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WO2024260562A1 - Adn aléatoire exploitable - Google Patents

Adn aléatoire exploitable Download PDF

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Publication number
WO2024260562A1
WO2024260562A1 PCT/EP2023/066962 EP2023066962W WO2024260562A1 WO 2024260562 A1 WO2024260562 A1 WO 2024260562A1 EP 2023066962 W EP2023066962 W EP 2023066962W WO 2024260562 A1 WO2024260562 A1 WO 2024260562A1
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Prior art keywords
sequence
composition
dsodns
random
sequencing
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English (en)
Inventor
Robert N. Grass
Wendelin Jan Stark
Reinhard Heckel
Anne M. LÜSCHER
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Eidgenoessische Technische Hochschule Zurich ETHZ
Technische Universitaet Muenchen
Original Assignee
Eidgenoessische Technische Hochschule Zurich ETHZ
Technische Universitaet Muenchen
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Application filed by Eidgenoessische Technische Hochschule Zurich ETHZ, Technische Universitaet Muenchen filed Critical Eidgenoessische Technische Hochschule Zurich ETHZ
Priority to PCT/EP2023/066962 priority Critical patent/WO2024260562A1/fr
Publication of WO2024260562A1 publication Critical patent/WO2024260562A1/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Definitions

  • the invention relates in general to compositions comprising a plurality of double-stranded oligodeoxynucleotides (dsODNs), methods of producing such compositions, the use of such compositions, a set comprising such a composition associated with an entity, and methods of verifying entities associated with such compositions.
  • dsODN compositions having a specific template structure, in which essentially random segments alternate with essentially identical segments, which results in partly random DNA, which can nevertheless be operated, e.g., for verification and/or authentication purposes.
  • Non-biological applications of DNA have gained importance due to the unique chemical properties of nucleic acids.
  • Single-stranded DNA contains 455 exabytes of information per gram, and molecular tools exist to write, read, and copy this information chemically.
  • the extraordinary storage density, synthetic accessibility, and structural properties of DNA open up new fields of application.
  • synthetic DNA has been used for digital data storage, barcoding and tracing, and steganography.
  • Techniques to encode information in synthetic DNA are known, wherein digital information is translated (using a given translation method) into a sequence combining the four natural deoxynucleotides (nucleobases: adenine, cytosine, guanine, and thymine). The sequence is then synthesized as DNA. In this form, the data can be stored in a highly compact way (with high storage density) and for long storage durations.
  • DNA computation has emerged as an interdisciplinary field that makes use of the tools provided by biology, which enable operations on a molecular level (e.g., copying, hybridization, extraction) and that can be exploited to perform calculations.
  • Nucleic acids have been successfully used to solve combinatorial problems as well as computationally hard tasks and were implemented in logic gates and for random number generation.
  • Cryptographic hash functions are "oneway" functions that calculate an output value from an input using mathematical operations that are relatively easy to perform in one direction, but very hard to invert. Moreover, such functions are designed in such a manner that "collisions" are very improbable. I.e., it is very unlikely to find two distinct inputs that produce the same output through such a function.
  • PUFs physical unclonable functions
  • Such functions exploit random features occurring naturally, or according to a non-deterministic process.
  • a PUF is a physical object, which, for a given input and conditions (i.e., the challenge), provides a given output (i.e., the response to the challenge).
  • PUFs are characterized by their ability to translate an input (challenge) into an output (response) through a physical system that is unique and cannot be replicated, such that the challengeresponse pairs (CRPs) are very difficult or impossible to predict.
  • the outputs of a PUF can accordingly serve as a "digital fingerprint".
  • a fingerprint can notably serve as a unique identifier.
  • PUFs are similar to cryptographic hash functions, except that they rely on a physical source of disorder instead of number theory. PUFs have been proposed for applications in intellectual property protection, public key cryptography, and anticounterfeiting of goods and services, amongst other examples.
  • the present inventors set themselves the challenge to design unclonable objects that are more versatile than usual PUF objects and allow new functionalities.
  • the present invention is embodied as a composition including a plurality of double-stranded oligodeoxynucleotides (dsODNs).
  • This composition is sometimes referred to as a dsODN composition in this document.
  • the dsODNs of said plurality have a same length of between 47 and 300 bp, preferably between 80 and 150 bp, and more preferably between 85 and 130 bp.
  • the dsODNs are structured according to a same template structure.
  • the template structure consists of an orderly set of sequence portions having respective lengths that are constant across all the dsODNs of said plurality.
  • the orderly set of sequence portions includes a first random segment, a first sequencing adapter, a second random segment, a second sequencing adapter, and a third random segment. Such segments are consecutively arranged to form a sequence.
  • Each of the first random segment, the second random segment, and the third random segment of the dsODNs of said plurality consists of essentially random permutations of nucleotides, whereas the first sequencing adapter and the second sequencing adapter of the dsODNs of said plurality consist, independently of each other, of essentially a same sequence of nucleotides.
  • a template structure as proposed herein gives rise to partly random DNA, which can be operated for verification and/or authentication purposes.
  • the underlying DNA sequence structure is sometimes referred to as relating to "operable random DNA” (or orDNA for short).
  • This terminology may similarly refer to dsODNs having said template structure, and which form a DNA pool or a DNA composition.
  • the proposed template structure of the dsODNs results in the second random segments of the dsODNs of said plurality of dsODNs being simultaneously sequenceable, or co- sequenceable, thanks to the sequencing adapters. That is, the partly (if not fully) defined sequencing adapters flanking the second random segments ensure that the latter can be simultaneously sequenced (i.e., are co-sequenceable) across the plurality of dsODNs, using a same sequencing primer having a defined sequence (as opposed to a randomized primer).
  • the composition can be used in a similar way as a mathematical one-way function and as a physical unclonable function (PUF).
  • PUF physical unclonable function
  • the composition can be used as a physical fingerprint, which can be challenged to verify or authenticate a product, an object, or any entity, with which the composition is paired.
  • the random (or quasi-random) information it contains results in that the composition cannot be re-generated from scratch, nor be copied and distributed by a malicious actor.
  • the same composition can be subjected to multiple challenges, hence providing higher certainty as to an associated item or entity.
  • the underlying technology is scalable, and samples of the composition can be distributed to multiple users, unlike usual PUF objects.
  • the proposed composition is more versatile than usual PUF objects and enable more functionalities. Accordingly, the proposed composition can adequately be used for securing objects or entities, in particular objects having high stakes in authenticity.
  • each of the first sequencing adapter and the second sequencing adapter has, independently of each other, a length of between 13 and 30 bp, preferably between 18 and 22 bp.
  • an average Levenshtein distance between first sequencing adapters of the dsODNs of said plurality is less than f a x Li, where Li is the length of the first sequencing adapters and f a is equal to 0.45.
  • an average Levenshtein distance between second sequencing adapters of the dsODNs of said plurality is less than f a x L 2 , where L2 is the length of the second sequencing adapters.
  • f a is equal to 0.30 or 0.10.
  • the first random segment has a length / x of between 5 and 25 bp (preferably between 6 and 10 bp)
  • the second random segment has a length / 2 of between 11 and 200 bp (preferably between 15 and 50 bp, more preferably between 18 and 22 bp)
  • the third random segment has a length I3 of between 5 and 25 bp (preferably between 6 and 10 bp).
  • an average Levenshtein distance between first random segments of the dsODNs of said plurality is larger than g a x l lr where /1 is the length of the first random segments.
  • an average Levenshtein distance between second random segments of the dsODNs of said plurality is larger than g a x / 2 , where I2 is the length of the second random segments
  • an average Levenshtein distance between third random segments of the dsODNs of said plurality is larger than g a x / 3 , where I3 is the length of the third random segments.
  • g a is equal to 0.55.
  • g a is equal to 0.70, or 0.90.
  • the orderly set of sequence portions of each of the dsODNs of said plurality further comprises two outer handle sequences, these including a first handle sequence and a second handle sequence, whereby the first handle sequence, the first random segment, the first sequencing adapter, the second random segment, the second sequencing adapter, the third random segment, and the second handle sequence, are consecutively arranged in the sequence.
  • each of the first handle sequence and the second handle sequence consist, independently of each other, of essentially a same sequence of nucleotides.
  • each of the first handle sequence and the second handle sequence has, independently of each other, a length of between 1 and 30 bp, preferably between 3 and 30 bp, and more preferably between 3 and 22 bp.
  • each of the first handle sequence and the second handle sequence has, independently of each other, a length of between 10 and 30 bp, preferably between 18 and 22 bp.
  • the first handle sequence and the second handle sequence have, independently of each other, a length of between 1 and 9 bp, preferably between 4 and 7 bp.
  • terminal base pairs (bp) of the dsODNs of said plurality are at least partly random, whereby no more than 55% of the dsODNs of said plurality have the same terminal base pairs.
  • terminal base pairs of the dsODNs are deoxynucleotide - dideoxynucleotide base pairs, preferably selected from a group consisting of ddA-dT base pairs, ddC-dG base pairs, ddG-dC base pairs, and ddT-dA base pairs.
  • ddA 2',3'-dideoxyadenosine
  • ddC 2', 3'- dideoxycytidine
  • ddG 2',3'-dideoxyguanosine
  • ddT 2',3'-dideoxythymidine
  • dA, dC, dG, and dT refer to the respective 2'-deoxynucleotides.
  • the composition comprises a total amount of DNA in the range of 0.05 ng to 1000 ng, preferably 1 ng to 25 ng.
  • the invention is embodied as a method of producing a dsODN composition as defined above.
  • the method comprises a first step of (a) providing an initial composition including a plurality of single-stranded oligodeoxynucleotides (ssODNs).
  • the initial composition is also referred to as an ssODN composition in this document.
  • the ssODNs of said plurality have a same length of between 47 and 300 bp, preferably between 80 and 150 bp, and more preferably of between 85 and 130 bp. They are structured according to a same template structure, the latter consisting of an orderly set of sequence portions having respective lengths that are, by design, constant across all the ssODNs of said plurality.
  • the orderly set of sequence portions includes a first handle sequence, a first random segment, a first sequencing adapter, a second random segment, a second sequencing adapter, a third random segment, and a second handle sequence, which are consecutively arranged to form a sequence.
  • Each of the first random segment, the second random segment, and the third random segment of the ssODNs of said plurality consists of essentially random permutations of nucleotides.
  • the first handle sequence, the second handle sequence, the first sequencing adapter and the second sequencing adapter of the ssODNs of said plurality consist, independently of each other, of essentially a same sequence of nucleotides.
  • the method further comprises a step (b) of subjecting the initial composition to a polymerase chain reaction to obtain a dsODN composition (orDNA pool or orDNA composition) as described earlier.
  • a dsODN composition orDNA pool or orDNA composition
  • the ssODN composition is amplified over its entire length using a pair of primers (forward and reverse primer) that anneal to the first handle sequence and the second handle sequence, respectively.
  • the method further comprises a step (c) of at least partly cleaving the first handle sequence and the second handle sequence to obtain a dsODN composition as described earlier, wherein residual portions of the first handle sequence and the second handle sequence, if any, have a length that is less than 10 bp, preferably less than 8 bp.
  • first handle sequence and the second handle sequence are only partly cleaved, whereby each of the first handle sequence and the second handle sequence has, independently of each other, a length of between 1 and 9 bp, preferably between 4 and 7 bp.
  • the invention is embodied as a set comprising an entity and a dsODN composition as defined earlier, wherein the dsODN composition is associated with the entity.
  • the composition is attached to an object, or a packaging thereof, corresponding to that entity.
  • the invention is embodied as a use of such a dsODN composition as a physical fingerprint of an entity of interest.
  • the invention is embodied as a method of verifying an entity of interest associated with the dsODN composition.
  • This method comprises performing a verification procedure, wherein this verification procedure first comprises performing a polymerase chain reaction (PCR), on the composition.
  • the PCR involves one or more pairs of PCR primers, wherein each of the one or more pairs includes a forward PCR primer and a reverse PCR primer, which are respectively adapted to bind the first random segment and the third random segment, respectively, of at least some of the dsODNs of said plurality, so as to amplify sequences of the dsODNs based on the one or more pairs of PCR primers.
  • PCR polymerase chain reaction
  • the verification procedure further comprises sequencing the amplified sequences to obtain a sequencing dataset containing sequencing reads.
  • the sequencing dataset is preferably obtained by filtering out sequencing reads that are inconsistent with said first sequencing adapter and said second sequencing adapter.
  • the verification procedure further comprises performing a k-mer extraction analysis of at least a subset of the sequencing reads, where 7 ⁇ k ⁇ 20, preferably 8 ⁇ k ⁇ 16.
  • the k-mer analysis is preferably restricted to a subset of most- frequently occurring ones of the sequencing reads.
  • the method further comprises: assigning a given portion of the composition to an entity; subsequently receiving the given portion assigned, or a part thereof, for verification purposes, whereby said verification procedure is performed based on the received portion, or the received part thereof, to obtain a test result; and verifying said portion of the composition by comparing the test result with a reference result as obtained by performing the same verification procedure on a reference portion of the composition.
  • comparing the test result with the reference result comprises measuring a statistical similarity between two outcomes of the -mer extraction analysis, as obtained by performing said verification procedure on the given portion and the reference portion of the composition.
  • Said two outcomes preferably consist of two sets of extracted features, which are more preferably extracted, each, in a form of a onedimensional array of numbers.
  • measuring the statistical similarity between said two outcomes comprises weighting -sequences reads obtained according to the k-mer analysis in accordance with respective frequencies of occurrence.
  • the similarity coefficient is preferably measured as a Jaccard coefficient.
  • the method further comprises mapping a set of input numbers to unique pairs of the PCR primers and mapping the input numbers to output numbers generated based on the sequencing reads obtained.
  • FIG. 1 is a schematic representation of the template structure of dsODNs according to embodiments
  • FIG. 2 is a schematic representation of an exemplary implementation of a chemical unclonable function using a template structure according to embodiments
  • FIG. 3 shows the relative frequency (%) of the four nucleobases A (top left), C (top right), G (bottom left) and T (bottom right) across the 21 positions of the second random segment (output sequence) as per counts in Illumina sequencing results with two different inputs and their replicates, as obtained in embodiments;
  • FIG. 4 shows the relative counts (y-axis) of the 10 most frequent output sequences (x- axis numbered by rank) of individual executions of a procedure as involved in embodiments
  • FIG. 5 shows the relative frequency (%) of the four nucleobases A (top left), C (top right), G (bottom left) and T(bottom right) across the 21 positions of the second random segment (output sequence) as per counts in Illumina sequencing results of an example according to embodiments;
  • FIG. 6 is a schematic of an example procedure for chemically synthesizing random DNA sequences
  • FIG. 7 is an illustrative sketch of a principle of random DNA synthesis, schematically showing growing chains on solid support employing an equimolar mix of the four DNA nucleotides, as involved in embodiments;
  • FIG. 8 is a sketch summarizing procedures involved in generating dsODN compositions according to embodiments and operating them as chemical unclonable functions;
  • FIG. 9 shows the Ct number plotted against the number of arbitrary bases in the reverse primer with sigmoidal fit, as obtained in embodiments.
  • FIG. 10 is a histogram of output similarity scores of like and unlike inputs across all challenges of an example, as obtained in embodiments;
  • FIG. 11 illustrates an experimental procedure to amplify dsODN compositions according to embodiments, using handle primers by making copies of copies, leading to multiple generations, as in embodiments;
  • FIG. 12 is a sketch of a procedure to remove the handles and thereby obtain a dsODN composition than cannot be copied (unclonable), as in embodiments;
  • FIG. 13 shows an exemplary AGE photograph showing a purified dsODN band after PCR amplification of a 121 nt long ssODN library, as in embodiments;
  • FIG. 14 shows exemplary AGE photographs depicting purified dsODN bands after various stages of challenge-response-pair generation, as involved in embodiments;
  • FIG. 15 is a sketch illustrating the general principle of a -mer extraction from sequencing data, as involved in embodiments.
  • FIG. 16 is a sketch illustrating a comparison performed based on a weighted Jaccard similarity, as used in embodiments.
  • FIG. 17 is a schematic representation of an exemplary implementation of a chemical unclonable function, as in embodiments.
  • FIG. 18 shows AGE photographs depicting dsODN bands after restriction digest, as obtained in embodiments
  • FIG. 19 shows a gel image showing that dsODNs comprising modified handle sequences containing terminal dideoxynucleotides, as obtained in embodiments, cannot be ligated;
  • FIG. 20 shows an expected number of sequences (x-axis) in a pool that perfectly matches with an input primer of a given length, as in embodiments.
  • compositions, methods, uses, and sets, embodying the present invention will now be described, by way of non-limiting examples.
  • Section 1 addresses particularly preferred embodiments.
  • Section 3 includes a detailed description of the appended drawings, including the numeral references therein.
  • compositions of double-stranded oligodeoxynucleotides dsODNs. 10
  • compositions of double-stranded oligodeoxynucleotides dsODNs
  • dsODNs doublestranded oligodeoxynucleotides
  • This composition is also referred to as a dsODN composition in the following.
  • the dsODNs are unmodified (i.e., they consist of deoxynucleotide base pairs).
  • the dsODNs may possibly be modified, as in other embodiments.
  • terminal base pairs of the dsODNs can be deoxynucleotide - dideoxynucleotide base pairs, for reasons that will become apparent later.
  • the composition includes a plurality of dsODNs.
  • the dsODNs of said plurality have a same length of between 47 and 300 base pairs (bp). Preferably, this length is of between 80 and 150 bp, and more preferably between 85 and 130 bp.
  • the dsODNs of said plurality are all structured according to a same template structure.
  • This structure consists of an orderly set of sequence portions having respective lengths. The lengths of such sequence portions are, by design, constant across all the dsODNs of said plurality.
  • the orderly set of sequence portions includes five segments, i.e., a first random segment, a first sequencing adapter, a second random segment, a second sequencing adapter, and a third random segment. Such segments are consecutively arranged and accordingly form a sequence such as shown in FIG. 1. Note, the first, second, and third random segments can also be referred to as first input sequence, output sequence, and second input sequence, respectively, for reasons that will become apparent later.
  • each of the first random segment, the second random segment, and the third random segment of the dsODNs of said plurality consists of essentially random permutations of nucleotides. That is, any two random segments, whether belonging to a same dsODN of said plurality or not, essentially differ. I.e., when comparing two random segments, most of the nucleotides of the compared segments are likely to differ, as further explained below.
  • the first sequencing adapter and the second sequencing adapter of the dsODNs of said plurality consist, independently of each other, of essentially a same sequence of nucleotides. That is, any two first sequencing adapters (i.e., belonging to two different dsODNs) are essentially equal. I.e., when comparing two first sequencing adapters of two distinct dsODNs of said plurality, most of the nucleotides of the compared segments are identical. The same holds for the second sequencing adapters of the dsODNs of said plurality.
  • the above specifications concern dsODNs of the plurality of dsODNs of the composition.
  • the dsODN composition may possibly include additional dsODNs (i.e., in addition to the plurality of dsODNs referred to above) having different characteristics.
  • the above specifications concern at least a given subset of dsODNs of the compositions.
  • the dsODNs can be subject to noise, meaning that distinct subsets of dsODNs may, in principle, be identified in the composition, where the dsODNs of such subsets have slightly different lengths, meaning also that lengths of the sequence portions may slightly vary across such subsets.
  • a substantially large number of dsODNs can in principle be identified in the composition, which have a same length and are structured according to a same template structure as described above.
  • the dsODNs refer to sequences of a same subset (i.e., corresponding to said plurality); they obey a same template structure, such that they have all a same length, and the respective lengths of their sequence portions are identical across all such dsODNs. Nevertheless, the lengths of the individual segments within a given template structure, i.e., the lengths of the first random segment vs. the length of the first sequencing adapter vs. the length of the second random segment vs. the length of the second sequencing adapter vs. the length of the third random segment may, and typically do, differ, as exemplified later.
  • a “sequence portion”, as used above, is a segment of a sequence. So, each of the above sequence portions are segments of a sequence spanned by a dsODN. According to the present template structure, the random permutations occurring in the random segments cause to increase the entropy of the dsODNs, while the partial order in the interspersed sequencing adapters cause to decrease it.
  • the proposed template structure of the dsODNs results in that the second random segments (also referred to as output sequences) of the dsODNs of said plurality of dsODNs are simultaneously sequenceable, or co-sequenceable.
  • “simultaneously sequenceable”, or “co- sequenceable” means that the dsODNs as a whole, or at least certain segments of the dsODNs, particularly the second random segments (output sequences), can be simultaneously sequenced using a same pair of sequencing adapter primers having a defined nucleotide sequence (as opposed to random primers, i.e., a mixture of primers essentially consisting of random permutations of nucleotides).
  • the above definitions exclude the presence of dsODNs consisting only of essentially random segments as such dsODNs are not simultaneously sequenceable, or co- sequenceable (i.e., simultaneously sequenceable), using a same sequencing primer having a defined nucleotide sequence, as further explained below.
  • the partly (if not fully) defined segments (the essentially identical sequencing adapters) flanking the second random segments of said plurality of dsODNs ensure that the second random segments can be simultaneously sequenced (i.e., are co-sequenceable) using a same sequencing primer having a defined nucleotide sequence.
  • the thereby obtained sequencing reads covering the second random segment (output sequence) may also be referred to as “output sequencing reads" or, in short, "outputs”.
  • the first sequencing adapter and the second sequencing adapter of the dsODNs of said plurality are adapted to bind to a same pair of sequencing adapter primers, to allow sequencing of all dsODNs of said plurality using that same pair of sequencing adapter primers, as in embodiments.
  • the first sequencing adapter and the second sequencing adapter are adapted for binding to each of the sequencing adapter primers with a melting temperature (T m ) of between 45°C to 60°C.
  • T m melting temperature
  • the first sequencing adapter is configured to be bound by a first sequencing adapter primer with a temperature T m of between 45°C to 60°C
  • the second sequencing adapter is configured to be bound by a second sequencing adapter primer with a temperature T m of between 45°C to 60°C, each of the first and second sequencing adapter primer independently of each other having a defined nucleotide sequence. Measuring and/or calculating T m is within the ordinary skill.
  • neither of the first random segment, the second random segment, and the third random segment of the dsODNs have a sufficient statistical identity (also in terms of average, relative Levenshtein distance, as explained below) across the plurality of dsODNs to be bound by a same oligonucleotide, such as a same primer. Accordingly, it is not possible to bind any one of the first random segments, the second random segments, and the third random segments, of all the dsODNs, using a same primer or same oligonucleotide having a defined sequence.
  • each of the first random segment, the second random segment, and the third random segment, of the dsODNs consists of essentially random permutations of nucleotides
  • a single primer having a defined nucleotide sequence i.e., not consisting of random permutations of nucleotides
  • a fraction of the first random segment, the second random segment, or the third random segment of the dsODNs will be bound by a primer or an oligonucleotide having a defined sequence, even though each of the first random segment, the second random segment, and the third random segment of the dsODNs, consists of essentially (i.e., substantially) random permutations of nucleotides.
  • the dsODNs of a certain subset of the composition may be amplified by PCR using a selected pair of PCR primers having a defined nucleotide sequence, also referred to as "input primers", as opposed to a pair of random primers, i.e., a mixture of primers essentially consisting of random permutations of nucleotides. That said, different subsets of dsODNs may be amplified, depending on the nucleotide sequence of the selected pair of defined PCR primers (input primers).
  • the first random segment and the third random segment (i.e., the first input sequence and the second input sequence, respectively) of a certain subset of dsODNs can be at least partially bound by the selected pair of defined PCR primers (input primers), whereby said subset is amplifiable by PCR using the selected pair.
  • any subset of dsODNs that happen to comprise a first random segment and a third random segment having sufficient complementarity to the selected pair of defined PCR primers can be amplified by PCR using that selected pair, thereby generating an amplified subset of dsODNs.
  • This PCR is also referred to as "selection PCR" (since a subset of dsODNs is amplified and thereby "selected").
  • Binding of the selected pair of PCR primers (input primers) to the first random segment and to the third random segment (i.e., the first input sequence and the second input sequence, respectively), as opposed to binding to the second random segment (output sequence), the first sequencing adapter or the second sequencing adapter, can be improved by using a selected pair of PCR primers having partial complementarity to a constant segment neighbouring the respective random segment.
  • the partial complementary of the selected pair directs the forward input primer towards binding to a sequence region partly spanning the first random segment and the first sequencing adapter, while directing the reverse input primer towards binding to a sequence region partly spanning the third random segment and the second sequencing adapter.
  • the first input primer may comprise a stretch of 1 to 20, e.g., 3 to 18, such as 4 to 15, 5 to 15 or 8 to 15, consecutive nucleotides that are complementary to the first sequencing adapter directly adjacent to a stretch of 3 to 20, e.g., 5 to 15, 5 to 12 or 5 to 10, consecutive nucleotides that are complementary to the first random segment of a subset of dsODNs.
  • the second input primer may comprise a stretch of 1 to 20, e.g., 3 to 18, such as 4 to 15, 5 to 15 or 8 to 15, consecutive nucleotides that are complementary to the second sequencing adapter directly adjacent to a stretch of 3 to 20, e.g., 5 to 15, 5 to 12 or 5 to 10, consecutive nucleotides that are complementary to the third random segment of a subset of dsODNs.
  • the forward input primer comprises a stretch of 14 consecutive nucleotides that are complementary to the first sequencing adapter directly adjacent to a stretch of 6 consecutive nucleotides that are complementary to the first random segment of a subset of dsODNs.
  • the reverse input primer comprises a stretch of 14 consecutive nucleotides that are complementary to the second sequencing adapter directly adjacent to a stretch of 7 consecutive nucleotides that are complementary to the third random segment of a subset of dsODNs.
  • performing the PCR on the dsODN composition using a selected pair of PCR primers having partial complementarity to a constant segment neighbouring the respective random segment e.g., first input primer partly spanning the first sequencing adapter and the first random segment and second input primer partly spanning the second sequencing adapter and the third random segment
  • a selected pair of PCR primers having partial complementarity to a constant segment neighbouring the respective random segment e.g., first input primer partly spanning the first sequencing adapter and the first random segment and second input primer partly spanning the second sequencing adapter and the third random segment
  • the amplified subset can be sequenced using a same sequencing adapter primer as defined above, thereby generating sequencing reads covering the second random segment (also referred to as output sequence).
  • a specific set of sequencing reads covering the second random segment i.e., the output sequence
  • the pair of PCR primers comprises a forward primer (forward input primer) and a reverse primer (reverse input primer).
  • said specific set of sequencing reads can be analysed to generate a specific sequencing fingerprint.
  • said specific fingerprint may be mapped to an output number, while said selected pair of PCR primers may be mapped to an input number, thereby linking input numbers to output numbers. This, in turn, enables a verification and/or authentication, as discussed later in detail.
  • dsODN compositions may be used to generate so-called challenge-response-pairs (CRPs).
  • CRPs challenge-response-pairs
  • a "challenge” may be a selected pair of input primers (defined PCR primers for selection PCR as described above) and the corresponding "response” being the sequencing dataset obtained after sequencing the amplified (selected) subset of dsODNs.
  • the input primers are mapped to an input number and the sequencing dataset is further analysed and mapped to an output number, the combination of input numbers and output numbers may be regarded as CRP.
  • the orderly set of sequence portions of each of the dsODNs of said plurality of dsODNs further comprises two outer handle sequences as described above.
  • binding of the selected pair of PCR primers (input primers) to the first random segment and to the third random segment (i.e., the first input sequence and the second input sequence, respectively), thereby mitigating binding of input primers to the second random segment (output sequence) can be improved by using a selected pair of PCR primers having partial complementarity to the first handle sequence and the second handle sequence, respectively.
  • the first input primer may comprise a stretch of 1 to 20, e.g. 3 to 18, such as 4 to 15, 5 to 15 or 8 to 15, consecutive nucleotides that are complementary to the first handle sequence directly adjacent to a stretch of 3 to 20, e.g. 5 to 15, 5 to 12 or 5 to 10, consecutive nucleotides that are complementary to the first random segment of a subset of dsODNs.
  • the second input primer may comprise a stretch of 1 to 20, e.g., 3 to 18, such as 4 to 15, 5 to 15 or 8 to 15, consecutive nucleotides that are complementary to the second handle sequence directly adjacent to a stretch of 3 to 20, e.g. 5 to 15, 5 to 12 or 5 to 10, consecutive nucleotides that are complementary to the third random segment of a subset of dsODNs.
  • the forward input primer comprises a stretch of 14 consecutive nucleotides that are complementary to the first handle sequence directly adjacent to a stretch of 6 consecutive nucleotides that are complementary to the first random segment of a subset of dsODNs.
  • the reverse input primer comprises a stretch of 14 consecutive nucleotides that are complementary to the second handle sequence directly adjacent to a stretch of 7 consecutive nucleotides that are complementary to the third random segment of a subset of dsODNs.
  • performing the PCR on the dsODN composition using a selected pair of PCR primers having partial complementarity to the first handle sequence and the second handle sequence i.e., first input primer partly spanning the first handle sequence and the first random segment and second input primer partly spanning the second handle sequence and the third random segment
  • first input primer partly spanning the first handle sequence and the first random segment and second input primer partly spanning the second handle sequence and the third random segment thus improves selective amplification of the subset of dsODNs that happen to comprise a first random segment and a third random segment having sufficient complementarity to the selected pair of defined PCR primers.
  • each of the first sequencing adapter and the second sequencing adapter consists of essentially a same sequence of nucleotides (whereby that sequence can differ between the first and second sequencing adapter) means that their average, relative Levenshtein distance (ARLD) is strictly smaller than 0.50. That is, the average LD across the relevant sequence portions is less than half their respective lengths. In preferred embodiments, however, the ARLD is smaller than 0.45, and preferably smaller than 0.30 (e.g., smaller than 0.20, 0.15 or even 0.10, on average).
  • the relative LD is the ratio of the LD to the length of the compared segments, which length is identical, by definition, for dsODNs of said plurality.
  • the average LD across the first and second sequencing adapters of the dsODNs of said plurality can be formulated as being strictly less than f a x /, where f a is equal to 0.50. In preferred variants, however, this average LD is less than f a x /, where f a is equal to 0.45, 0.30, or even 0.10.
  • the ARLD is calculated as an arithmetic mean of the relative LDs computed over each pair of dsODNs in a set of interest.
  • each of the first sequencing adapter and the second sequencing adapter are, independently of each other, strictly more than 50% identical, preferably at least 55% identical, more preferably at least 70% identical, and even more preferably at least 90% identical, across all the dsODNs of said plurality, on average.
  • the first sequencing adapters are at least 55% identical across all dsODNs of said plurality, on average.
  • the second sequencing adapters are at least 55% identical across all the dsODNs of said plurality, on average.
  • first sequencing adapter sequence and the second sequencing adapter sequence are meant to be identical by design, i.e., as per the fabrication method used (as discussed later in detail). However, they may, in practice, contain deviations and/or errors inherent to vagaries of the synthesis process used.
  • each of the first random segment, the second random segment, and the third random segment of the dsODNs of said plurality consists of essentially random permutations of nucleotides typically means that their average, relative LD is strictly larger than 0.50, preferably larger than 0.55, and more preferably larger than 0.70 (e.g., larger than 0.80 or 0.90), on average. That is, the average LD across the relevant sequence portions is more than half their respective lengths /. I.e., the average LD across each of the first, second, or third random segments in the dsODNs of said plurality strictly larger than 0.50 x /.
  • this average LD is larger than g a x /, where g a is equal to 0.55, 0.70, or 0.90.
  • the random segments have strictly less than 50% sequence identity across all the dsODNs of said plurality. Preferably, they have less than 45% sequence identity, more preferably less than 30%, and even more preferably less than 10% sequence identity.
  • the first sequencing adapter sequence and the second sequencing adapter sequence are essentially (i.e., substantially) identical across the dsODNs of said plurality, whereas the random segments of the dsODNs of said plurality consist of essentially (i.e., substantially) random permutations of nucleotides.
  • the Levenshtein distance is chosen for convenience, as the LD algorithm does not require any user input as to the alignment method, whereas the SWS and NWS methods rely on distinct alignment methods and can be specified different gap penalty parameters. That is, the LD metric is clearer.
  • nucleic acid sequence identity is known in the art. Typically, such techniques revolve around determining the nucleotide sequences of the DNA (or, in the present context, the dsODNs) and comparing these sequences to a second DNA sequence.
  • identity refers to an exact nucleotide-to-nucleotide correspondence of two DNA sequences, respectively. Accordingly, that a DNA sequence has a certain percent sequence identity to another DNA sequence means that, when aligned, the percentage of nucleobases are the same, and in the same relative position, when comparing the two sequences.
  • the percent identity of two DNA sequences is thus the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100.
  • sequence identity can generally be determined by alignment of two nucleic acid sequences using global or local alignment algorithms.
  • sequences of similar lengths are preferably aligned using a global alignment algorithm (e.g., Needleman Wunsch algorithm; cf. J. Mol. Biol. 48 (3) : 443-53) which aligns the sequences optimally over the entire length.
  • Sequences of essentially (i.e., substantially) different lengths are preferably aligned using a local alignment algorithm (e.g., Smith Waterman algorithm; cf. J. Mol. Biol. 147 (1) : 195-197).
  • sequence alignments may be performed using the EMBOSS Needle sequence alignment tool [accessible via https://www.ebi.ac.uk/Tools/psa/emboss_needle/; Madeira et al., Nucl. Ac. Res., 2022, Vol. 50, Web Server issue] using default settings as indicated below:
  • the percent identity can otherwise be computed directly from ARLD results, as exemplified above. That is, the percent identity can be approximated as being equal to 1 - the ARLD result, expressed as a percentage (i.e., multiplied by 100)
  • the maximal Levenshtein distance between any two sequencing adapters corresponding to the same dsODN segment is less that f x L, where L is the constant length of this sequencing adapter and f is a fraction, which is equal to 0.45, preferably equal to 0.30, and more preferably equal to 0.10. That is, the maximal LD between any pair of first sequencing adapters is less than f x Li, where Li is the constant length of this adapter, and the maximal LD between any pair of second sequencing adapters is similarly less than f x L 2 , where L 2 is the constant length of the second adapter. In other words, the maximal LD between any pair of first sequencing adapters or second sequencing adapters is less than a fraction f of the respective lengths of the first sequencing adapter and the second sequencing adapter, across all the dsODNs of said plurality.
  • Such maximal distances of the first sequencing adapter and the second sequencing adapter allow any two dsODNs of said plurality to be simultaneously sequenceable, or co- sequenceable, notwithstanding the essentially random permutations occurring in the random segments.
  • the minimal LD between any two random segments corresponding to the same dsODN segment is larger than g x I, where / is the constant length of this segment and g is a fraction, which is equal to 0.55, preferably equal to 0.70, and more preferably equal to 0.90. That is, the minimal LD between any pair of first random segments of length /1 is larger than g x l lr the minimal LD between any pair of second random segments of length I2 is larger than g x / 2 , and the minimal LD between any pair of third random segments of length I3 is larger than g x / 3 .
  • the minimal LD between any pair of first, second, or third random segments is larger than a fraction g of the respective lengths of such segments across the dsODNs of said plurality.
  • such values may be translated into maximal identity percentages.
  • a certain level of redundancy is expected, as in every DNA pool. I.e., in the present context, there are multiple copies of a same sequence in every orDNA pool.
  • the first sequencing adapter sequence and the second sequencing adapter sequence will often be meant to be identical by design, they may, in practice, contain deviations and/or errors, which will impact the extent to which dsODNs differ. Still, it remains that it is, in principle, possible to identify two dsODNs that differ by at least one permutation of nucleotides in any subset of dsODNs obeying the exact same template structure, notwithstanding the constant sequence portions.
  • the composition may include 10 6 to 10 13 , preferably 10 7 to 10 11 , more preferably 10 7 to 3 x IO 10 unique (i.e., distinct) dsODNs, as well as several copies of each dsODN, preferably between 5 to 1000 copies, more preferably 8 to 100 copies (e.g., 80 copies).
  • a template structure as proposed herein gives rise to partly random DNA, which can be operated for verification and/or authentication purposes.
  • the underlying DNA sequence structure is sometimes referred to as "operable random DNA", or orDNA for short, in this document.
  • operble random DNA and the acronym orDNA similarly refer to dsODNs having said template structure, and which form a DNA pool or a DNA composition.
  • the above sequence structure allows the composition to be used in a similar way as a mathematical one-way function (hereafter a "one-way function” for short) and as a physical unclonable function (PUF).
  • the composition can be used as a physical fingerprint, which can be challenged to verify or authenticate a product, an object, or any entity, with which the composition is paired, as in embodiments discussed below.
  • the proposed composition is unique, and subject to a very low, and therefore negligible, collision probability.
  • such a composition contains sufficient random information, so that it is practically impossible to fully analyse and reproduce it.
  • the proposed sequence structure of the composition is unclonable.
  • the composition cannot be copied, i.e., it is unclonable.
  • the composition can still not be copied. I.e., it is unclonable if the handle sequences are less than 10 bp long and are additionally modified, a concept referred to as "modified partial handle sequences", as discussed below in detail. Accordingly, this composition cannot be copied and distributed by a malicious actor.
  • the dsODNs are still operable. In particular, they can still be used as a one-way function.
  • a copiable (clonable) - but operable - dsODN composition may be transformed into an unclonable (and still operable) dsODN composition, provided the full handle sequences comprise a restriction enzyme cut site, particularly a type IIS restriction enzyme cut site, e.g., a Pie I cut site, thereby rendering the full handle sequences cleavable (cf. example 5, FIG. 12).
  • the same composition (irrespective of the presence and length of outer handle sequences) can be subjected to multiple challenges, hence providing higher certainty as to an associated item or entity, as discussed later in reference to other aspects of the invention (cf. example 2).
  • the proposed composition is more versatile than usual PUF objects and enable more functionalities.
  • the technological barrier required to process DNA-based materials is considerably higher than that required to process numbers and functions in silico, making the proposed solution virtually unbreakable. Therefore, the present approach is suitable for securing objects or entities, in particular objects having high stakes in authenticity.
  • the proposed orDNA structure exploits a chemical process as a source of entropy, whereby the resulting orDNA can reliably be used to determine outputs from given inputs, as with one-way functions.
  • the underlying CUF technology is scalable in terms of the potential input-output pairs it supports, and can be distributed to multiple users, unlike usual PUF objects. For this other reason, the proposed orDNA structure is more versatile than usual PUF objects.
  • the orDNA can be designed so that, e.g., a manufacturer may decide in advance how many times the function can be operated by limiting the amount of dsODN composition (i.e., total amount of dsDNA) that is deposited on an item, something that is not possible with conventional one-way functions, or PUFs.
  • a relatively low amount of dsODN composition e.g., a dsODN composition comprising a total amount of DNA in the range of 0.05 ng to 1000 ng, preferably 1 ng to 25 ng
  • the present description discusses concrete use cases of verifications/authentications, which notably enable multiple simultaneous users to verify each other in a decentralized manner.
  • each of the first sequencing adapter and the second sequencing adapter has, independently of each other, a length of between 13 and 30 bp. Preferably, this length is of between 18 and 22 bp. That is, the first sequencing adapter has a length L x of between 13 and 30 bp (or preferably between 18 and 22 bp), and the second sequencing adapter similarly has a length L2 of between 13 and 30 bp (preferably between 18 and 22 bp), although L x does not necessarily need to be equal to L2. It has been found that the above lengths of the first and second sequencing adapters (L and L2, respectively) are particularly suitable for selectively binding sequencing adapter primers.
  • the average LD between first sequencing adapters of the dsODNs of said plurality is typically less than f a x / .
  • the average LD between second sequencing adapters of the dsODNs of said plurality is typically less than f a x / 2 , where f a is equal to 0.45 (or preferably equal to 0.30 or 0.10).
  • the first random segment in each of the dsODNs of said plurality, has a length / of between 5 and 25 bp, preferably of between 6 and 10 bp. Meanwhile, the second random segment has a length / 2 of between 11 and 200 bp, preferably between 15 and 50 bp, and more preferably between 18 and 22 bp. Finally, the third random segment has a length / 3 of between 5 and 25 bp, preferably between 6 and 10 bp.
  • the average LD an average LD between first random segments of the dsODNs of said plurality is typically larger than g a x / .
  • the average LD between second random segments of the dsODNs of said plurality is typically larger than g a x / 2
  • the average LD between third random segments of the dsODNs of said plurality is typically larger than g a x / 3 , where g a is equal to 0.55, preferably equal to 0.70, or more preferably equal to 0.90.
  • the orderly set of sequence portions of each of the dsODNs of said plurality may further comprises two outer handle sequences.
  • the outer handle sequences include a first handle sequence and a second handle sequence.
  • the first handle sequence, the first random segment, the first sequencing adapter, the second random segment, the second sequencing adapter, the third random segment, and the second handle sequence are consecutively arranged in the sequence.
  • each of the first handle sequence and the second handle sequence consist, independently of each other, of essentially a same sequence of nucleotides.
  • each of the first handle sequence and the second handle sequence has, independently of each other, a length of between 1 and 30 bp.
  • this length is of between 3 and 30 bp, and more preferably of between 3 and 22 bp.
  • Each of the first handle sequence and the second handle sequence preferably consist, independently of each other, of essentially a same sequence of nucleotides.
  • the orderly set of sequence portions may include a first handle sequence and a second handle sequence, where such sequence portions are at least 55% identical, preferably at least 70%, and more preferably at least 90% identical across all the dsODNs in said plurality, or even in the whole composition, as discussed earlier. Note, such percentages may again be calculated from relative LDs. If present, the first handle sequence is located upstream and directly adjacent to the first input sequence, while the second handle sequence is located downstream and directly adjacent to the second input sequence.
  • the present dsODN compositions can be formed as an orDNA pool containing full handle sequences.
  • the dsODN composition contains partial handle sequences.
  • handle sequences having a length of between 10 and 30 bp preferably between 18 and 22 bp
  • full handle sequences preferably between 18 and 22 bp
  • handle sequences having a length of between 1 and 9 bp are referred to as "partial handle sequences", as now discussed in detail.
  • Each of the first handle sequence and the second handle sequence may possibly have, independently of each other, a length of between 1 and 9 bp. Preferably, this length is of between 4 and 7 bp.
  • a handle sequence refers to any one of the two outer handle sequences that the present dsODNs may optionally include, as in preferred embodiments.
  • Modified partial handle sequences Partial handle sequences may possibly be modified, a concept that is referred to as "modified partial handle sequences", which is now discussed in detail.
  • Modified partial handle sequences contain a base pair formed by a dideoxynucleotide and a standard deoxynucleotide, where the dideoxynucleotide is present on the 3' end of each strand of the dsODN cf. example 5).
  • modification of the outer handle sequences prevent ligation, and consequently render the dsODNs containing such modified handle sequences unclonable. Note, the full handle sequences do not contain this modification.
  • This modified base pair is formed by a dideoxynucleotide and a standard deoxynucleotide. It can be introduced by first digesting dsODNs having full handle sequences with a restriction enzyme (preferably a type IIS restriction enzyme, such as Piel), which produces a 5' overhang, and then using a DNA polymerase to blunt the sticky end with a dideoxynucleotide, e.g., a T7 polymerase without 3'-5' exonucleoase activity, which is preferably configured to incorporate ddATP, ddGTP, ddCTP, ddTTP with equal preference.
  • a restriction enzyme preferably a type IIS restriction enzyme, such as Piel
  • a commercially available example of such a modified T7 polymerase is the so-called Sequenase (commercially available from Thermo Fisher).
  • Sequenase commercially available from Thermo Fisher.
  • the full handle sequence must include a suitable restriction site, i.e., a restriction site that is recognized by the respective restriction enzyme, e.g., Piel.
  • full handle sequences are initially employed to generate a modified partial handle sequence that contains a randomized base pair at the restriction enzyme cut site. This has the advantage that, after cleavage with a type IIS restriction enzyme, the 5' overhang resulting from the cleavage is randomized.
  • the resulting base pair formed by the dideoxynucleotide and the standard deoxynucleotide is random, i.e., the 3' terminal position of each strand may consist of a matching base pair that is randomly selected from all four nucleotides, preferably a random selection from ddA-dT, ddC-dG, ddG-dC and ddT-dA base pairs, where the 3'-terminal position of each strand is a dideoxynucleotide.
  • terminal base pairs of the dsODNs of said plurality are at least partly random, whereby no more than 55% of the dsODNs of said plurality have the same terminal base pairs.
  • Ns e.g., Ns > 1000 sequences are arbitrarily selected from the pool, a given sequence position is understood as being random if no more than 55% of the analysed sequences contain the same base at that position. This way, it is also possible to use only two of the four bases to generate a function (which would reduce entropy by half, but would otherwise still work the same), or of mixing randomly synthesized with non-randomly synthesized strands.
  • terminal base pairs of the dsODNs are deoxynucleotide - dideoxynucleotide base pairs, preferably selected from a group consisting of ddA-dT base pairs, ddC-dG base pairs, ddG-dC base pairs, and ddT-dA base pairs.
  • the present compositions may advantageously comprise a total amount of DNA in the range of 0.05 ng to 1000 ng, preferably 1 ng to 25 ng. That is, the amount of DNA present in the composition limits the number of times the DNA pool can be analysed.
  • a composition as described in section 1.1.1 may be verified by detecting the nucleotide composition using PCR and sequencing.
  • a composition as described in section 1.1.1 comprises a first sequencing adapter and a second sequencing adapter (which can also be referred to as 'forward' and 'reverse' sequencing adapters, respectively), a first and a second input sequence and an output sequence.
  • first sequencing adapter and the second sequencing adapter consist, independently of each other, of essentially a same sequence of nucleotides (e.g., the average Levenshtein distance across the dsODN composition is small, as discussed earlier). This is the case for example, if a pair of primers exists that are complementary to the large majority of the adapter sequences across the dsODN composition and with which a PCR can be performed. Furthermore, it has to be shown that the first input sequence and the second input sequence (first and third random segments, respectively) consist of essentially random permutations of nucleotides (e.g., their average Levenshtein distance is large, as discussed earlier).
  • the output sequences (second random segments) consist of essentially random permutations of nucleotides (e.g., this corresponding to large average Levenshtein distances). This may be achieved by performing PCR with primers that bind to the first and second sequencing adapters and subjecting the thus amplified sequences to next generation sequencing.
  • qPCR quantitative PCR
  • primer sets i.e., various pairs of primers
  • Each of the qPCR reactions is run under equal conditions, except that the sequence of the sequencing adapter primers varies for each qPCR (in one series of qPCR reactions, the first sequencing adapter primer remains constant, while the second sequencing adapter primer is varied, whereas in a second series of qPCR reactions, the second sequencing adapter primer remains constant, while the first sequencing adapter primer is varied).
  • a common three-step qPCR protocol can be used for each qPCR reaction, and the qPCR reactions can be performed on a commercially available qPCR platform (e.g., Roche Lightcycler 480 system). Each qPCR programme should include at least 35 cycles.
  • primers with perfect complementarity to the first and second sequencing adapter portions are used (i.e. the primers only bind to the sequencing adapter portions and amplify the portion in between).
  • the first sequencing adapter primer i.e., the forward sequencing adapter primer
  • the second sequencing adapter primer i.e., the reverse sequencing adapter primer
  • the nucleotide arbitrarily added on the 5' end should not be the same one as the one removed from the 3' end.
  • the first sequencing adapter primer remains unchanged, while the second sequencing adapter primer is shortened by one nucleotide on the 3' end and lengthened by an arbitrary nucleotide on the 5' end, compared to the second qPCR (step 3.2), whereby the nucleotide arbitrarily added on the 5' end should not be the same one as the one removed from the 3' end.
  • the first sequencing adapter primer remains unchanged, while the second sequencing adapter primer is shortened by one nucleotide on the 3' end and lengthened by an arbitrary nucleotide on the 5' end compared to the third qPCR (step 3.3), whereby the nucleotide arbitrarily added on the 5' end should not be the same one as the one removed from the 3' end.
  • a plateau is here defined as follows:
  • the mean Ct value change over at least 3 consecutive PCRs as per the description of changing primers above is ⁇ 0.5, after having been >0.5 for at least 3 consecutive PCRs as per the description of changing primers.
  • Any adapted primer should not differ by more than 15% in GC-content relative to the original primer, i.e. the primer used for the first PCR.
  • the T m (melting temperature) of all used primers is within a range of ⁇ 3 °C.
  • step 3 tests the fraction of random permutations of nucleotides in the third random segments (second input sequences) of dsODNs.
  • steps 3.2-3.5 are repeated analogously, wherein the second sequencing adapter primer remains constant in each qPCR reaction, whereas the first sequencing adapter primer is varied in each qPCR reaction as described above with respect to the second sequencing adapter primer.
  • the tested dsODN composition complies with the template structure as defined in section 1.1.1 if the following criteria are fulfilled:
  • the average relative LDs of sequence reads for the portion amplified in between the sequencing adapters, as per step 1 of the exemplary test protocol, is above 0.45.
  • the p-parameter of the fitted curve as described in step 3.7 of the exemplary test protocol for sampling the first input is between 0.15 and 0.4.
  • the p-parameter of the fitted curve as described in step 3.7 of the exemplary test protocol for sampling the second input is between 0.15 and 0.4.
  • Another aspect of the invention concerns a method of producing a dsODN composition as described in section 1.1 (cf. examples 1 and 4; step 202 in FIG. 2, step 802 in FIG. 8).
  • this method comprises two steps (a) and (b).
  • First the method comprises (a) providing an initial composition, which includes a plurality of single-stranded oligodeoxynucleotides, or ssODNs for short (for example referred to as "Library 1" in example 2; cf., e.g., reference 201 in FIG. 2 and reference 801 in FIG. 8; synthesis of an ssODN composition is illustrated, e.g., in FIG. 6 and FIG. 7).
  • the method comprises (b) subjecting the initial composition to a PCR.
  • the ssODNs of said plurality have a same length of between 47 and 300 bp. Preferably, this length is of between 80 and 150 bp, and more preferably between 85 and 130 bp.
  • the ssODNs are structured according to a same template structure, the latter consisting of an orderly set of sequence portions having respective lengths that are constant across all the ssODNs of said plurality.
  • the orderly set of sequence portions of the ssODNs includes a first handle sequence, a first random segment, a first sequencing adapter, a second random segment, a second sequencing adapter, a third random segment, and a second handle sequence. All such segments are consecutively arranged to form a sequence.
  • each of the first random segment, the second random segment, and the third random segment of the ssODNs of said plurality consists of essentially random permutations of nucleotides
  • the first sequencing adapter and the second sequencing adapter of the ssODNs of said plurality consist, independently of each other, of essentially a same sequence of nucleotides.
  • the first and second handle sequences consist, independently of each other, of essentially a same sequence of nucleotides.
  • the initial composition provided is then subjected to a PCR (step b) to obtain a dsODN composition as described earlier.
  • this PCR (“production PCR") is performed using a pair of primers that anneal to the first and second handle sequences, respectively (also referred to as handle primers; cf. example 1).
  • handle primers also referred to as handle primers; cf. example 1.
  • modification reactions may possibly be involved, as usual in the art.
  • handle primers having certain mismatches with the ssODNs to be amplified, e.g., in order to introduce restriction enzyme cut sites that may not be present in the ssODN template.
  • the initial set of sequence portions defining the ssODN sequences and the set of sequence portions defining the dsODN sequence are structurally similar, subject to the lengths, possible modification, and possible absence of the outer handle sequences in the dsODNs, as in embodiments discussed earlier. Since the ssODNs refers to single strands, while the dsODNs refer to corresponding double strands, the dimensions and properties of the ssODNs remain consistent with the dsODNs'.
  • each of the ssODNs may have a length of between 47 and 300 bp and form an orderly set of sequence portions including a first random segment, a first sequencing adapter, a second random segment, a second sequencing adapter, and a third random segment, which are consecutively arranged to form a sequence.
  • each of the ssODNs contain outer handle sequences, i.e., a first handle sequence and a second handle sequence, as discussed above.
  • the first random segment has a length of between 5 and 25 bp, preferably between 6 and 10 bp.
  • the first sequencing adapter has a length of between 13 and 30 bp, preferably between 18 and 22 bp.
  • the second random segment has a length of between 11 and 200 bp, preferably between 15 and 50 bp, more preferably between 18 and 22 bp.
  • the second sequencing adapter has a length of between 13 and 30 bp, preferably between 18 and 22 bp
  • the third random segment has a length of between 5 and 25 bp, preferably between 6 and 10 bp.
  • each of the first sequencing adapter and the second sequencing adapter will preferably be at least 55% identical, preferably at least 70% identical, more preferably at least 90% identical across all the ssODNs, on average, as discussed earlier. Again, such percentage values may be derived from average relative LDs.
  • the dsODNs obtained still include handle sequences.
  • the handle sequences may be cleaved, at least partly, as in preferred embodiments. That is, the method may further comprise a step (c) of at least partly cleaving the first handle sequence and the second handle sequence cf. example 5).
  • the residual portions of the first handle sequence and the second handle sequence if any, may for instance have a length that is less than 10 bp, preferably less than 8 bp.
  • another possibility is to only partly cleave the first handle sequence and the second handle sequence to obtain a composition, in which each of the first handle sequence and the second handle sequence, as partly cleaved, has a length of between 1 and 9 bp, preferably between 4 and 7 bp.
  • the main differences between the initial ssODN library and the dsODN composition is the double- (instead of single-) stranded feature.
  • the resulting dsODNs are, on average, present in multiple copies and are subject to an inevitable PCR bias.
  • the outer handle sequences are present in the ssODN library but are only optional in the dsODN composition (i.e., the orDNA composition or orDNA pool).
  • the length of the outer handle sequences may differ between the ssDNA library and the dsODN composition (i.e., the orDNA).
  • the lengths (or even presence/absence) of the handle sequences in the dsODN composition depend on the handle PCR primers used for amplifying the ssDNA library.
  • PCR amplification of the ssDNA library using a pair of handle PCR primers covering (i.e., binding to) only a part of the handle sequences that are proximal to the first and the third random segment, respectively (as opposed to covering the full handle sequences including the 5' and 3' terminal nucleotides of the ssDNA) yields a dsODN composition where each dsODN comprises partial handle sequences.
  • the lengths of the partial handle sequences depend on the lengths of the sequence regions of the handle sequences present in the ssDNA that is covered (bound) by the handle PCR primers.
  • such partial handle sequences may contain a dideoxy nucleotide at the 3'-ends of both strands.
  • This modification can for instance be introduced by digesting the dsODNs obtained after PCR amplification of the ssDNA library with a type IIS restriction enzyme, such as Piel, which produces a 5' overhang, and then using Sequenase to blunt the sticky end with a dideoxy nucleotide.
  • this position is also random, meaning that in the final dsODN composition, the terminal base pairs (both ends) of the dsODNs vary between dA-ddT, dC-ddG, dT-ddA and dG-ddC base pairs (the dideoxynucleotide being present on the 3'-end of each strand). Randomisation of the terminal position of each strand is achieved by designing the ssDNA library such that a random nucleotide is present at the restriction enzyme cut site.
  • a dsODN composition as described in section 1.1. is associated (i.e., paired) with an object (e.g., a jewel, an item of goods, a consumer goods, such as a car, an electronic device), a person, or, more generally, an entity.
  • an entity can be any object, a human being, an animal, or an organization (e.g., a legal entity).
  • the composition is typically associated with a physical medium used by this abstract entity, i.e., a physical carrier.
  • a further aspect of the invention concerns a set including an entity (e.g., an object) and a dsODN composition as described earlier, wherein the composition is associated with the entity.
  • the composition can be attached to a physical object, or any item, or a packaging thereof.
  • the composition can be used as a physical fingerprint for the corresponding entity.
  • it can be used as a physical fingerprint for physical objects, consumer goods, or other types of objects or items of goods.
  • a sample of the composition may be packaged and attached (e.g., glued) to an object or its packaging.
  • a sample of dsODN composition may be encapsulated in a matrix, made of, e.g., silica, calcium phosphate, or lipid nanoparticles.
  • the encapsulated sample may subsequently be incorporated into (or coated onto) a product or packaging material, for example a paint or a dye, a solid, semi-solid or liquid pharmaceutical, a food product, a polymer-based product, a jewel, e.g., a ring or a gemstone.
  • Another aspect of the invention concerns a method of verifying an entity of interest (e.g., a person or an object as defined above), where this entity is associated with the dsODN composition.
  • entity of interest e.g., a person or an object as defined above
  • the verification method revolves around performing a verification procedure comprising: (i) performing a PCR on the composition; and (ii) sequencing the amplified sequences.
  • the PCR involves one or more pairs of PCR primers (input primers), where each pair of primers includes a forward PCR primer and a reverse PCR primer (each pair of input primers corresponds to a given challenge).
  • the forward and reverse PCR primers are respectively adapted to at least partially bind the first random segment and the third random segment of at least some of the dsODNs of said plurality. This, in turn, makes it possible to amplify sequences of the dsODNs based on said one or more pairs of PCR primers. That is, the PCR causes to amplify sequences of the dsODNs that are being bound by the forward PCR primer and the reverse PCR primer of each selected pair of PCR primers.
  • the amplified sequences are then sequenced to obtain a sequencing dataset, i.e., a set containing sequencing reads.
  • a sequencing dataset i.e., a set containing sequencing reads.
  • the sequencing dataset will advantageously be obtained by filtering out sequencing reads that are inconsistent with the first sequencing adapter and the second sequencing adapter.
  • sequencing reads which can subsequently be used to challenge and verify the physical fingerprint.
  • Various methods can be contemplated to operationalize this, such as methods based on feature extractions (i.e., feature extractors in a machine learning sense) and/or k-mer analyses.
  • feature extraction can be applied to the sequencing reads or, even, to the results of the k-mer analysis.
  • any mapping function can be used to map the sequencing reads onto numbers (or arrays of numbers), something that enables easy comparisons.
  • Preferred embodiments of the verification procedure rely on performing a k-mer extraction analysis, where, typically, 7 ⁇ k ⁇ 20.
  • the value of k is between 8 and 16.
  • the k- mer analysis can advantageously be restricted to a subset of most-frequently occurring sequencing reads. That is, the sequencing dataset considered in the analysis may be restricted to the top-k sequences, e.g., the K sequences that are the most frequently occurring, where K is typically between 1 and 1000.
  • the verification procedure is normally performed at least twice, i.e., to identify reference results and to verify entities to which the same composition is assigned, as further discussed below.
  • the reference results can for instance be identified in the form of output numbers (e.g., arrays of numbers, such as vectors and matrices), with which respective pairs of PCR primers are associated.
  • This verification may notably be used for authentication purposes, as noted earlier.
  • the above composition can be used as a PUF and play the role of a physical fingerprint.
  • embodiments of the verification method involve assigning a given portion of the dsODN composition to an entity of interest, which typically is the entity one wants to verify, though not necessarily. I.e., an intermediate carrier may be used, as noted earlier.
  • a verifier receives the assigned portion of the dsODN, or a part thereof, for verification purposes. From this moment on, the verifier may perform the verification procedure based on the received portion (or part thereof). This yields a test result, which can then be compared with a reference result.
  • the reference result is obtained by performing the same verification procedure as described above, albeit on a reference portion of the composition. Importantly, the reference result can be obtained before (i.e., ex-ante) or after receiving the assigned composition (or part thereof) for verification purposes. In both cases, the comparison makes it possible to verify the (portion of the) composition as initially assigned to the entity of interest.
  • the verification procedure may be performed by directly comparing given output numbers (as obtained for the test composition) with reference output numbers, as obtained by performing the same procedure on the reference composition (or sample thereof). In that case, the comparison is performed so as to verify that the given output numbers match the reference output numbers.
  • the reference composition is assumed to be statistically equivalent to the assigned portion. E.g., the assigned portion is a sufficiently large sample of a reference composition or, conversely, both the assigned portion and the reference composition are sufficiently samples of an initial composition.
  • the verification method further comprises mapping a set of input numbers to unique pairs of the PCR primers. Moreover, the same set of input numbers is mapped to output numbers generated based on the sequencing reads obtained thanks to such primers. In that case, verifying that the given output numbers match the reference output numbers may be performed by: selecting one or more input numbers of the input set; identifying the corresponding one or more unique pairs of primers; and performing the verification PCR reaction based on the identified pairs of PCR primers. Eventually, one verifies whether the output numbers obtained in accordance with the selected input numbers match the reference output numbers.
  • the verification procedure can possibly be performed based on reference results obtained ex-ante for the reference composition.
  • the reference results Once the reference results have been obtained, one can assign the residual portion of the composition to the target entity, or only a sample thereof and, in that case, eliminate the unused part of the composition, for more security. I.e., the composition can no longer be "hacked", at least not at the manufacturer site. Only the target entity is in possession of the composition.
  • the initial composition can be safely stored and then used to perform verifications a posteriori, when and if necessary. In that case, there is no need to map input numbers to output numbers a priori.
  • the advantage of this variant is that it does not require systematically obtaining the reference results in the first place. This may be advantageous in scenarios where entities do not systematically need to be verified.
  • the comparison between the test result and the reference involves a statistical similarity metric. That is, one measures a statistical similarity between two outcomes of the k-mer extraction analysis, as obtained by performing said verification procedure on the given portion (i.e., as corresponding to the position assigned to the entity of interest) and the reference portion of the composition.
  • the two outcomes at issue may for instance consist of two sets of extracted features.
  • the two sets of features can notably be extracted, each, in the form of a one-dimensional array of numbers, i.e., a vector.
  • Various statistical analysis methods can be contemplated. In general, one will seek to favour methods that allow stable arrays (i.e., keys) to be extracted from noisy data.
  • a convenient option is to use fuzzy extractors, as these have a tolerance for noise.
  • autoencoders can be used, too, which can be trained to filter out noise and extract noise-free features. Noise-free features can then safely be compared to authenticate a composition sample.
  • the statistical similarity between said two outcomes is measured by weighting k-sequence reads obtained according to the k-mer analysis in accordance with respective frequencies of occurrence.
  • the similarity coefficient is preferably measured as a Jaccard coefficient J, as assumed in FIG. 16, which can be weighted or unweighted.
  • PCR and sequencing are noisy channels with error sources, in particular due to off-target amplification caused by similarity with other sequences, or low template concentration, relative to background.
  • a particularly suitable procedure is one that computes a similarity score between 0 and 1 for the sequence sets obtained from individual experiments, which considers the presence or absence of given sequences in the compared sets, as well as their frequency.
  • the reads are filtered for constant adapter regions, which are expected to be present in all correctly amplified sequences. I.e., only those sequences exhibiting the expected sequencing adapter regions are considered for further analysis.
  • the output sections e.g., 21 bp
  • This filter procedure aims at removing the sequence information that is either artefactual or belongs to constant adapter regions.
  • Example 1 Synthesis of dsODN composition (orDNa composition, orDNA pool)
  • Example 1 comprised generating orDNA compositions comprising random and constant portions.
  • a single-stranded library (Library 1, SEQ ID NO 1) was ordered from an external supplier. The library layout is shown in FIG. 2 (A-G and 201). Synthesis was performed on solid support using standard phosphoramidite chemistry, which is schematically described in FIG. 6. To generate the random segments (first random segment, second random segment and third random segment), the four nucleobases were added in equimolar amount, leaving it to stochastics which base is incorporated. This procedure is schematically described in FIG. 7 and results in a combinatorial library. From this ssODN library, approx. 10 8 sequences were arbitrarily extracted to generate a doublestranded orDNA pool via PCR (Library 1 orDNA pool, SEQ ID NO 14).
  • ATGCGATGCAGTAAGCACTCN N N N N N N N N ACACGACGCTCTTCCGATCTN NNNNNNNNN N N N N N N NGCTCAGG ATACCAAGCTGTCCN N N N N N N N N NGATATCTGCTCGG ACCGCTA with SEQ ID NO 1 was ordered from Microsynth AG (Balgach, Switzerland). The synthesis yielded around 4 mg of DNA, equivalent to ca. 6 10 16 sequences. A 5 nmol aliquot of the dried library as received from the supplier was dissolved in PCR-grade water (type 1, 18.2 MQ-cm at 24°C, Milli-Q®; Merck, Darmstadt, Germany).
  • Dilution series were performed to achieve the concentration needed to pipette the amount corresponding to the desired pool size of approx. 10 8 sequences into a PCR reaction.
  • the final PCR mix contained lx KAPA SYBR FAST qPCR master mix (KAPA Biosystems, Wilmington, USA) and 0.5 pM of each primer.
  • the primers were Handle 1.1 (SEQ ID NO 45, ATGCGATGCAGTAAGCACTC) and Handle II.
  • I SEQ ID NO 46, TAGCGGTCCGAGCAGATATC
  • the final reaction volume comprised 20 pl. Dilutions and reaction mixes were prepared under laminar flow.
  • Thermal cycling consisted of 180 s pre-incubation at 95 °C, followed by 30 cycles of 15 s denaturing at 95 °C, 30 s annealing at 56 °C and 30 s elongation at 72 °C, performed on a LightCycler 96 platform (Roche Diagnostics, Rotnch, Switzerland). The reaction was completed with 180 s of final elongation at 72 °.
  • DNA purification and work-up was performed using the DNA Clean & Concentrator kit (Zymo Research, Irvine, CA, USA) according to the manufacturer's protocol.
  • the purified product was eluted in 50 pl PCR-grade water (type 1, 18.2 MQ-cm at 24°C, Milli-Q® ; Merck, Darmstadt, Germany).
  • Analytical agarose gel electrophoresis (AGE) was performed to confirm product size, using E-Gel EX gels (2%, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) on a Power Snap Electrophoresis Device (Thermo Fisher Scientific).
  • n denotes the number of synthetic cycles performed with the nucleotide mixture.
  • approx. 66 kg of single-stranded DNA would have to be produced. This estimate is based on a molecular weight of ca. 40'000 g/mol, given by an average molecular weight of 330 g/mol/nt x 121 nt. It follows that 4 mg of ssDNA are equivalent to ca. 6 x 10 16 single-stranded sequences.
  • Example 1 shows that a subset with a size of approx. 10 8 sequences of the single-stranded Library 1 composition can be PCR- amplified using handle 1.1 and handle II. I primers to produce double-stranded orDNA of the structure described in FIG. 2 (step 203). This double-stranded composition was named Library 1 orDNA pool
  • Example 2 Challenge response pair generation and evaluation (operation of orDNA composition; verification method)
  • Example 2 was performed to show that an orDNA composition such as the one generated in example 1 can be used to generate challenge-response-pairs (CRPs).
  • a challenge- response-pair refers to the use of a pair of primers (challenge) to selectively amplify sequences from the orDNA composition, the output portions of which are sequenced to give an overall output (the response). The response is unique to a given challenge, and different challenges consistently produce different responses.
  • CRPs To generate different CRPs, different primer pairs were used, each primer pair binding to and amplifying a different subset of sequences out of the Library 1 orDNA pool in a PCR reaction.
  • Library 1 orDNA pool (SEQ ID NO 14) as produced in example 1 comprises approx. 10 8 unique sequences.
  • the number of selective nucleotides is given by the equation 4 X ⁇ 10 8 , meaning x ⁇ 8/log(4). This relationship is shown in FIG. 20.
  • 13 selective nucleotides (complementary to the first and second random segments of a subset of dsODNs) distributed over the two primers (6 and 7 nt, respectively) were used. The remaining nucleotides of both primers were chosen to overlap with the adjacent constant handle segments in order to stabilize the primer-template pair during PCR and to guide the primers to the correct binding position.
  • the sequences amplified in this reaction are termed Library 1 orDNA selection (SEQ ID NO 15). They were then processed further and prepared for sequencing. This comprises 3 consecutive PCR reactions ('trimming PCR', 'Illumina preparation T and 'Illumina preparation IT). These three steps serve the sole purpose of making the orDNA sequences emerging from selection PCR compatible with the Illumina iSeq 100 platform for Next Generation Sequencing, which was chosen as an exemplary sequencing platform for this example. The procedure is further explained in a separate section ("note to sequencing preparation" further below). The procedure for example 2 starting from selection PCR and ending at the composition for sequencing is schematically shown in FIG. 2 (steps 204-209).
  • FIG. 15 provides a further overview of the procedure, with steps 803-812 describing the process of challenge-response pair generation starting with the ds orDNA pool and ending with the processed sequencing data.
  • steps 803-812 describe the process of challenge-response pair generation starting with the ds orDNA pool and ending with the processed sequencing data.
  • the steps implemented in example 2 show that repetitions with the same input primers lead to similar challenge response pairs and changing the input primer distinguishably changes the output (response).
  • the Illumina NGS system requires the presence of specific DNA segments adjacent to the segment of interest that is to be sequenced. These segments serve as the starting points for the sequence reads. Furthermore, the Illumina system works with indices, which are 6-nt long sequence segments to identify which sequences belong to which sample. In case of example 2, a different index was used for the processing of each challenge as a unique identifier, which allows to run several challenges in parallel and still distinguish which response belongs to which challenge.
  • indices which are 6-nt long sequence segments to identify which sequences belong to which sample. In case of example 2, a different index was used for the processing of each challenge as a unique identifier, which allows to run several challenges in parallel and still distinguish which response belongs to which challenge.
  • These Illumina sequence segments summarized under the term 'Illumina adapters', have to be added to the orDNA sequences after the selection PCR.
  • the first step in this procedure amplifies the orDNA selection further and ensures that only sequences of the correct format, i.e., containing the second random segment (output sequence) flanked by the first sequencing adapter and the second sequencing adapter (essentially constant segments), are processed further. They are then subjected to Illumina preparation I, which is an overhang PCR.
  • Overhang PCR uses primers that are longer than necessary and lead to a template-primer duplex with a single-stranded overhang formed by the primer.
  • the polymerase then extends in both directions (the primer overhang and the template), leading to a product that is longer than the original PCR template by the number of base pairs corresponding to the length of the primer overhang).
  • Illumina preparation II is again an overhang PCR and adds another short portion to both ends.
  • PCR reaction mixes contained 10 pl KAPA SYBR FAST qPCR master mix (KAPA Biosystems, Wilmington, USA), 1 pl of the desired input I (forward) and input II (reverse) primer (10 pM), respectively, 1 pl template (1 ng/pl Library 1 orDNA from example 1) and 7 pl PCR- grade water.
  • the challenge-response pairs differ from each other by the primer combinations that were used.
  • the different input I and input II primer combinations are listed in Table 1 below.
  • Thermal cycling consisted of 180 s pre-incubation at 95 °C, followed by 30 cycles of 15 s denaturing at 95 °C, 30 s annealing at 62 °C and 30 s elongation at 72 °C with 180 s final elongation, performed on a LightCycler 96 platform (Roche Diagnostics, Rotnch, Switzerland). DNA purification and work-up was performed using the DNA Clean & Concentrator kit (Zymo Research, Irvine, CA, USA) according to the manufacturer's protocol. The purified product was eluted in 50 pl PCR-grade water (type 1, 18.2 MQ-cm at 24°C, Milli-Q®; Merck, Darmstadt, Germany).
  • Table 1 List of challenges with input I and input II primers used within example 2. Sequence segments in italic overlap with the forward strand of the first handle segment (SEQ ID NO 2) of the Library 1 orDNA pool, underlined sequence segments overlap with the reverse strand of the second handle segment (SEQ ID NO 9) of the Library 1 orDNA pool. The nucleotides in bold characters bind to the complementary random input I and input II segments (first and third random segment) adjacent to the first and second handle segments, respectively. These nucleotides are termed the 'input sequences' and are also separately listed in the fourth column.
  • Trimming PCR Reaction mixes contained 10 pl KAPA SYBR FAST qPCR master mix (KAPA Biosystems, Wilmington, USA), 1 pl of forward and reverse primers (both 10 pM), 1 pl of a 1 ng/pl template solution (DNA purified from selection PCR) and 7 pl PCR-grade water.
  • Thermal cycling consisted of 180 s pre-incubation at 95 °C, followed by 12 cycles of 15 s denaturing at 95 °C, 30 s annealing at 56 °C and 30 s elongation at 72 °C with 180 s final elongation, performed on a LightCycler 96 platform (Roche Diagnostics, Rotnch, Switzerland). DNA purification and work-up was performed using the DNA Clean & Concentrator kit (Zymo Research, Irvine, CA, USA) according to the manufacturer's protocol. The purified product was eluted in 50 pl PCR-grade water (type 1, 18.2 MQ-cm at 24°C, Milli-Q®; Merck, Darmstadt, Germany).
  • Illumina preparation I Illumina preparation I reaction mix contained 10 pl KAPA SYBR FAST qPCR master mix (KAPA Biosystems, Wilmington, USA), 1 pl each of forward and reverse primer (both 10 pM), 1 pl of a 1 ng/pl template solution (purified DNA from previous step) and 7 pl PCR-grade water.
  • the primers used were Illumina primer I forward (SEQ ID NO 61, ACACTCTTTCCCTACACGACGCTCTTCCGATCT) and Illumina primer I reverse (SEQ ID NO 62,
  • a 50 bp ladder (GeneRuler 50 bp DNA Ladder, ready-to-use, Thermo Fisher Scientific, Waltham, MA, USA) was used.
  • the desired product band was extracted and purified using the Zymoclean Gel DNA recovery kit (Zymo Research, Irvine, CA, USA) according to the manufacturer's instructions, eluting the purified product in 50 pl PCR-grade water (type 1, 18.2 MQ-cm at 24°C, Milli-Q®; Merck, Darmstadt, Germany). Concentration measurements were performed using Qubit fluorometric quantification (Thermo Fisher Scientific, Waltham, MA, USA).
  • Illumina preparation II Illumina preparation II reaction mix contained 10 pl KAPA SYBR FAST qPCR master mix (KAPA Biosystems, Wilmington, USA), 1 pl each of forward and reverse primer (both 10 pM), 1 pl of a 1 ng/pl template solution (purified DNA from previous step) and 7 pl PCR-grade water (type 1, 18.2 MQ-cm at 24°C, Milli-Q®; Merck, Darmstadt, Germany).
  • the primers used were Illumina primer II forward (SEQ ID NO 63, AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGC) and Illumina primer II reverse (SEQ ID NO 64-71, depending on the sample).
  • Illumina primer II reverse exists in several variants, each with a different 6-base index segment. Which Illumina primer II reverse was used for which challenge is indicated in Table 2 below. Illumina primer II forward is identical for all challenges. Thermal cycling consisted of 180 s pre-incubation at 95 °C, followed by 12 cycles of 15 s denaturing at 95 °C, 30 s annealing at 56 °C and 30 s elongation at 72 °C with 180 s final elongation, performed on a LightCycler 96 platform (Roche Diagnostics, Rotnch, Switzerland).
  • AGE Preparative agarose gel electrophoresis
  • E-Gel EX gels 2%, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA
  • Power Snap Electrophoresis Device Thermo Fisher Scientific
  • a 50 bp ladder GeneRuler 50 bp DNA Ladder, ready-to-use, Thermo Fisher Scientific, Waltham, MA, USA
  • the desired product band was extracted and purified using the Zymoclean Gel DNA recovery kit (Zymo Research, Irvine, CA, USA) according to the manufacturer's instruction, eluting the purified product in 50 pl water (type 1, 18.2 MQ-cm at 24°C, Milli-Q®; Merck, Darmstadt, Germany). Concentration measurements were performed using Qubit fluorometric quantification (Thermo Fisher Scientific, Waltham, MA, USA).
  • Table 2 List of challenges with their corresponding Illumina primer II reverse sequences that were used for Illumina preparation. Nucleotides in italic partially overlap with Illumina primer I reverse. The nucleotides in bold are the Illumina indices, which are used to assign each read to the correct sample when several samples are run in parallel.
  • a python pipeline was used to filter the sequencing reads, including only the reads containing the sequencing adapter I portion at the expected position. The output portions were then analysed by their read count for each challenge using MS Excel. Relative counts of the 10 most frequently read output portions for challenges 1.1, 1.2, 4.1 and 4.2 were plotted using the Origin 2021b software.
  • a python pipeline was used to filter the sequencing reads, including only the reads containing the first sequencing adapter segment.
  • the output segments (second random segments) were then analysed by the relative frequencies of the four nucleobases A, C, G and T across the 21 positions of the output portion over all included reads using a MATLAB pipeline, and plotted using the Origin 2021b software.
  • the FASTQ files returned by the sequencing platforms were processed with a python pipeline. This pipeline first filtered the reads, only including the sequences containing the sequencing adapter segments at the expected position, with a maximum allowed hamming distance of 3. In a secondary filtering step, only sequences were included that had a 21-mer insert (comprising the 'output' portion, or second random segment) between the sequencing adapter segments. In a third filtering step, only sequences with read counts comprising at least 0.1% of the overall index reads were included in further analysis.
  • FIG. 14 Representative AGE quality control gel images for all the PCR stages are shown in FIG. 14. For each stage, the expected band sizes of 110 bp, 62 bp, 109 bp, and 164 bp are present.
  • An example of read frequency analysis is displayed in FIG. 4 and an example for analysis of base content by position is given in FIG. 3.
  • Table 3 shows the similarity scores assigned to the responses generated from the different challenges in the form of a matrix. The table indicates the log-weighted Jaccard similarity score (rounded to the first decimal position) as calculated after -mer extraction.
  • FIG. 10 shows a histogram of the same data, indicating the normalized counts of like and unlike output comparisons given similarity scores.
  • Table 3 Similarity matrix between responses to challenges performed in example 2.
  • Example 3 was performed to demonstrate that an orDNA pool with full handles can be copied multiple times and retain its properties regarding challenge-response-pair (CRP) generation.
  • An orDNA pool comprising 10 8 dsDNA sequences was copied by PCR, whereby the copy was again used as a template to produce more copies for a total of 5 'generations'.
  • a selection PCR using the same input primers was performed to show that the resulting outputs matched across all generations.
  • Each generation was synthesized by using 1 ng (approx. 80 copies of the Library 1 orDNA pool, SEQ ID NO 14) of the purified previous generation as a PCR template, as illustrated in FIG. 11.
  • each PCR mix contained lx KAPA SYBR FAST qPCR master mix (KAPA Biosystems, Wilmington, USA) and 0.5 pM of each primer (Handle 1.1 primer, SEQ ID NO 45, ATGCGATGCAGTAAGCACTC and Handle II.
  • the final reaction volume comprised 20 pl. Dilutions and reaction mixes were prepared under laminar flow.
  • Thermal cycling consisted of 180 s pre-incubation at 95 °C, followed by 10 cycles of 15 s denaturing at 95 °C, 30 s annealing at 56 °C and 30 s elongation at 72 °C, performed on a LightCycler 96 platform (Roche Diagnostics, Rotnch, Switzerland). The reaction was completed with 180 s of final elongation at 72 °C.
  • DNA purification and work-up was performed using the DNA Clean & Concentrator kit (Zymo Research, Irvine, CA, USA) according to the manufacturer's protocol. Concentration measurements were performed using Qubit fluorometric quantification (Thermo Fisher Scientific, Waltham, MA, USA).
  • the purified product was eluted in 50 pl PCR-grade water (type 1, 18.2 MQ x cm at 24°C, Milli-Q®; Merck, Darmstadt, Germany).
  • Analytical agarose gel electrophoresis (AGE) was performed to confirm product size, using E-Gel EX gels (2%, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) on a Power Snap Electrophoresis Device (Thermo Fisher Scientific).
  • a 50 bp ladder GeneRuler 50 bp DNA Ladder, ready-to-use, Thermo Fisher Scientific, Waltham, MA, USA
  • Generating challenqe-response-pairs The procedure to generate and compare challenge- response-pairs was identical to the procedure described in example 2. The entire procedure from steps 'selection PCR' to 'Illumina preparation IT was performed separately for generations 0-5 of Library 1 orDNA pool. For selection PCR, 1 ng of the respective Library 1 orDNA pool generation was used, with input I primer infw4 (TGCAGTAAGCACTCTACGAC, SEQ ID NO 49) and input II primer inrv5TCCGAGCAGATATCGGCAACG, SEQ ID NO 50). This was followed by the consecutive steps of trimming PCR, Illumina preparation I, Illumina preparation II, and sequencing.
  • Table 4 List of challenges with their corresponding Illumina primer II reverse sequences that were used for Illumina preparation. Nucleotides in italic partially overlap with Illumina primer I reverse. The nucleotides in bold are the Illumina indices, which are used to assign each read to the correct sample when several samples are run in parallel.
  • FIG. 5 shows the base content by position across the output sequencing data resulting from example 3.
  • Table 5 shows the similarity scores assigned to the outputs generated from a constant input across the Library 1 orDNA pool generations in the form of a matrix. Shown is the log-weighted Jaccard similarity score as calculated after -mer extraction.
  • Table 5 Similarity matrix between responses given by an orDNA pool and five of its copy generations to the same challenge.
  • Example 4 Upscaling of orDNA compositions (orDNA pools, dsODN compositions)
  • Example 4 comprised generating larger orDNA pools and subjecting them to challenges.
  • the orDNA pools were synthesized analogous to the procedure described in example 1, using a composition extracted from Library 1 as a template. Approx. 1.6 x 10 9 and 2.6 x IO 10 sequences were arbitrarily extracted from ssODN Library 1 as described in example 1 to generate two new double-stranded orDNA pools via PCR. These pools are identically structured as the Library 1 orDNA pool but contain a 16-fold and 256-fold higher amount of unique sequences. Both pools were afterwards challenged with two inputs each, which were evaluated to show that orDNA pools can be scaled up and still successfully and reproducibly generate challenge-response-pairs that can be separated from each other.
  • Generating orDNA pools A 5 nmol aliquot of the dried ssODN Library 1 as received from the supplier (SEQ ID NO 1,
  • both of the final PCR mixes contained lx KAPA SYBR FAST qPCR master mix (KAPA Biosystems, Wilmington, USA) and 0.5 pM of each primer (Handle 1.1 primer, SEQ ID NO 45, ATGCGATGCAGTAAGCACTC, and Handle II. I primer, SEQ ID NO 46, TAGCGGTCCGAGCAGATATC, Microsynth AG, Balgach, Switzerland).
  • the final reaction volume comprised 20 pl for the 1.6 x 10 9 pool size and 10x50 pl for the 2.6 x IO 10 pool size. Dilutions and reaction mixes were prepared under laminar flow.
  • Thermal cycling consisted of 180 s pre-incubation at 95 °C, followed by 21 cycles of 15 s denaturing at 95 °C, 30 s annealing at 56 °C and 30 s elongation at 72 °C, performed on a LightCycler 96 platform (Roche Diagnostics, Rotnch, Switzerland). The reaction was completed with 180 s of final elongation at 72 °C.
  • DNA purification and work-up was performed using the DNA Clean & Concentrator kit (Zymo Research, Irvine, CA, USA) according to the manufacturer's protocol.
  • Analytical agarose gel electrophoresis (AGE) was performed to confirm product size, using E-Gel EX gels (2%, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) on a Power Snap Electrophoresis Device (Thermo Fisher Scientific).
  • AGE agarose gel electrophoresis
  • E-Gel EX gels 2%, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA
  • Power Snap Electrophoresis Device Thermo Fisher Scientific
  • Concentration measurements were performed using Qubit fluorometric quantification (Thermo Fisher Scientific, Waltham, MA, USA).
  • the resulting Library 1 orDNA pools are of the structure
  • Generating challenqe-response-pairs The procedure to generate and compare challenge- response-pairs was analogous to the procedure described in example 2. Two inputs per Library 1 orDNA pool size were tested, each in duplicates. For selection PCR with the 1.6 x 10 9 pool size, 1 ng of the respective orDNA pool (SEQ ID NO 14) was used. For selection PCR with the 2.6 x 10 10 pool size, 23 ng of the respective orDNA pool (SEQ ID NO 14) was used. The concentration of the primers and mastermix were identical in both cases. However, the total reaction volume was 20 pl for the 1.6 x 10 9 pool size and 50 pl for the
  • Table 6 List of challenges with primers and corresponding input sequences used for the
  • the primers used for the four selection PCRs of the 2.6 x IO 10 orDNA pool are shown in Table 7 below.
  • Table 7 List of challenges with primers and corresponding input sequences used for the 2.6 x 1O 10 orDNA pool within example 4.
  • the Illumina primer II reverse sequences used for Illumina preparation II for each challenge are listed in Table 8 below.
  • Table 9 shows the similarity scores assigned to the outputs generated from the four challenges by the 1.6 x 10 9 orDNA pool in the form of a matrix. Shown is the weighted Jaccard similarity score as calculated after -mer extraction. Table 9: Similarity matrix between responses to challenges performed in example 4 with the 1.6 x 10 9 orDNA pool.
  • Table 10 shows the similarity scores assigned to the outputs generated from the four challenges by the 2.6 x IO 10 orDNA pool in the form of a matrix. Shown is the weighted Jaccard similarity score as calculated after -mer extraction.
  • Table 10 Similarity matrix between responses to challenges performed in example 4 with the 2.6 x 1O 10 orDNA pool.
  • Example 5 was performed to show that orDNA pools can be switched from a clonable, i.e., copiable, to an unclonable, i.e., uncopiable, state.
  • the procedure is schematically described in FIG. 12.
  • an ssODN composition with cleavable handles as shown in Figure 17 (steps 1701-1708) was synthesized using a procedure equivalent to the chemical DNA synthesis described in example 1. From this ssODN library (Library 2, SEQ ID NO 32), approx. 10 8 sequences were arbitrarily extracted to generate a double-stranded orDNA pool via PCR (Library 2 orDNA pool, SEQ ID NO 41).
  • the thus generated copies all contained first handle and second handle segments with Piel restriction enzyme recognition sites at both ends. Following treatment with Piel, a large portion of both handles was removed, leaving only 5 and 6 constant nucleotides on each side, respectively, and a 5'N-overhang (N being dA, dC, dG or dT, determined by the random synthesis process for each individual sequence).
  • This composition was termed 'Library 2 orDNA digested' (SEQ ID NOs 42 and 43, respectively). This procedure disables PCR amplification of the composition in its entirety, as the remaining constant nucleotides are insufficient for successful primer annealing due to the low melting temperature.
  • ddNTPs 2',3'-dideoxy nucleotides
  • SEQ ID NO 44 2',3'-dideoxy nucleotides
  • an ssODN composition (Library 2, SEQ ID NO 32, ATGCGAGTCAG ATNGCACTCN N N N N N N N N N ACACGACGCTCTTCCGATCTN NNNNNNNNNNN N N N N N N N GOTO AGG ATACC AAGCTGTCC N NNNNNNN G AC ATNGG ACG ACTCAGCTA) was ordered from an external supplier (Microsynth AG, Balgach, Switzerland). The library layout is shown in FIG. 17 (1701-1708). The composition differs from the one synthesized in example 1 in that it contains Piel restriction sites in both handle segments, with the cut site being located next to a randomly synthesized position. The synthetic approach was equivalent to the procedure described in example 1.
  • PCR-grade water type 1, 18.2 MQ-cm at 24°C, Milli-Q®; Merck, Darmstadt, Germany. Dilution series were performed to achieve the concentration needed to pipette the amount corresponding to the desired pool size of approx. 10 8 sequences into a PCR reaction.
  • the final PCR mix contained lx KAPA SYBR FAST qPCR master mix (KAPA Biosystems, Wilmington, USA) and 0.5 pM of each primer (handle I. II primer, SEQ ID NO 47, ATGCGAGTCAGATNGCACTC, and handle II.
  • the final reaction volume comprised 20 pl. Dilutions and reaction mixes were prepared under laminar flow. Thermal cycling consisted of 180 s pre-incubation at 95 °C, followed by 30 cycles of 15 s denaturing at 95 °C, 30 s annealing at 56 °C and 30 s elongation at 72 °C, performed on a LightCycler 96 platform (Roche Diagnostics, Rotnch, Switzerland). The reaction was completed with 180 s of final elongation at 72 °C.
  • DNA purification and work-up was performed using the DNA Clean & Concentrator kit (Zymo Research, Irvine, CA, USA) according to the manufacturer's protocol.
  • the purified product was eluted in 50 pl PCR-grade water (type 1, 18.2 MQ-cm at 24°C, Milli-Q® ; Merck, Darmstadt, Germany). Concentration measurements were performed using Qubit fluorometric quantification (Thermo Fisher Scientific, Waltham, MA, USA).
  • Analytical agarose gel electrophoresis (AGE) was performed to confirm product size, using E-Gel EX gels (2%, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) on a Power Snap Electrophoresis Device (Thermo Fisher Scientific).
  • a 50 bp ladder GeneRuler 50 bp DNA Ladder, ready-to-use, Thermo Fisher Scientific, Waltham, MA, USA
  • composition was thereafter further amplified, using 40 x 20 pl reaction volume, each containing 1 ng of the purified composition, lx KAPA SYBR FAST qPCR master mix (KAPA Biosystems, Wilmington, USA) and 0.5 pM of each primer (handle I. II primer, SEQ ID NO 47, and handle II. II primer, SEQ ID NO 48, as above).
  • primer handle I. II primer, SEQ ID NO 47, and handle II. II primer, SEQ ID NO 48, as above.
  • Library 2 orDNA pool (SEQ ID NO 41, ATGCGAGTCAG ATNGCACTCN N N N N N N N N N N ACACGACGCTCTTCCGATCTN NNNNNNNNNNN N N N N N N N N GOTO AGG ATACC AAGCTGTCC N NNNNNNN G AC ATNGG ACG ACTCAGCTA) .
  • Preparative agarose gel electrophoresis was performed using a 2% agarose (Ultrapure, Thermo Fisher Scientific, Waltham, MA, USA) gel stained with gel red nucleic acid stain (Biotium, Fremont, CA, USA).
  • the AGE was run at 100 mA/130 V for 90 min on a PowerEase 90 W device (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA).
  • a 50 bp ladder GeneRuler 50 bp DNA Ladder, ready-to- use, Thermo Fisher Scientific, Waltham, MA, USA
  • As a negative control an undigested sample of Library 2 orDNA was run.
  • the product bands were excised and purified using the Zymoclean Gel DNA recovery kit (Zymo Research, Irvine, CA, USA) according to the manufacturer's instruction. Concentration measurements were performed using Qubit fluorometric quantification (Thermo Fisher Scientific, Waltham, MA, USA). The purified digested product was termed 'Library 2 orDNA digested'.
  • the forward strand of the composition (SEQ ID NO 42,
  • the product was purified using the DNA Clean & Concentrator kit (Zymo Research, Irvine, CA, USA) according to the manufacturer's protocol and eluted in 10 pl PCR-grade water (type 1, 18.2 MQ-cm at 24°C, Milli-Q®; Merck, Darmstadt, Germany).
  • the blunted product was termed Library 2 orDNA blunted (SEQ ID NO 44).
  • the product was purified using the DNA Clean & Concentrator kit (Zymo Research, Irvine, CA, USA) according to the manufacturer's protocol and eluted in 10 pl PCR-grade water (type 1, 18.2 MQ-cm at 24°C, Milli-Q®; Merck, Darmstadt, Germany).
  • the blunted product was termed Library 2 orDNA dNTP blunted (SEQ ID NO 78).
  • the digested and ddNTP blunted product (Library 2 orDNA blunted, SEQ ID NO 44) was tested for ligation using standard analysis and quality controls. As controls, a composition blunted with dNTPs (Library 2 orDNA dNTP blunted, SEQ ID NO 78, NGCACTCNNNNNNNACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNNNNNGCTCA GGATACCAAGCTGTCCNNNNNNNNNNGACATN) and the undigested product (Library 2 orDNA pool, SEQ ID NO 41,
  • Example 6 Execution of an exemplary test procedure to determine that a dsODN composition complies with a template structure as in section 1.1.1
  • composition of interest contained a first and a second sequencing adapter with an essentially same sequence of nucleotides, as well as a first and a second input sequence (first and third random segment) consisting of essentially random permutations of nucleotides.
  • composition used for example 6 was Library 3 orDNA pool (SEQ ID NO 79, ATGCGATGCAGTAAGCACTCN N NNNNNNNNNNNNNNNN N ACACGACGCTCTTCCGATCTN N NNNNNNNNNNNNNGCTCAGGATACCAAGCTGTCCNNNNNNNNNNNNNNNNN GATATCTGCTCGGACCGCTA) .
  • the performed experiment comprises 20 PCR reactions using forward primers that partially bind to the first sequencing adapter and the first random segment, whereby for each PCR a different ratio of the primer nucleotides binding to either of the two template portions was chosen.
  • the reverse primer was sequencing adapter II primer (SEQ ID NO 60, GGACAGCTTGGTATCCTGAGC) and constant over all reactions.
  • the number of arbitrary nucleotides (which bind to the first input sequence) in the forward primer is increased from one PCR to the next and,s concurrently, the number of constant nucleotides (which bind to the first sequence adapter) is decreased.
  • the first input sequence consists of essentially random permutations of nucleotides
  • increasing the number of arbitrary nucleotides in one of the primers decreases the number of sequences in the composition that can be bound by the primer at a constant annealing temperature, thus increasing the Ct-value of the quantitative PCR readout. This dependence would not occur if the thus probed sequence region consisted of essentially a same sequence of nucleotides across the composition.
  • this experiment is suitable to verify that a randomly synthesized sequence portion is adjacent to a constantly synthesized sequence portion.
  • PCR with different primer pairs All 20 PCR reactions were conducted under equal conditions, with the single exception of the varying forward primer.
  • the reverse primer was always sequencing adapter II primer (SEQ ID NO 60, GGACAGCTTGGTATCCTGAGC) and constant over all reactions, while the forward primer changed with each PCR as per Table 11.
  • Each PCR mix contained 1 ng of the orDNA composition (Library 3 orDNA pool, SEQ ID NO 79,
  • Thermal cycling consisted of 180 s pre-incubation at 95 °C, followed by 30 cycles of 15 s denaturing at 95 °C, 30 s annealing at 62 °C and 30 s elongation at 72 °C, performed on a LightCycler 96 platform (Roche Diagnostics, Rotnch, Switzerland). Data analysis: Ct values were determined automatically by the LightCycler 96 software (Roche Diagnostics, Rotnch, Switzerland). In cases where the programme failed to call a Ct value because the initial slope was too high, a Ct value of 1 was assumed per default.
  • Table 11 List of forward primers used in example 6. Arbitrary nucleotides binding to the first input sequence are marked in bold, while nucleotides binding to the first sequencing adapter are underlined.
  • Test procedure primer 4 SEQ ID NO 83 4
  • Test procedure primer 5 SEQ ID NO 84 5
  • the probed orDNA composition is used in a high initial concentration (0.05 ng/pl in the reaction) and is highly diverse, i.e. contains approx. 10 8 unique sequences. As all the sequences in the composition are expected to contain the first sequencing adapter portion and the second sequencing adapter portion, primer pairings where the forward primer largely overlaps with the first sequencing adapter (i.e. most primer nucleotides bind to said adapter) will bind and amplify nearly all sequences in the pool. This leads to an immediate and steeply increasing fluorescence signal, which registers as a Ct-value of 1.
  • the amount of amplifiable template sequences in the composition decreases, as the forward primer can only bind and amplify the sequences with full complementarity to both primers.
  • this effect is not immediately reflected in the Ct-value, as the Ct- value has a lower limit of 1, and forward primers with only a low number of arbitrary nucleotides still find sufficient template sequences to cause a steep fluorescence increase from the start.
  • FIG. 1 Schematic representation of the template structure of dsODNs according to embodiments.
  • First random segment (first input sequence), e.g., comprising between 5-25 essentially randomly permutated base pairs
  • Second random segment (second input sequence), e.g., comprising between 5-25 essentially randomly permutated base pairs across the plurality of dsODNs
  • FIG. 2 Schematic representation of an exemplary implementation of a Chemical Unclonable Function using a template structure according to embodiments. Shown are the template structure, a method for producing a dsODN composition (orDNA composition), and an exemplary implementation of its use as a Chemical Unclonable Function (method of verification I authentication; i.e., operation of the dsODN composition) as performed in examples 1-4.
  • ssODN composition as synthesized and described, e.g., in example 1 (Library 1), e.g., having a length of 121 nt.
  • Amplified subset of dsODN composition e.g., comprising 110 bp. Subset as amplified by selection PCR using input primer I and input primer II, as described in example 2.
  • 206 Trimming PCR with primers annealing to the first sequencing adapter and the second sequencing adapter (sequencing adapter primer I and sequencing adapter primer II).
  • 207 Amplified ("selected") and trimmed dsODN obtained by PCR with sequencing adapter primer I and sequencing adapter primer II, as described in example 2.
  • First random segment (first input sequence; input I), e.g., comprising 9 nt, e.g., having the sequence 5'NNNNNNNNN3'
  • Sequence adapter I First sequencing adapter (sequencing adapter I), e.g., comprising 20 nt, e.g., having the sequence 5'ACACGACGTCTTCCGATCT3' (SEQ ID NO: 3)
  • D Second random segment (output sequence), e.g., comprising 21 nt, e.g., having the sequence 5'NNNNNNNNNNNNNNNNNNNNNNN3'
  • Second sequencing adapter (sequencing adapter II), e.g., comprising 21 nt, e.g., having the sequence 5'GCTCAGGATACCAAGCTGTCC3' (SEQ ID NO: 4)
  • Second random segment (second input sequence; Input II), e.g., comprising 10 nt, e.g., having the sequence 5'NNNNNNNNNN3'
  • G Second handle sequence (handle II), e.g., comprising 20 nt, e.g., having the sequence 5'GATATCTGCTCGGACCGCTA3' (SEQ ID NO: 5)
  • H-N denote the reverse complementary segments corresponding to A-G:
  • Handle I reverse e.g., comprising 20 nt, e.g., having the sequence 3TACGCTACGTCATTCGTGAG5' (SEQ ID NO: 6)
  • Input I reverse e.g., comprising 9nt, e.g., having the sequence 3'NNNNNNNNN5'
  • Sequencing Adapter I reverse e.g., comprising 20 nt, e.g., having the sequence 3TGTGCTGCGAGAAGGCTAGA5' (SEQ ID NO: 7)
  • K Output sequence reverse, e.g., comprising 21 nt, e.g., having the sequence 3'NNNNNNNNNNNNNNNNNNNNN5'
  • L Sequencing Adapter II reverse, e.g., comprising 21 nt, e.g., having the sequence 3'CGAGTCCTATGGTTCGACAGG5' (SEQ ID NO: 8)
  • M Input II reverse, e.g., comprising 10 nt, e.g., having the sequence 3'NNNNNNNN5'
  • N Handle II reverse, e.g., comprising 20 nt, e.g., having the sequence 3'CTATAGACGAGCCTGGCGAT5' (SEQ ID NO: 9)
  • Handle I after selection PCR e.g., comprising 15 nt, e.g., having the sequence 5'ATGCAGTAAGCACTC3' (SEQ ID NO: 10)
  • P Handle II after selection PCR, e.g., comprising 14 nt, e.g., having the sequence 5'GATATCTGCTCGGA3' (SEQ ID NO: 11)
  • Handle I reverse after selection PCR e.g., comprising 15 nt, e.g., having the sequence 3 ACGTCATTCGTGAG5' (SEQ ID NO: 12)
  • R Handle II reverse after selection PCR, e.g., comprising 14 nt, e.g., having the sequence 3'CTATAGACGAGCCT5' (SEQ ID NO: 13)
  • S Illumina adapter I forward
  • T Illumina adapter II forward
  • U Illumina adapter I reverse
  • V Illumina adapter II reverse.
  • S, T, U and V are the sequences arising from preparation for Illumina sequencing.
  • FIG. 3 Relative frequency (%) of the four nucleobases A (top left), C (top right), G (bottom left) and T (bottom right) across the 21 positions of the second random segment (output sequence) as per counts in Illumina sequencing results with two different inputs (challenge 1.1 and challenge 4.1) and their replicates (challenge 1.2 and challenge 4.2), as described in example 2.
  • the position-dependent frequencies show high similarity between replicates but a clear difference between the two inputs.
  • FIG. 4 Relative counts (y-axis) of the 10 most frequent output sequences (x-axis numbered by rank) of individual executions of the procedure as described in example 2, with challenges 1.1, 1.2, 4.1 and 4.2. There is a clear similarity between repetitions with the same input, and a clear difference between different inputs.
  • FIG. 5 Relative frequency (%) of the four nucleobases A (top left), C (top right), G (bottom left) and T(bottom right) across the 21 positions of the second random segment (output sequence) as per counts in Illumina sequencing results of example 3.
  • the position- dependent frequencies show high similarity between all generations of the Library 1 orDNA pool (cf. reference 209 in FIG. 2).
  • FIG. 6 Schematic of an example procedure for chemically synthesizing random DNA sequences as supplied by several commercial suppliers. Shown is the synthetic process for a single growing chain, whereby each cycle as illustrated by the arrows adds a single nucleotide, which is incorporated at random by the entropy of a mix of nucleotides.
  • 61 Start of the cycle; 62: Detritylation; 63: Incoming nucleotide, from a mix of nucleotides; 64: Coupling; 65: Oxidation; 66: Cleavage from solid support and deprotection
  • FIG. 7 Illustrative sketch of the principle of random DNA synthesis, schematically showing growing chains on solid support employing an equimolar mix of the four DNA nucleotides.
  • 71 Synthesis chamber; 72: Solid support; 73: First synthetic cycle adding a nucleotide at random; 74: Second synthetic cycle adding the next nucleotide at random; 75: Subsequent synthetic cycles 3 to n adding the next n - 2 nucleotides at random; 76: 4 n possible sequences arising from the combinatorial possibilities of n random nucleotides
  • FIG. 8 Summarizing sketch of the procedures involved in generating orDNA pools (dsODN compositions) and operating them as chemical unclonable functions. Shown are the various steps from generating a dsODN composition (orDNA composition) to receiving responses (outputs) to challenges (inputs).
  • dsODN composition serving as chemical unclonable function (optionally comprising first and second handle sequences)
  • 804 Numeric input to the function (optional); 805: Mapping of numeric input to primer sequence (optional); 806: Input primers
  • 809 Data processing using k-mer extraction (optional); 810: Set of k-mers with frequency of occurrence (optional)
  • FIG. 9 Ct number plotted against the number of arbitrary bases in the reverse primer with sigmoidal fit, as described in test procedure steps 3.6-3.7.
  • Al is equal to 0.5956 ⁇ 0.32332.
  • A2 is equal to 18.57025 ⁇ 0.38632.
  • Log(xo) is equal to 11.28173 ⁇ 0.17121, and p is equal to 0.25948 ⁇ 0.0245.
  • the reduced chi-square obtained is 0.46704, the reduced R- square is 0.99305, and the adjusted R-square is 0.99175. Parameters are unrounded as returned for the fit by the Origin2021b software.
  • FIG. 10 Histogram of output similarity scores of like and unlike inputs across all challenges of example 2. White bars represent normalized comparisons between challenges conducted with unlike inputs, black bars represent normalized comparisons between challenges with like inputs.
  • 1001 Range of similarity scores assigned to unlike inputs; 1002: Range of similarity scores assigned to like inputs; 1003: Threshold between similarity scores assigned to unlike/like inputs
  • FIG. 11 Experimental procedure to amplify dsODN compositions using handle primers by making copies of copies, leading to multiple generations, as described in example 3. Starting with approx. 16'000 copies of a composition comprising approx. 10 8 sequences (generation 0), the first generation was obtained by performing PCR of approx. 0.5% of generation 0 with the handle primers, yielding again approx. 16'000 copies on average. This procedure was repeated 5 times in total, each time using the previous generation as a template.
  • FIG. 12 Sketch of the procedure as described in example 5 to remove the handles, to thereby obtain a dsODN composition than cannot be copied (unclonable); i.e., no PCR amplification or ligation being possible.
  • 1201 Handle I; 1202: Input I; 1203: Sequencing Adapter I; 1204: Output; 1205: Sequencing Adapter II; 1206: Input II; 1207: Handle II; 1208: Restriction site; 1209: Restriction digest using restriction enzyme; 1210: End fill-in with dideoxy nucleotides; 1211 : Incorporated dideoxy nucleotides
  • FIG. 13 Exemplary AGE photograph showing the purified dsODN band (Library 1 orDNA pool) after PCR amplification of a 121 nt long ssODN library in example 1. For size comparison a 50 bp ladder is shown.
  • 131 50 bp marker; 132: 100 bp marker; 133: 150 bp marker; 134: Sample band, 121 bp; 135: Ladder lane; 136: Sample lane
  • FIG. 14 Exemplary AGE photographs showing the purified dsODN bands after the various stages of challenge-response-pair generation as described in example 2. For size comparison a 50 bp ladder is shown on all gels.
  • FIG. 15 Schematic sketch illustrating the general principle of k-mer extraction from sequencing data.
  • FIG. 16 Sketch showing the comparison via weighted Jaccard similarity. Two k-mer sets as extracted from the filtered sequence reads stemming from two challenges are compared by calculating their weighted Jaccard similarity.
  • 1601 k-mer set 1; 1602: k-mer set 2; 1603: Formula for calculating the Jaccard coefficient
  • FIG. 17 Schematic representation of an exemplary implementation of a Chemical Unclonable Function using a sequence composition structure as described in example 5.
  • 1701 Template structure of the ssODN composition as synthesized and described in example 5 (121 nt) .
  • Sequencing adapter I 5'ACACCACGCTCTTCCGATCT3' (SEQ ID NO: 34)
  • dsODN composition (orDNA pool) according to example 5 (121 bp), 5'ATGCGAGTCAGATNGCACTCN N N N N N N N N ACACGACGCTCTTCCGATCTN NNNNNNNNNN N N N N N N N NGCTCAGGATACCAAGCTGTCCN N N N N N N N N NG ACATNGG ACGACTCAGCTA3' (SEQ ID NO: 41)
  • 1737 Digested dsODN composition with 5'N-overhangs, forward (SEQ ID NO: 42) and reverse strand (SEQ ID NO: 43) 1738: 5'N-overhang; 1739: 5'NGCACT3'; 1740: 5'GACAT3'; 1741 : 3'CGTGAG5'; 1742: 3'CTGTAN5'; 1743: 5'N-overhang
  • FIG. 18 AGE photographs showing the dsODN bands after restriction digest. For size comparison a 50 bp ladder is shown.
  • 1801 Ladder lane; 1802: Sample lane; 1803: 150 bp marker; 1804: 100 bp marker; 1805: 50 bp marker; 1806: Sample band digested product, 92 bp double-stranded with 5'N-overhangs (forward strand with SEQ ID NO: 42, reverse strand with SEQ ID NO: 43)
  • FIG. 19 Gel image showing samples 1-3 of example 5 after ligation.
  • Sample 2 composition Piel digested and blunted with dNTPs (SEQ ID NO: 78)
  • FIG. 20 Expected number of sequences (x-axis) in a pool that perfectly match to an input primer of a given length, measured by the number of variable base pairs within a primer pair (y-axis), for the pool sizes as indicated by the three curves.

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Abstract

L'invention concerne notamment une composition comprenant une pluralité d'oligodésoxynucléotides double brin (ODNdb). Les ODNdb de ladite pluralité ont une même longueur comprise entre 47 et 300 pb (par exemple, entre 80 et 150 pb, ou entre 85 et 130 bp). Les ODNdb sont structurés selon une même structure de matrice, qui consiste en un ensemble ordonné de portions de séquences présentant des longueurs respectives constantes dans tous les ODNdb de ladite pluralité. L'ensemble ordonné de parties de séquence comprend un premier segment aléatoire, un premier adaptateur de séquençage, un deuxième segment aléatoire, un deuxième adaptateur de séquençage et un troisième segment aléatoire. De tels segments sont agencés consécutivement pour constituer une séquence. Chacun des segments aléatoires des ODNdb de ladite pluralité est constitué de permutations essentiellement aléatoires de nucléotides, tandis que les premier et deuxième adaptateurs de séquençage des ODNdb de ladite pluralité sont constitués, indépendamment l'un de l'autre, essentiellement d'une même séquence de nucléotides. La structure de la matrice ci-dessus donne naissance à un ADN partiellement aléatoire, qui peut être fonctionnel à des fins de vérification et/ou d'authentification. La structure de la séquence ci-dessus permet d'utiliser la composition comme une fonction mathématique à sens unique et comme une fonction physique non clonable (PUF). En d'autres termes, elle peut être utilisée comme une empreinte digitale physique, qui peut être examinée pour vérifier ou authentifier un produit, un objet ou toute autre entité à laquelle la composition est associée. La même composition peut être soumise à plusieurs interrogations, ce qui permet d'avoir une plus grande certitude quant à l'entité qui lui est associée. La technologie sous-jacente est évolutive. Des échantillons de la composition peuvent être distribués à de multiples utilisateurs, contrairement aux objets PUF habituels. Par conséquent, la composition proposée peut être utilisée de manière adéquate pour sécuriser des objets ou des entités. L'invention concerne en outre des procédés de production d'une telle composition, l'utilisation d'une telle composition, un ensemble comprenant une telle composition associée à une entité, et des procédés de vérification d'entités associées à de telles compositions.
PCT/EP2023/066962 2023-06-22 2023-06-22 Adn aléatoire exploitable Pending WO2024260562A1 (fr)

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