016] In some embodiments, the predetermined sequence that is quantified is proportional to the amount of target sequence (x). In some embodiments, the plurality of target sequences are simultaneously transduced into corresponding predetermined sequences for multiplexed detection in a single assay. In some embodiments, the at least one inner arm comprises a universal primer binding site to enable amplification with a single primer pair. In some embodiments, the predetermined sequence is selected from a predefined set of sequences configured for optimal amplification and detection performance.

[0017] In another aspect, there is provided a system for substituting a molecule of a predetermined sequence for a molecule of a target sequence. The system comprises (a) a sequence transduction module configured to identify a target sequence, and (b) a molecular substitution module configured to substitute, the target sequence with a predetermined sequence, such that one or more molecules of the predetermined sequence serve to represent the absence, the presence and the amount of one or more molecules of the target sequence. The system is configured to perform molecular operations using the predetermined sequence rather than the target sequence.

[0018] In another aspect, there is provided a method of substituting a molecule of a predetermined sequence for a molecule of a target sequence in an assay. The methodcomprise identifying a target sequence, and substituting, the target sequence with a predetermined sequence, such that one or more molecules of the predetermined sequence serve to represent the absence, the presence and the amount of one or more molecules of the target sequence. The sequence-transduction process comprises generating a molecular construct that specifically associates with the target sequence and generates, directly or indirectly, the corresponding predetermined sequence that is independent of the target sequence, thereby enabling subsequent molecular operations to be performed using the predetermined sequence rather than the target sequence.

[0019] Embodiment here provides a method for accelerated development of multiplexed detection assays using sequence transduction and universal detection. A sequence transducer, such as a padlock probe, is used to substitute known sequences (e.g. DNA, RNA or protein sequences) into predetermined sequences. The padlock probe hybridizes to the known sequence, undergoes circularization upon ligation, and is available as input for downstream molecular mechanisms like detection. The universal detection method utilizes shared primer-binding regions, distinct melting curves, optimized hybridization properties, and error correction mechanisms to enhance performance across PCR, microarrays, Next-Generation Sequencing (NGS), and high-resolution melting analysis. This method reduces cross-reactivity, simplifies assay design, and allows for assay reuse across different applications. By enabling the reuse of detection systems, this method significantly decreases development time, improves specificity and sensitivity, and provides a scalable, cost-effective solution for multiplexed assays.

[0020] These and other aspects of the embodiments herein will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. It should be understood, however, that the following descriptions, while indicating preferred embodiments and numerous specific details thereof, are given by way of illustration and not of limitation. Many changes and modifications may be made within the scope of the embodiments herein without departing from the spirit thereof, and the embodiments herein include all such modificationsBRIEF DESCRIPTION OF THE DRAWINGS

[0021] The embodiments herein will be better understood from the following detaileddescription with reference to the drawings, in which:

[0022] FIG. 1 illustrates a typical hydrolysis probe based qPCR assay;

[0023] FIG. 2 illustrates a padlock probe that has been designed to function as a transducer during sequence transduction according to an embodiment herein;

[0024] FIG. 3 illustrates a system for substituting a molecule of a predetermined sequence for a molecule of a target sequence according to an embodiment herein;

[0025] FIG. 4 illustrates a method for substituting a molecule of a predetermined sequence for a molecule of a target sequence according to an embodiment herein;

[0026] FIGS. 5A-5C illustrate the execution of sequence transduction using the padlock probe of FIG. 2 for enabling accelerated development of highly multiplexed assays according to an embodiment herein;

[0027] FIG. 6A illustrates a sequence transduction process of converting / substituting a target sequence into a predetermined sequence, which is then detected through fluorescence measurement using a qPCR and direct detection of a predetermined sequence without qPCR according to an embodiment herein;

[0028] FIG. 6B illustrates the concordance and reproducibility of the predetermined sequences with sequence transduction and without sequence transduction of FIG. 6A according to an embodiment herein;

[0029] FIG. 7A illustrates a post-PCR melt curve output of a sequence transduction reaction performed during a sequence transduction process according to an embodiment herein;

[0030] FIG. 7B shows a graph illustrating the derivative fluorescence versus temperature data obtained post PCR for sequence transduction and direct amplification reactions according to an embodiment herein; and

[0031] FIG. 8 illustrates a method of developing an accelerated multiplexed assay using sequence transduction and universal detection according to an embodiment herein.DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0032] The embodiments herein and the various features and advantageous details thereof are explained more fully with reference to the non-limiting embodiments that areillustrated in the accompanying drawings and detailed in the following description. Descriptions of well-known components and processing techniques are omitted so as to not unnecessarily obscure the embodiments herein. The examples used herein are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein.

[0033] As mentioned, there remains a need for an assay that combines the sequence transduction and universal detection to address specificity and sensitivity challenges. The embodiments herein provide a design that reduces assay development time from 3-4 years to under 6 months and enables faster and cost-effective development of multiplexed assays. Referring now to the drawings, and more particularly to FIGS. 1 through 8, where similar reference characters denote corresponding features consistently throughout the figures, preferred embodiments are shown.

[0034] Sequence Transduction: A molecular mechanism that substitutes a known sequence with a predetermined sequence such that: 1. If the known sequence is present in the sample then a proportional amount of the predetermined sequence is present at the end of the molecular mechanism; 2. If the known sequence is absent in the sample then the predetermined sequence is absent at the end of the molecular mechanism; and 3. Both known sequence and predetermined sequence can be specified arbitrarily.

[0035] Universal Detection: A detection method optimized for a predefined set of sequences to allow reuse across multiple assays.

[0036] Cross-Reactivity: Cross-Reactivity refers to unintended interactions between assay components that lead to false-positive results.

[0037] Competitive Amplification: Competitive Amplification refers to unequal amplification efficiencies among targets in a multiplexed reaction, which causes false negatives.

[0038] Target sequence: Target sequence refers to a sequence of interest whose absence, presence, or quantity is to be determined in a biological or analytical sample.

[0039] Predetermined sequence: Predetermined sequence refers to a non-nativesequence that is selected and used as a surrogate molecular representation of the target sequence.

[0040] FIG. 1 illustrates a typical hydrolysis probe based qPCR assay. The figure shows two primers (e.g. forward primer (FP) 102 and reverse primer (RP) 104) that bind to two different strands of a target double stranded DNA. The region between binding sites of these primers is amplified using a typical PCR reaction. The amplification of this target double stranded DNA is detected using a hydrolysis probe (DET) 106. The hydrolysis probe 106 is attached with a fluorophore (F) at 5’ end and a quencher (Q) at 3’ end for detection. Thus, three oligonucleotide sequences including 2 primers (FP, RP) and one hydrolysis probe (DET) are typically required to perform a qPCR assay against a specific double stranded DNA target.

[0041] FIG. 2 illustrates a padlock probe that has been designed to function as a transducer during sequence transduction according to an embodiment herein. The padlock probe is a single stranded DNA sequence with distinct regions such as a target binding sequence at 5’ end (Fl), a target binding sequence at 3’ end (F2), a primer binding site for forward primer (FP’), reverse primer (RP) sequence, a hydrolysis probe sequence (DET) and a short gap sequence (G). Each of these distinct regions executes a specific function. For example, the target binding sequence at 5’ end (Fl) and the target binding sequence at 3’ end (F2) of the padlock probe recognises and binds to a target / predetermined DNA sequence. The target binding sequence at 5’ end (Fl) and the target binding sequence at 3’ end (F2) can be modified based on a target / predetermined DNA of interest. The region of the padlock probe in between Fl and F2 is programmable, and in this example, are constructed using sequences corresponding to the 2 primers (i.e. the forward primer (FP) and the reverse primer (RP)) and one hydrolysis probe sequence (DET) from an existing qPCR assay, as shown on the right side of the figure. Specifically, the padlock probe includes a binding site for the forward primer (FP) followed by the reverse primer (RP) sequence, a short gap region (G) and a hydrolysis probe sequence (DET). This padlock probe can be used for the sequence transduction to enable accelerated development of highly multiplexed assays.

[0042] FIG. 3 illustrates a system 300 for substituting a molecule of a predetermined sequence for a molecule of a target sequence according to an embodiment herein. The system300 comprises (a) a sequence transduction module 302 that is configured to identify a target sequence, and (b) a molecular substitution module 304 that is configured to substitute, the target sequence with a predetermined sequence, such that one or more molecules of the predetermined sequence serve to represent the absence, the presence and the amount of one or more molecules of the target sequence. The system 300 is configured to perform molecular operations using the predetermined sequence rather than the target sequence.

[0043] In some embodiments, the system 300 generates a molecular construct that specifically associates with the target sequence and generating, directly or indirectly, the predetermined sequence that is independent of the target sequence, thereby enabling subsequent molecular operations to be performed using the predetermined sequence rather than the target sequence. In some embodiments, the target sequence is transduced / substituted into the predetermined sequence, using a sequence transduction process, by hybridizing a padlock probe having at least two outer arms complementary to the target sequence and at least one inner arm encoding the predetermined sequence, followed by ligation to generate a circularized padlock probe in the presence of the target sequence. The predetermined sequence is detected or quantified to determine the presence or quantity of the corresponding target sequence. In some embodiments, the predetermined sequence is detected or quantified to determine the presence or quantity of the corresponding target sequence. In some embodiments, the method comprises digesting non-circularized probes and linear nucleic acids using a plurality of exonucleases to selectively retain only the circularised padlock probe, and amplifying the predetermined sequence from the circularized padlock probe using a plurality of universal primers shared across multiple predetermined sequences before detecting or quantifying the predetermined sequence.

[0044] In some embodiments, the predetermined sequence is detected by performing a quantitative real-time PCR using a shared primer set and hydrolysis probe, wherein the predetermined sequence is selected to generate distinct melting curves to enable high-resolution melting analysis. In some embodiments, the plurality of exonucleases degrades linear single-stranded and double-stranded DNA, other than the circularized padlock probe. In some embodiments, the predetermined sequences are configured to have a large pairwise Hamming distance to enable error correction during Next-Generation Sequencing.

[0045] In some embodiments, the predetermined sequence that is quantified is proportional to the amount of target sequence (x). In some embodiments, the plurality of target sequences are simultaneously transduced into corresponding predetermined sequences for multiplexed detection in a single assay. In some embodiments, the at least one inner arm comprises a universal primer binding site to enable amplification with a single primer pair. In some embodiments, the predetermined sequence is selected from a predefined set of sequences configured for optimal amplification and detection performance.EXAMPLES:EXAMPLE 1: Multiplexed Detection of Pathogenic Nucleic Acid Targets Using Predetermined Sequences

[0046] In this Example, a system according to FIG. 3 is used to simultaneously detect and quantify multiple pathogenic nucleic acid targets in a single biological sample. A biological sample comprising extracted nucleic acids is provided. The sample is contacted with a plurality of padlock probes, each padlock probe comprising two outer arms complementary to flanking regions of a corresponding target sequence and an inner arm encoding a predetermined sequence. Each predetermined sequence is selected from a predefined library of non-naturally occurring sequences having a minimum pairwise Hamming distance of at least four relative to other predetermined sequences in the library.

[0047] The padlock probes are hybridized to the extracted nucleic acids under conditions permitting sequence- specific hybridization. In the presence of a corresponding target sequence, the outer arms of a padlock probe hybridize to the target sequence and a ligase enzyme circularizes the padlock probe to form a circularized probe. Padlock probes that do not hybridize to a target sequence remain linear. Following ligation, the reaction mixture is treated with a plurality of exonucleases configured to degrade linear singlestranded and double-stranded DNA, thereby selectively retaining the circularized padlock probes.

[0048] The predetermined sequences encoded within the circularized padlock probes are amplified using a single pair of universal primers common to all predetermined sequences. Quantitative real-time PCR is performed using a shared hydrolysis probe. High-resolution melting analysis is then performed to distinguish the predetermined sequences based on distinct melting curve profiles. Detection of a predetermined sequence indicates the presence of the corresponding target sequence in the biological sample, and the quantity of the predetermined sequence is proportional to the amount of the corresponding target sequence.EXAMPLE 2: Quantification of Rare Mutant Alleles in Cell-Free DNA

[0049] In this Example, the system of FIG. 3 is used to detect and quantify rare somatic mutations in cell-free DNA. Cell-free DNA is isolated from a plasma sample. A plurality of padlock probes is provided, each padlock probe being specific to a mutant allele of interest. The ligation junction of each padlock probe is designed such that circularization occurs only when the mutant allele is present. Each padlock probe comprises an inner arm encoding a predetermined sequence uniquely associated with the corresponding mutant allele. The predetermined sequences are selected to be independent of the genomic context of the mutant allele and to include universal primer binding sites.

[0050] Following hybridization and ligation, linear nucleic acids and noncircularized probes are digested using exonucleases. The retained circularized padlock probes are amplified using universal primers and subjected to next-generation sequencing. Sequencing reads corresponding to each predetermined sequence are counted to determine the number of mutant alleles present in the plasma sample. The predetermined sequences are configured to have large pairwise Hamming distances, enabling correction of sequencing errors.

[0051] This Example demonstrates sensitive detection and digital quantification of low-abundance mutations independent of wild-type genomic background.EXAMPLE 3 : Multiplexed Environmental DNA Detection

[0052] In this Example, the system is used for detection of multiple environmental DNA targets in a water sample. Nucleic acids are extracted from a water sample suspected of containing DNA from multiple biological species. The extracted nucleic acids are contacted with a plurality of padlock probes, each padlock probe being specific to a targetsequence associated with a different species.

[0053] Upon hybridization and ligation, target sequences present in the sample are transduced into corresponding predetermined sequences encoded within circularized padlock probes. Linear nucleic acids and non-circularized probes are enzymatically degraded. The predetermined sequences are amplified using universal primers and detected using quantitative PCR. Presence or absence of each species is determined based on detection of the corresponding predetermined sequence, and relative abundance is inferred from the amount of amplified predetermined sequence.EXAMPLE 4: High-Throughput Gene Expression Profiling Using Sequence Substitution

[0054] In this Example, the system is applied to gene expression analysis. RNA is isolated from a biological sample and converted to complementary DNA (cDNA). A plurality of padlock probes is provided, each padlock probe being complementary to a target cDNA sequence corresponding to a gene of interest. Each padlock probe encodes a predetermined sequence serving as a molecular surrogate for the gene of interest.

[0055] Hybridization, ligation, and exonuclease digestion are performed to generate circularized padlock probes encoding predetermined sequences corresponding to expressed genes. The predetermined sequences are amplified using universal primers and detected by next-generation sequencing. The number of sequencing reads corresponding to each predetermined sequence is proportional to the expression level of the corresponding gene. This Example enables multiplexed gene expression profiling using a unified amplification and detection workflow.

[0056] FIG. 4 illustrates a method for substituting a molecule of a predetermined sequence for a molecule of a target sequence according to an embodiment herein. At a step 402, a target sequence is identified. At a step 404, the target sequence is substituted with a predetermined sequence, such that one or more molecules of the predetermined sequence serve to represent the absence, the presence and the amount of one or more molecules of the target sequence. The method significantly reduces development time from years to months, making it possible to respond to emerging applications / diagnostic needs. An assay design generated using the method minimizes the need for repeated processes and enhancesscalability and adaptability across a wide range of targets. This combination of speed, costefficiency, and accuracy makes the method a transformative solution for multiplexed assay development.

[0057] In some embodiments, the method comprises generating a molecular construct that specifically associates with the target sequence and generating, directly or indirectly, the predetermined sequence that is independent of the target sequence, thereby enabling subsequent molecular operations to be performed using the predetermined sequence rather than the target sequence.

[0058] In some embodiments, the target sequence is transduced / substituted into the predetermined sequence, using a sequence transduction process, by hybridizing a padlock probe having at least two outer arms complementary to the target sequence and at least one inner arm encoding the predetermined sequence, followed by ligation to generate a circularized padlock probe in the presence of the target sequence. The predetermined sequence is detected or quantified to determine the presence or quantity of the corresponding target sequence.

[0059] In some embodiments, the predetermined sequence is detected or quantified to determine the presence or quantity of the corresponding target sequence. In some embodiments, the method comprises digesting non-circularized probes and linear nucleic acids using a plurality of exonucleases to selectively retain only the circularised padlock probe, and amplifying the predetermined sequence from the circularized padlock probe using a plurality of universal primers shared across multiple predetermined sequences before detecting or quantifying the predetermined sequence.

[0060] In some embodiments, the predetermined sequence is detected by performing a quantitative real-time PCR using a shared primer set and hydrolysis probe, wherein the predetermined sequence is selected to generate distinct melting curves to enable high-resolution melting analysis.

[0061] In some embodiments, the plurality of exonucleases degrades linear singlestranded and double-stranded DNA, other than the circularized padlock probe. In some embodiments, the predetermined sequences are configured to have a large pairwise Hamming distance to enable error correction during Next-Generation Sequencing.

[0062] In some embodiments, the predetermined sequence that is quantified is proportional to the amount of target sequence (x). In some embodiments, the plurality of target sequences are simultaneously transduced into corresponding predetermined sequences for multiplexed detection in a single assay. In some embodiments, the at least one inner arm comprises a universal primer binding site to enable amplification with a single primer pair. In some embodiments, the predetermined sequence is selected from a predefined set of sequences configured for optimal amplification and detection performance.

[0063] FIGS. 5A-5C illustrate the execution of sequence transduction using the padlock probe of FIG. 2 for enabling accelerated development of highly multiplexed assays according to an embodiment herein. The sequence transduction provides a mechanism to substitute a known sequence (x) (e.g. DNA, RNA or amino acid sequence) into a predetermined sequence (y). In particular, the sequence transduction accommodates scenarios where the predetermined sequence (y) is shorter in length than the known sequence (x). Failure to meet this condition disqualifies an implementation from being considered a valid implementation of sequence transduction. A sequence transducer ST(x, y) functions as a molecular gate where the presence of nucleic acid sequence x results in the proportional production of nucleic acid sequence y. In a molecular implementation, the sequence transduction is performed using the padlock probe. The padlock probe is designed with two outer arms that are complementary to the known sequence x and an inner arm encoding the predetermined sequence y. Upon hybridization to the known sequence x, the padlock probe undergoes circularization through a ligation process using a ligase enzyme. This implementation of the sequence transducer ensures that ligation and subsequent circularization of the padlock probe occur only in the presence of x. In the absence of x, the padlock probe remains linear and cannot circularize. After the ligation process, noncircularized probes and single DNA strands are then digested using exonucleases, which leaves only the circularized probe for subsequent analysis. As a result, the transduced predetermined sequence which includes y is preserved only if x is present in the sample being tested. In the absence of x, all DNA containing y is degraded using exonucleases. This ensures that the predetermined sequence y is specifically generated in response to the presence of the known sequence x.

[0064] In other words, the circularized padlock probe serves as a template for enabling exponential amplification of the predetermined sequence y. This method enhances sensitivity and specificity and enables multiplexed PCR to detect targets such as genes from E. coli by transducing sequences from unrelated markers like EGFP gene. By leveraging the padlock probe for the sequence transduction, the development of multiplexed assays can be accelerated.

[0065] FIG. 5 A illustrates a padlock probe that binds in a sequence specific manner to a predetermined DNA sequence according to an embodiment herein. The binding of the padlock probe is configured so that its two ends align to form a junction, which can be ligated using a ligase enzyme, as illustrated in the figure. Upon ligation, the padlock probe is converted into a circularized single-stranded DNA (ssDNA) molecule. The circularization of the padlock probe only occurs in the presence of a specific predetermined DNA sequence that it is designed to recognise.

[0066] Typically, the padlock probe is implemented to capture and analyze a target DNA sequence bound by the ends of the probe, with the linking region either completely ignored or at most acting like a sample-level barcode. In contrast, the padlock probe used in the present implementation represents a significant difference from the prior approach by utilizing the padlock probe for sequence transduction, not, with opportunity for the linking region to subsequently be analyzed through detection technologies like sequencing, PCR, or microarrays.

[0067] The padlock probe enables sequence transduction by substituting known sequences (e.g., xl, x2, ..., xN) into predetermined sequences (e.g., ul, u2, ..., uN). This sequence transduction focuses on detecting the linker region of the padlock probe, which serves as the transduced sequence, thereby enabling the universal detection independent of the known sequence. The sequence transduction also ensures compatibility with a universal detection assay by maintaining shared end sequences / primer for PCR amplification, generating distinct melting curves for high-resolution melting (HM) analysis, optimizing hybridization properties for microarrays, and enabling an error correction mechanism for accurate Next-Gen Sequencing. For example, during high-resolution melting analysis, the predetermined sequences are selected with distinct melting curves to maximizedistinguishability. For Next-Gen Sequencing (NGS), the predetermined sequences are selected to maximize Hamming distance, which allows for error correction even when minor sequencing errors occur.

[0068] In other words, the embodiment herein provides a method that substitutes a known sequence (xl, x2, ..., xN) with a predetermined sequence ul, u2, ..., uN) using the sequence transduction and the padlock probe. The presence or absence of the known sequence is determined by detecting the predetermined sequence. The amount of the known sequence is determined by measuring the quantity of the predetermined sequence. The predetermined sequence or the known sequence represents a nucleic acid sequence. The predetermined sequence or the known sequence may each represent a nucleic acid sequence, an amino acid sequence, or other synthetic or natural polymer that may be represented as a sequence. The method ensures that the detection or measurement is performed via polymerase chain reaction (PCR), Loop-Mediated Isothermal Amplification (LAMP), Mass Spectrometry, NGS, Melting Curves, Transcription-Mediated Amplification (TMA), ELISA or Fluorimetry or Spectroscopy.

[0069] FIG. 5B illustrates the selection of circularized padlock probes using exonuclease enzymes according to an embodiment herein. Exonuclease enzymes used in this assay specifically degrade linear single stranded DNA (e.g. linear padlock probe) and double stranded DNA (e.g. a target DNA) and have no effect on circularized single stranded DNA (ssDNA). Furthermore, the linker region in the circularized padlock probe serves as a product of sequence transduction that enables the detection and quantification of a known sequence. The circularized padlock probe acts as a sequence transducer for enabling the reuse of detection assays.

[0070] For example, a sequence transducer ST(x, y) substitutes a known sequence x into a predetermined sequence y. This substitution enables detection systems to measure fluorescence proportional to the substituted predetermined sequence. As a result, the known sequence can be quantitatively analyzed with minimal changes to the assay. The process ensures that only circularized probes corresponding to specific known sequences are retained, thereby enabling accurate downstream analysis. Furthermore, this approach supports multiplexed detection and allows for simultaneous analysis of multiple known sequences (x 1 ,x2, xN) by substituting them into universal predetermined sequences (ul, u2, uN).

[0071] FIG. 5C illustrates the detection of circularized padlock probes using a qPCR assay according to an embodiment herein. In this example, the qPCR assay employs the same primers and hydrolysis probe as described in FIG. 1. These components, in combination with a DNA polymerase enzyme such as Taq polymerase, perform the exponential amplification and real-time quantification of the circularized padlock probe. During the amplification of the circularized padlock probe, the fluorophore is released from the 5’ end of the Taqman probe (i.e. hydrolysis probe). This circularized padlock probe is generated only in the presence of a specific predetermined DNA, thereby ensuring specificity in the qPCR assay. The qPCR assay employs the universal detection method by transducing known sequences(x 1 , x2, ..., xN) into a predefined set of predetermined sequence (ul, u2, ..., uN) with shared primer-binding regions. This universal detection method ensures that the detection mechanism remains consistent across different assays, thereby significantly reducing development time and effort. The universal detection method begins with the known sequences (xl, x2, ..., xN) identification and their transduction into the predetermined sequence (ul, u2, ..., uN).

[0072] The universal detection is built and optimised first, before targets are even specified. Each subsequent assay to be built reuses the same universal detection scheme. For example, a detector for a known sequence y, such as a Taqman probe TP(y), can be reused for another known sequence x by employing a transducer ST(x, y) that substitutes x to y and then reuse TP(y) to detect the known sequence y. This enables quantitative detection on the same fluorescence channel and ensures the qPCR assay remains adaptable, and scalable for various diagnostic needs.

[0073] The universal detection method involves designing an assay capable of detecting a predefined set of sequences (ul, u2, ..., uN). These sequences may be chosen for improved performance in detection. For PCR, the sequences (ul, u2, ..., uN) are designed to share the same end sequences which enables the use of a single primer-co-primer pair to amplify all the sequences. For microarrays, the sequences are selected to be sufficiently distinct to avoid long runs of identical bases and to maintain balanced AT-to-GC ratios to ensure effective hybridization with minimal cross-interference. For Next-GenerationSequencing (NGS), the sequences are selected to have a large Hamming distance between them which allows for error correction even if some bases are misread. These sequences also avoid long base runs and maintain balanced AT-to-GC ratios for optimal performance. For high-resolution melting curve analysis, the sequences are selected to produce distinct melting curves. This enables the algorithms to separate / differentiate them and minimizes the risk of misidentification / chance of error.

[0074] In an embodiment, when detecting a set of known sequences (xl, x2, ..., xN), the targets are first transduced into the predetermined sequences (ul, u2, ..., uN) using sequence transduction that is incorporated with the padlock probe, and then the universal detection method is employed. This detection assay is referred to as "universal" because it remains unchanged regardless of specific targets being detected. Further, the same universal detection method may be reused across different applications and different targets. While developing the detection assay may still require significant time and effort, this assay is effectively amortized across numerous assays for various targets.

[0075] FIG. 6A illustrates a sequence transduction process of converting / substituting a target sequence into a predetermined sequence, which is then detected through fluorescence measurement using a qPCR and direct detection of a predetermined sequence without qPCR according to an embodiment herein. In the Sequence Transduction process, a reaction including 4 different amounts of the target sequence is carried out. The products obtained, after the sequence transduction process, include the predetermined DNA sequence, which is then amplified and detected using real-time PCR. This reaction is set up in three replicates. Similarly, a reaction including the 4 different amounts of the predetermined DNA, corresponding to each target amount in the sequence transduction reaction, are directly amplified and detected using real-time PCR.

[0076] The data below shows the efficiency and the reproducibility of Sequence Transduction process. Table 1 show the Ct values obtained from the real-time PCR of the predetermined sequence with and without sequence transduction, respectively, for each amount.

[0077] Table 1: It shows the Ct values obtained from the real-time PCR of the predetermined sequence with and without sequence transduction, for each amount."

[0078] The concordance between the Ct values of the predetermined sequences obtained with and without sequence transduction shows that the efficiency of sequence 5 transduction is 100%. The concordance across the replicates for the Ct values of the predetermined sequences obtained with sequence transduction and without sequencetransduction depicts the reproducibility of the data. FIG. 6B illustrates the concordance and reproducibility of the predetermined sequences with sequence transduction and without sequence transduction, as discussed above.

[0079] FIG. 7A illustrates a post-PCR melt curve output of a sequence transduction reaction performed during a sequence transduction process according to an embodiment herein. FIG. 7 A shows the post-PCR melt profiles that are generated by a sequence transduction reaction / process and a control real-time PCR reaction. The sequence transduction process depicts the different types of reactions (e.g., Positive sequence transduction reaction and Negative sequence transduction) that are carried out. Positive sequence transduction reactions are those where the target sequence / DNA is added to the reaction and the sequence transduction is performed. Negative sequence transduction reactions are those where no target DNA is added and the sequence transduction is performed. The sequence transduction products obtained, after the sequence transduction process, are then amplified and detected using real-time PCR. Real-time PCR reaction, where the predetermined sequence / DNA is directly amplified, serves as a positive control reaction. The Real-time PCR reaction, where no DNA is added, serves as a negative control real-time PCR reaction.

[0080] Table 2: Table 2 shows melting temperature peaks obtained for all reactions tested.

[0081] FIG. 7B shows a graph illustrating the derivative fluorescence versustemperature data obtained post PCR for sequence transduction and direct amplification reactions according to an embodiment herein. The graph shows that the post-PCR melt profiles of the predetermined sequence / DNA and the sequence transduction reactions in the presence of target sequence / DNA are identical (as shown by dotted black and solid black curves). The graph also shows that the post-PCR melt profile of sequence transduction reactions in the absence of target sequence is distinct and easily differentiated from melt profiles generated by the reactions that included target sequence.

[0082] FIG. 8 illustrates a method of developing an accelerated multiplexed assay using sequence transduction and universal detection according to an embodiment herein. At a step 802, the method begins with the identification and selection of known sequences (x 1 , x2, ..., xN) based on the specific application (e.g. diagnostic or research application). At a step 804, these known sequences are then transduced / substituted into predetermined sequences (ul, u2, ..., uN) using sequence transducers such as padlock probes. At a step 806, the transduced sequences are then detected using the universal detection method. For example, a detector for a known sequence y, such as a Taqman probe TP(y), can be reused for another known sequence x by employing a transducer ST(x, y) that substitutes x to y and then reuse TP(y) to detect the known sequence y. At a step 808, the method provides us a quantitative readout of the known sequence x based on the detection since the sequence x and y are equimolar.

[0083] The method enhances specificity in PCR assays by using a single primer pair, which reduces cross-reactivity and simplifies limit-of-detection validation as a one-time process. The method enables the reuse of existing microarrays to detect a set of new targets and facilitate high-quality sequencing reads even from noisy machines. Additionally, the method uses high-resolution melting curve analysis for detecting new targets without performing any experimental characterization of the new targets. This method reduces development time and costs, while being scalable across multiple platforms such as PCR, microarrays, sequencing, and melting curve analysis, making it an efficient solution for various applications.

[0001] By leveraging the sequence transduction and the universal detection assay, the method significantly reduces development time from years to months, making it possible torespond to emerging applications / diagnostic needs. The assay design generated using the method minimizes the need for repeated processes and enhances scalability and adaptability across a wide range of targets. This combination of speed, cost-efficiency, and accuracy makes the method a transformative solution for multiplexed assay development.

[0002] The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and / or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the scope of appended claims.

Claims

CLAIMS1 / We Claim:

1. A method for substituting a molecule of a predetermined sequence for a molecule of a target sequence, comprising:identifying a target sequence;characterized in that, substituting the target sequence with a predetermined sequence, such that one or more molecules of the predetermined sequence serve to represent the absence, the presence and the amount of one or more molecules of the target sequence.

2. The method as claimed in claim 1 , wherein the method comprises generating a molecular construct that specifically associates with the target sequence and generating, directly or indirectly, the predetermined sequence that is independent of the target sequence, thereby enabling subsequent molecular operations to be performed using the predetermined sequence rather than the target sequence.

3. The method as claimed in claim 1, wherein the target sequence is transduced / substituted into the predetermined sequence, using a sequence transduction process, by hybridizing a padlock probe having at least two outer arms complementary to the target sequence and at least one inner arm encoding the predetermined sequence, followed by ligation to generate a circularized padlock probe in the presence of the target sequence, wherein the predetermined sequence is detected or quantified to determine the presence or quantity of the corresponding target sequence.

4. The method as claimed in claim 3, wherein the predetermined sequence is detected or quantified to determine the presence or quantity of the corresponding target sequence.

5. The method as claimed in claim 3, wherein the method comprises:digesting non-circularized probes and linear nucleic acids using a plurality of exonucleases to selectively retain only the circularized padlock probe; andamplifying the predetermined sequence from the circularized padlock probe using a plurality of universal primers shared across multiple predetermined sequences before detecting or quantifying the predetermined sequence.

6. The method as claimed in claim 3, wherein the predetermined sequence is detected by performing a quantitative real-time PCR using a shared primer set and hydrolysis probe, wherein the predetermined sequence is selected to generate distinct melting curves to enable high-resolution melting analysis.

7. The method as claimed in claim 4, wherein the plurality of exonucleases degrades linear single-stranded and double-stranded DNA, other than the circularized padlock probe.

8. The method as claimed in claim 1, wherein the predetermined sequences are configured to have a large pairwise Hamming distance to enable error correction during Next-Generation Sequencing.

9. The method as claimed in claim 1, wherein the predetermined sequence that is quantified is proportional to the amount of target sequence (x).

10. The method as claimed in claim 1, wherein the plurality of target sequences are simultaneously transduced into corresponding predetermined sequences for multiplexed detection in a single assay.

11. The method as claimed in claim 3, wherein the at least one inner arm comprises a universal primer binding site to enable amplification with a single primer pair.

12. The method as claimed in claim 1, wherein the predetermined sequence is selected from a predefined set of sequences configured for optimal amplification and detection performance.

13. A system for substituting a molecule of a predetermined sequence for a molecule of a target sequence, comprising:(a) a sequence transduction module configured to identify a target sequence; and (b) a molecular substitution module configured to substitute, the target sequence with a predetermined sequence, such that one or more molecules of the predetermined sequence serve to represent the absence, the presence and the amount of one or more molecules of the target sequence,wherein the system is configured to perform molecular operations using the predetermined sequence rather than the target sequence.

14. A method of substituting a molecule of a predetermined sequence for a molecule of a target sequence in an assay, comprising:identifying a target sequence; andcharacterized in that, substituting, the target sequence with a predetermined sequence, such that one or more molecules of the predetermined sequence serve to represent the absence, the presence and the amount of one or more molecules of the target sequence, wherein the sequence-transduction process comprises generating a molecular construct that specifically associates with the target sequence and generates, directly or indirectly, the corresponding predetermined sequence that is independent of the target sequence, thereby enabling subsequent molecular operations to be performed using the predetermined sequence rather than the target sequence.

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