Nanites: smart, programmable polymer composite nucleic-acid nanostructures for sensitive, targeted, molecular nucleic acid detection

Abstract

The present invention relates to Nanites, a programmable aptameric polymer composite characterised by a hybrid silica / iron structure with embedded folded reconfigurable DNA aptamers. The nanites are preferably designed to remain self-folded and inert in the absence of a target molecule. In the presence of a target molecule, the sentry module component activates and binds the target molecule. The activation initiates a hybridisation cascade with other stabiliser and signaller modules resulting a fluorescent signal increase. Nanite is designed with specific lengths and bonding setups and is designed to change its three-dimensional structure upon interaction with the specific target sequence. Also disclosed is a method for designing nanites and a method of utilising programmable polymer-DNA nanostructure composites paired with micro-compartmentalisation for enzyme-free, digital single molecule nucleic acid detection.

Description

The present invention pertains to the field of nanotechnology, molecular diagnostics, and DNA origami, particularly focusing on the design and functionality of programmable, computationally and rationally designed polymer Aptameric composite structures named "Nanites" for dynamic molecular interactions and amplified target detection. Summary of the Invention The invention describes ‘Nanites’, sophisticated polymer Aptameric composites characterized by hybrid silicon / iron structures embedded with programmed DNA aptamers. Designed for dynamic behaviour, these Nanites can self-fold in the absence of a target and activate upon target detection, triggering a unique hybridization chain reaction, resulting in an amplified fluorescent signal. The nanites' surfaces are adorned with aptamer "arms" that remain selffolded, akin but not identical to hairpin structures, in the absence of the target molecule of interest. However, in the presence of the target molecule, the aptamer arms unwind, allowing the nanite to capture the target molecule through sequence-specific hybridization controlled by thermodynamics. These Nanites are uniquely programmed for dynamic behaviour in response to specific environmental triggers. The Nanites' distinguishing feature is their ability to self-fold;, remain inactive in the absence of a target, and activate upon target presence, initiating a chain reaction yielding a detectable fluorescent signal. This targeted capture initiates a chain reaction recognition event involving multiple nanites, setting off a highly efficient hybridization cascade. The cascade results in exponential signal amplification, making the detection of the target nucleic acid rapid and sensitive. Detailed Description of the Invention The present invention pertains to Nanites, advanced programmable DNA nanostructures, and the method for their production. Nanites, defined as smart, programmable DNA nanostructures, are composed of aptameric polymer composites tailored for next-generation detection. These Nanites offer a new approach to molecular detection without the reliance on enzymes. Given their advanced design, Nanites can detect and bind to specific target molecules in complex samples, distinguishing them from surrounding molecules. Their programmability ensures adaptability, making them an essential tool for various molecular diagnostics scenarios. Disclosed herein are methods and systems for producing and utilizing Nanites. Nanites are rationally engineered nanostructures, designed to fold into specific configurations based on situational requirements &exhibit situational ‘behavioural’ changes in response to particular stimuli - such as the presence of a particular sequence target molecule. Each Nanite encompasses polymeric composites, produced through a proprietary in-house process. The nanites consist of iron oxide crystals dispersed in a polymer matrix, with a functional polymer overcoating for the encapsulation of magnetite and the introduction of reactive groups. The core structure of nanites is primarily composed of short, programmed DNA aptamers designed to form the Nanite framework and contain complementary sequences for particular target recognition. Preferably, at least three separate programmed aptamers are encompassed within one Nanite. These are the Sentry(Nl) or primary Nanite, the Stabiliser(N2) or Auxiliary nanite 1, and the Signaller,(N3) or Auxiliary Nanite 2. Nanites, at their core, are exemplary models of DNA Origami, where DNA fragments are controlled to assume specific shapes. The entirety of this spherical structure, encapsulating foldable aptamer complexes of DNA with embedded iron and silica, is termed a single Nanite. The surfaces of the Primary Nanites are adorned with aptamer "arms" , while resembling a hairpin structure with a long unpaired toehold, they are designed to remain self-folded in the absence of a target molecule, with only the exposed toe-hold region unpaired. Primary Nanite aptamer arms can in some instances possess a fluorophore molecule which differs from those of the auxiliary nanites which preferentially possess a single fluorophore molecule each and no quencher molecules. Primary Nanites are preferably magnetic, so that they may be manipulated and confined using magnetic fields, especially within micro fluidic arrays. Upon encountering the target nucleic acid, these Nl aptamer arms demonstrate high sensitivity and specificity, recognizing and binding the target through sequence-specific hybridization by way of toe-hold mediated strand displacement, a process governed by thermodynamic shifts in the free-energy of folding. Upon capturing the target molecule, the Nl Primary Nanite which anchors the target DNA strand to its surface, facilitates a landing pad for the smaller auxiliary nanites N2 and N3 to begin the cascade. N2, which is also self-folded save for its own small toe-hold region, hybridises with a particular portion of the target sequence, unwinding from its self-folded state through toehold mediated strand displacement. At least one portion of the Auxiliary Nanite(Nl) is complementary to the Auxiliary Nanite(N2) itself self-folded until the specific sequence interaction domain of Nl is exposed through its unwinding to the target, which facilitates the unwinding of N2 to preferentially bind the complementary portion of Nl. Through the unwinding and sequence-specific hybridisation of N1 to N2, at least one other portion of N2 unwinds to provide a complementary landing pad for a further Nl Auxiliary Nanite to hybridise to. As each Auxiliary nanite possesses at least one single fluorophore molecule each, concomitant linear hybridisation chains of Nl &N2 nanites occur originating from and anchored to the primary Nanite through the captured, anchored target molecule. The nanites initiate a chain reaction known as a hybridization cascade. Hybridisation Cascading involves multiple nanites and results in the formation of a signal amplification cascade. This cascade culminates in a rapid, pronounced fluorescent signal, ensuring a robust and easily detectable signal, marking the presence of the target molecule. The Nanite-capture complex can be manipulated using a magnet in combination with custom large number, low-volume microchamber, microfluidic chip arrays to confine each Nanite in femtoliter-sized reaction array wells, courtesy of their magnetic properties. Their uniqueness lies in their engineered capability to change configurations; self-folding, unfolding, bridging, re-orienting, based on the presented stimuli. Upon successful target capture, Nanites are programmed to exhibit colour-changes - from at least one colour to another wavelength, offering a distinct detection signal when housed within specialized wells. Unlike conventional methods, Nanites offer enzyme-free amplification capabilities. When paired with a specific reader instrument, a separate invention, they enable single-molecule detection. The Nanites can bind to specific target molecules, making them ideal probes for a broad range of applications. Their programmable nature allows for adaptability in various detection scenarios, revolutionizing the field of molecular diagnostics Apparatus, System, and Method for Nanites Production and Utilization The present invention provides an ensemble of Nanite formations specifically designed for precise molecular detection. Detailed aspects of the invention include: (a) A Sentry Module (Nl): This module is specifically engineered to recognize and bind to certain target molecules, acting as the primary interface. (b) A Stabilizer Module (N2): This module's primary function is to establish a stable environment that reinforces molecular capture. It inhibits the potential release or degradation of the target and sets the groundwork for the Signaler, amplifying detection through fluorescence arising from simultaneous hybridizations. (c) A Signaler Module (N3): Upon assimilating information from the Sentry and Stabilizer modules, the Signaler produces an identifiable output signal. This is achieved through sequence-specific hybridization. The module interacts with presented complementary domains and further exposes its own domains, enhancing subsequent interactions with the N2 molecule. This design guarantees effective communication of the detection process. In virtue of the structured sequence of these Nanite modules, which delineate a detection pathway, the system exhibits exceptional precision and regulatory control. The integrated operation of the Sentry, Stabilizer, and Signaler modules culminates in heightened molecular detection sensitivity. In a preferred embodiment, a minimum of two Nanite modules are anchored to a common structure, ideally the Primary Nanite. This ensures streamlined interactions and effective signal propagation among modules. It is particularly advantageous for the Stabilizer and Signaler modules to be affixed to the aforementioned Primary Nanite structure. The linkage can be achieved via specialized tethering techniques or direct attachment. This configuration guarantees close proximity and efficient inter-module communication, enhancing their combined efficiency. In terms of adaptability and precision in design, the universal structure for the Nanites could adopt a DNA-based formation. For user interface efficiency and cohesive design, this structure might manifest as a nanoparticle. To broaden its compatibility with various systems or apparatuses, the structure might also be conceived as a substrate. For synchronized module interactions, it's advantageous for at least two Nanite modules to be anchored to the universal structure in a predetermined configuration. An example would be the strategic placement of the Stabilizer module between the Sentry and Signaler modules. The Sentry module is adept at detecting specific target molecules., spanning from DNA to RNA to proteins. Upon successful target identification, the module triggers the subsequent Nanite system modules. This recognition proficiency heightens the Nanites' selective detection capability, refining its specificity. Sentry Module For optimal sensitivity, the Sentry module is configured to recognize multiple target molecules, thereby expanding its detection range. This multifaceted interaction capability can be attributed to specialized Nanite arms that are present on its surface. Each arm is designed to match specific target sequences, allowing for selective and broad-spectrum detection. For enhanced precision, the Sentry module might be designed to interact with multiple target molecules, thus broadening its detection spectrum. It may do this through different, specific Nanite arms adorning its surface, tailored to particular target sequences. The Sentry module can comprise distinct DNA sequences. These sequences might be fixed to a shared architecture, preferably a DNA formation. Their arrangement is ideally predetermined, potentially utilizing a tethering technique for efficient anchorage. Upon successful interaction with a target molecule, the Sentry module can undergo structural alterations. This allows it to effectively engage subsequent modules. A secondary sequence within the Sentry can further solidify this recognition, ensuring a durable and amplified initial detection. Mechanisms, such as competitive hybridization, might be integrated into the design. Strategies like toe-hold-mediated strand displacement or toe-hold exchange can be leveraged to facilitate this process. Stabilizer Module The Stabilizer module plays a pivotal role in upholding the integrity of the signal instigated by the Sentry module. It acts as a shield, ensuring that the initial detection is unaffected by external influences or disruptions. It ensures that the initial detection is preserved and not degraded by external factors or interferences. This protective function underscores the reliability of the entire Nanite system. In a preferred embodiment, the Stabilizer module consists of multiple subunits. Each subunit is crafted to specifically support the signal initiated by the Sentry module. When these subunits operate in concert, they guarantee that the signal remains unaffected, even in the face of potential interferences or varying environmental conditions. Several versions of the Stabilizer subunits can be integrated, with each variant performing nuanced roles, yet collectively ascertaining signal consistency. Signaller Module Upon assimilation of inputs from both the Sentry and Stabilizer modules, the Signaller module generates a discernible output. This output can manifest in various forms, tailored to the specific application—be it visual, electronic, or other relevant modalities. A fluorescent output is especially favoured due to its clear visibility and ease of detection. The module's design might incorporate principles akin to competitive hybridization, ensuring potent signaling. The Signaller module can possess distinct DNA sequences that, upon activation by the Sentry and Stabilizer modules, yield a detectable output. These sequences might be affixed to a unified structure, preferably a DNA formation, in a predetermined layout, possibly utilizing a tethering strategy for secure attachment. Enhancements and Considerations To augment the accuracy of detection and minimize potential false positives, the Nanite system may integrate mechanisms to neutralize interfering molecules. This ensures that only the designated target molecules activate the Nanite system, thus preserving detection fidelity. To mitigate potential false positives and enhance the accuracy of detection, Nanites might incorporate mechanisms to counter interfering molecules. Such mechanisms would ensure that only the intended target molecules trigger the Nanite system, thus maintaining high fidelity in detection. Ideally, the Stabiliser module encompasses multiple subunits, each one specifically tailored to sustain the integrity of the signal initiated by the Sentry module. These subunits work in unison, ensuring the signal remains consistent and doesn't degrade amidst potential interferences or environmental factors. Multiple iterations of the Stabiliser subunits might be present, each performing slightly varied functions but collectively ensuring signal stability. The Sentry module may comprise multiple instances of specific DNA sequences (potentially anchored to a shared structure, optimally a DNA formation, ideally in a pre-defined arrangement, possibly using a tethering mechanism) designed to recognize and bind to target molecules. Alongside, the Signaller module may have specific DNA sequences (also potentially anchored to a common structure, optimally a DNA formation, in a pre-defined arrangement, and possibly tethered) that produce a detectable output upon activation by the Sentry and Stabiliser modules. Upon recognition of the target molecule, the Sentry module might undergo conformational changes, enabling it to activate the subsequent modules. A secondary sequence within the Sentry could further stabilize or amplify this recognition, ensuring a robust initial detection. The design might include mechanisms such as competitive hybridization, facilitated by strategies like toe-hold-mediated strand displacement or toe-hold exchange. The Signaller module, equipped with its specific DNA sequences, gets activated in response to the cues from the Sentry and Stabiliser. The Signaller sequences might be pre-hybridized, and upon activation, produce a detectable output. This output generation might involve mechanisms similar to competitive hybridization or other conformational changes, ensuring effective signaling and clear detection. For enhanced accuracy and to mitigate potential false positives., the Stabiliser module might consist of multiple pathways tailored to sustain various signals initialized by the Sentry module. Multiple iterations of the Stabiliser subunits might be incorporated, each fine-tuned to maintain specific signals. To achieve optimal amplification, these pathways could be designed to mutually enhance each other's functions. Preferably, the Signaller module includes a DNA capture mechanism, adept at securely holding a reporter molecule in an initial state and releasing it in a subsequent state, ensuring a clear transition from non-detection to detection. The DNA capture mechanism in the Signaller could be designed to transition between its states upon interaction with specific sequences relayed by the Sentry and Stabiliser modules. Such interactions could be based on competitive hybridization strategies, potentially employing techniques like toe-hold-mediated strand displacement or toe-hold exchange. In another aspect of the Nanites system, there's a method for detecting specific target molecules by introducing the ensemble of Nanite modules to a sample, ideally conducted in vitro. Another aspect details the detection process using Nanites: The Sentry module recognizes and binds to the target molecule, initiating a landing pad signal for the stabilizer, signal. This signal is then acted upon by the Stabiliser module, through the form of sequence-specific hybridisation, providing capture reinforcement and ensuring the integrity of the target molecule. Subsequently, the Signaller module, upon receiving cues from the preceding stabilizer module, produces a detectable output, ideally changing its internal DNA structure to embody, deliver and facilitate a specific reporting signal, which in tandem with other signallers exponentially renders the detection evident. The Stabilizer module may include several instances of particular sequences, each specifically engineered to safeguard the integrity of signals initiated by the Sentry module. Concurrently, the Signaller module may encompass sequences tailored to yield a detectable outcome upon being activated. The aforesaid Sentry, Stabilizer, and Signaller modules may be constituents of a more expansive Nanite formation ensemble. Each module might be methodically positioned upon a shared foundation, ideally leveraging a DNA-centric structure, to foster efficient inter-module communication. Potential applications of Nanites involve introducing the aforementioned DNA structures to diverse sample types, with a preference for in vitro procedures. In another embodiment, a computer program product can be devised to emulate the interactions and functionalities of the Nanite ensemble. Such a digital model assists in predicting, understanding, and refining the operational dynamics of Nanites in practical settings. Moreover, an approach may encompass digitally simulating the Nanite ensemble's behaviour prior to its physical assembly. Post-simulation, distinct DNA sequences for each module are identified, followed by the physical construction of the Nanite ensemble based on these sequences. For the sake of clarity: "DNA sequence" or "DNA strand" within this context typically alludes to specific DNA configurations with predetermined sequences. "Stabilization" primarily refers to the process where the initial signal from the Sentry is maintained and shielded from potential disturbances through sequence-specific hybridisation to favourable free-energy and increased stability of the bound duplex. "Ensemble of Nanite formations" signifies an aggregated system of Nanite modules, potentially in a solution, affixed to a substrate, or anchored to a common foundation. "Nanite formation" generally denotes a designated DNA configuration fashioned for a specific role within the Nanite architecture. The design of the Nanites can manifest in varied configurations, contingent upon distinct module activations and their interactions. A distinct embodiment entails a computer program product equipped with specific software code to simulate and analyse interactions of the Nanite modules. Furthermore, a non-transitory computer-readable medium is provided, storing instructions tailored for the simulation and optimization of the Nanite system. Another facet of the Nanite invention includes a digital signal depiction outlining the operational schematic of Nanites. Such a representation is transmittable and can be integrated into computer systems with operating systems conducive to Nanite simulations and analytics. Furthermore, another approach entails the creation of the Nanite ensemble by first simulating its behaviour. Post-simulation, specific DNA sequences for each module are determined. The Nanite ensemble is then physically assembled based on these identified sequences. Features described for any given Nanite module can potentially be repurposed as a methodological procedure, and vice versa. The features across various Nanite modules are malleable and can be executed in a multitude of combinations, bestowing the system with adaptability. Distinct combinations of features across Nanite modules can be independently conceptualized, implemented, and utilized to suit specific applications. Considering the diverse applications of Nanites, it's conceivable that features described as software implementations might also be translated to hardware forms and vice versa. All references to software and hardware functionalities should be interpreted in this light. Nanites are preferably designed for digital counting rather than bulk intensity measurements, therefore Nanites offer enhanced sensitivity and specificity, however in some instantiations they may be tailored for intensity measurements. Features and functionalities across the different Nanite modules are interchangeable and can be applied in diverse combinations, ensuring flexibility and adaptability. Specifically, methodologies pertaining to one module can be adapted to functionalities of another, enhancing the holistic efficiency of the Nanite system. It's essential to acknowledge that unique combinations of features across the Nanite modules can be designed, deployed, and utilized independently, catering to specific applications and requirements. As used herein, means plus function features of Nanites may be expressed alternatively in terms of their corresponding structure, such as a suitably programmed molecular matrix and associated memory within the nanite's core. Applications and Advantages: Beyond mere target detection, Nanites facilitate extraction, purification, and concentration seamlessly integrated into the detection process, eliminating the need for cumbersome preprocessing steps . Nanites are tailored for microfluidic microchamber containment arrays, emphasizing isolated detection over bulk ensemble reactions, as seen with traditional HCR hairpins, CHA, PCR and other systems of detection, enabling the potential for unprecedented sensitivity and specificity at high-speed through employment of Digital Counting and Bioassay principles. Simulations and experimental data, as elaborated later, showcase the superior sensitivity and functionality of Nanites compared to conventional methods. In the illustrations DNA strands are depicted as lines, with the 5’ and 3’ ends indicated. Subsections of nucleotides as indicated by their bases(ATCG) are grouped into domains and given designations based on groups of corresponding lower-case letters - I.e. - (a) represents a domain and (a*) represents a region complementary. Domains are included as a means of illustrating modular, discrete regions which hybridise with one-another and is used as an aid to understanding the molecular interplay. These and other aspects of the present invention will become apparent from the following exemplary embodiments that are described with reference to the following figures in which: Figure 1: Overview of Nanite components A schematic representation showcasing the entire Nanite ensemble, including the Sentry, Stabilizer, and Signaller modules alongside representative target DNA. Corresponding domains are marked. The Nanite is designed to allow unparalleled molecular detection capabilities when paired with microchamber compartmentalisation and scanned with our reader instrument. The Nanite is precise enough to distinguish between molecular compositions with minute differences, akin to discerning between different DNA or RNA fragments. When a target molecule is detected, the Nanite causes a signal that is observable to a user (e.g., a fluorescent signal), facilitating instant feedback, preferably for research purposes. Because the Nanite identifies specific molecular structures, it can provide insight into the molecular composition and potential anomalies. The Nanite is formed of aptameric polymer composites through a custom laboratory protocol involving a self-assembly process, as is cutting-edge in the field. The target molecule to be detected is broken into domains a*, c* and b* respectively. Note, the target molecule is usually self-folded to varying degrees dependent upon the particular sequence composition of each individual target. For clarity, it is depicted unfolded in this illustration for ease of understanding. Domain a of the Sentry hybridises sequence specifically with the domain denoted a* on the target sequence to be detected. The aptameric arm of the Sentry begins self-folded with a small toehold region unpaired in the absence of the target molecule. The Stabilizer nanite possesses a single fluoro pho re molecule (F) and no quencher moieties and is organised to self-fold into a hairpin like structure (c* and c, with d as the unpaired loop region in between) in the absence of the target-molecule, with a long, exposed, single-stranded toehold region denoted b. The Signaller module comprises a single fluorophore molecule(F) and a long, unpaired single-stranded toehold region (d*), followed by a hairpin( c and c* with b* as the unpaired loop region in between.) Figure 2: Detailed Anatomy of the Sentry -Target-Stabilizer interaction A zoomed-in illustration of the Sentry-target bound complex aligning to a Stabilizer module Which is expanded to detail its multiple subunits which encompass specific DNA sequences designed for target molecule recognition. Appropriate co-hybridising domains are marked with the corresponding letter. The 5’ portion of the target sequence is sequence-specifically hybridised to the 3’ portion of a nanite arm on the surface of a Sentry. Following this initial attachment, the Sentry provided sufficient contact and access points (c*, which becomes exposed through at least the partial anchoring by the Sentry, exposing the c* domain and the b* domain of the target. The b* domain serves as the landing pad for the Stabilizer module whose single-stranded toehold region(denoted b) finds, and sequence specifically hybridises initiating a hybridisation through strand displacement of any present secondary structure of the target. Portions b and c of the Stabilizer sequence-specifically hybridise with the corresponding b* and c* portions of the target. Figure 3: Signaller Module Subunits and association with Sentry-Target-Stabilizer complex A zoomed-in illustration of the Sentry-target-stabilizer bound complex aligning to a Signaller module which is expanded to detail its multiple subunits. With the Stabilizer anchoring the target to the Sentry, the remaining unpaired portion of the Stabilizer d provides the landing pad for Signaller module to hybridise sequence-specifically through its toehold domain d* -resulting in a sequence-specific hybridisation and strand displacement of the Signallers c* and c domain in favour of the signaller c hybridising to the stabilizers own c* module. The result of this association is a bound signaller which remains anchored to the Sentry and its captured target by way of the stabilizer module. The bound signaller leaves exposed a b* and c* domain which serves as the anchoring point for a second Stabilizer module. Figure 4: Signal Production from the signaller module schematic A detailed portrayal of the Signaller module, emphasizing the sequences responsible for producing a detectable output (e.g., fluorescent) upon activation. In scheme i the Sentry Nanite is inactive, with no target interaction occurring. Scheme ii denotes a target by itself with the corresponding sections of interest annotated as domains. In scheme iii the Sentry anchors the target to its surface by way of toehold mediated strand-displacement and / or sequence specific hybridisation of the a* domain of the target with the a domain of the Sentry aptameric arm. Anchoring of the target molecule to the Sentry through domain a-a* hybridisation facilitates interaction of the Stabilizer module’s b and c domains which hybridise preferentially with the target over hybridising with itself in its self-folded state owing to favourable thermodynamics which drive the unwinding of the Stabiliser which minimises its free energy through preferential hybridisation to the target. In doing so, the stabiliser leaves unpaired its d and c* domains which facilitate the sequence-specific hybridisation of a signaller module through its unpaired d* overhang and the correspondingly complementary c domain. Once a first signaller module is hybridised to a first stabilizer, the remaining unpaired portion of the first signaller with domains b* and c* provides the necessary anchoring point for a second Stabilizer module to hybridise to. In this fashion, a sequence-specific hybridisation cascade can occur which results in the anchoring of the target to the Sentry and in parallel the production of a pronounced fluorescence increase through sequential addition of alternating Stabilizer and signaller modules. Figure 5: Nanite use process An illustration detailing the main steps in Nanite use experimentally. Scheme 1 Denotes the starting point which begins with a sample containing both the target molecule of interest in addition to other non-specific components. Scheme 2 involves simply the addition of Nanites(given to mean Sentry, Stabilizer and Signaller modules). Scheme 3 denotes a zoomed out depiction of the reaction as illustrated in more detail in figures 4 and 3. Once the reaction has occurred, the sample can be manipulated as in scheme 4 whereby a magnet is applied to the reaction tube to sequester the sentry and by proxy any captured target molecule and associated bound Stabilizer and signaller. Scheme 5 illustrates the simple wash-steps which can be performed through utilisation of the magnet, followed by decanting supernatant and resuspension in wash buffer. Scheme Figure 6: Compartmentalization of Nanites Mechanism A focused illustration of Nanite compartmentalisation into Large arrays of Femtoliter reaction chambers to confine a single Nanite as illustrated in scheme 6 of figure 5. The Nanite is here depicted anchoring a single target molecule to which Auxiliary Nanites Stabilizer and Signaller have initiated a hybridisation cascade upon. The small volume of the Femtoliter Array serves to house at most one Nanite-capture complex (Sentry-Target-and n amount of stabilizer and signaller respectively. Confinement in this small volume enables a digital counting methodology to be applied rendering single-molecule sensitivity and specificity through Nanites achievable. Figure 7: Digital Simulation Framework A flowchart and schematic representing the computer program product designed to simulate Nanite interactions. The software simulates experimental conditions and takes specific user-entered inputs to produce a tailored nanite sequence composition to the given target of interest. Inputs include the target sequence, reaction conditions such as ion concentration or workable temperature range as well as specified sub-domains of interest on the target. The software simulations provide an outputted Nanite sequence composition which can then be experimentally verified, and feedback / validations fed back into the software. Figure 8 : Alternative Sentry Nanite aptamer arm configuration An alternate design for the Nanite aptamer arms which features a different length toehold region with respect to a varying length self-folded hairpin-like component. The mechanism itself of the Nanite with respect to Sentry, Stabilizer and Signaller interactions remains unchanged from figure 3 and 4.

Claims

1. A programmable Aptameric polymer composite termed "Nanite", characterized by its hybrid silica / iron structure with embedded folded, reconfigurable DNA aptamers for the purposes of targeted Molecular Nucleic-acid detection2. The Nanite as per claim 1, designed to remain self-folded and inert in the absence of a target molecule.

3. The Nanite as claimed in claim 1 or 2, wherein upon presence of a target molecule, the Sentry module component activates and binds to the target molecule in a 1:1 molecular ratio.

4. The Nanite as claimed in any preceding claim, wherein activation of the Nanite initiates a hybridization cascade with other Stabilizer and Signaller modules, resulting in a pronounced fluorescent signal increase.

5. The Nanite as per any of the preceding claims, whose activation is characterized by its dynamic fluorescent signal increase including but not limited to a shifting from red only (inactive) to additionally green (active) upon target capture.

6. The Nanite as per any preceding claim, showcasing magnetic properties, enabling precise manipulation and confinement within in femtoliter-sized microfluidic chamber arrays for the purpose of detection digitally.

7. A method for designing Nanites using custom-developed algorithm / software tools, optimizing sequence design, thermodynamic stability, and functional performance.

8. A method of utilising programmable polymer-DNA nanostructure composites paired with microcompartmentalisation across large arrays for enzyme-free, digital singlemolecule nucleic-acid detection.

9. The Nanite as per any preceding claim, facilitating target extraction, purification, and concentration as part of the detection workflow, negating the need for external preprocessing.

10. A system comprising the Nanite as per any of claims 1 to 6, paired with microfluidic microchamber arrays, enabling enhanced sensitivity and specificity in target detection through reduced signal to noise ratio compared with bulk, ensemble methods of detection.

11. A programmable Nanite as per any of the preceding claims, wherein its DNA aptamers are specifically designed for dynamic behaviour in response to environmental stimuli.

12. The Nanite as per any preceding claim, designed with specific sequence lengths and bonding setups to enhance specificity and minimize off-target bindings.

13. The Nanite as per any preceding claim, wherein its activation and subsequent chain reaction initiation begins in seconds upon target detection.

14. The Nanite as per claim 3, wherein the hybridization involves three dynamic Nanite components of Sentry, Stabilizer and Signaller, emphasizing an enzyme-free, thermodynamically driven mechanism of signal amplification without prior target amplification.

15. The Nanite as per any preceding claim, designed to change its three-dimensional structure upon interaction with specific target sequences, facilitating target capture.

16. A method for detecting target molecules using the Nanite as per any of claims 1 to 10, wherein the Nanites' shift in fluorescence signal provides a clear indication of target presence in a binary format within a digital assay.

17. The Nanite as per any preceding claim, wherein its arms are self-folded, rearrangeable aptamer sequences capable of specific hybridization and dynamic reconfiguration.

18. The Nanite as per any preceding claim, devoid of Fluorophore-Quencher pairing, focusing on single fluorophores and subsequent fluorescence additions for signal detection.

19. The Nanite as per any preceding claim, designed for applications in microfluidic arrays, ensuring each Nanite is physically isolated from others for precise individual target detection.

20. The Nanite as per any preceding claim, where the iron core serves a dual purpose of imparting magnetic properties and adding weight to the structure, facilitating its confinement in the micro fluidic arrays when spread across the surface.

21. A system comprising the Nanite as per any of claims 1 to 10, and a magnet, facilitating the precise positioning and confinement of the Nanites.

22. The Nanite as per any preceding claim, wherein its design and function differ significantly from traditional standard ensemble amplification hairpins measured in bulk, showcasing enhanced sensitivity and specificity in molecular detection.

23. The Nanite as per any preceding claim, wherein the thermodynamic equilibrium shifts in the presence of the target molecule, ensuring specific and precise target capture.

24. A method utilizing the Nanite as per any of claims 1 to 10, which avoids heavy extraction and purification steps, simplifying the process of target molecule detection.

25. The Nanite as per any preceding claim, designed for digital counting methodologies, distinguishing it from traditional bulk intensity measurement techniques.

26. A method of detecting specific sequence nucleic-acid target comprising: a Nanite as per claim 3, wherein the Sentry component captures the target, enabling Stabilizer and Signaller module components to sequentially bind initiating a chain-reaction hybridization cascade emphasizing a single-molecule sensitive, specific enzyme-free, thermodynamically driven mechanism of signal amplification without any target amplification.Conclusion:The present invention provides a novel and efficient approach to nucleic acid detection through the use of nanites. These programmable DNA nanostructures offer highly specific and sensitive target molecule recognition, along with exponential signal amplification through a hybridization chain reaction. The applications of nanites extend to various fields, including life sciences research and clinical diagnostics.

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