WO2021197610A1 - Molecular experimentation device, apparatus and method - Google Patents

Abstract

Molecular experimentation device (1) for studying a thermally activated chemical reaction, the molecular experimentation device comprising a cladding (2) including at least one confinement (5) provided with a well (6) configured to receive a reaction medium, the confinement (5) being configured to photothermally heat the reaction medium upon exposure to a heating light having an heating light wavelength, wherein the cladding (2) further includes an insulating member (7) surrounding the confinement (5), the insulating member (7) being configured to thermally insulate the confinement (5) from a cladding remaining portion (8) of the cladding (2).

Description

Molecular experimentation device, apparatus and method

Technical field

The invention relates to a molecular experimentation device, a molecular experimentation apparatus and a molecular experimentation method.

Background

The principal goals of biophysics are to understand the function of the biological macromolecules and mechanisms of the biological processes. Kinetic measurements are among the most important experimental tools for elucidating reaction mechanisms. Sequences and the order of the elementary reaction steps and their rates can be inferred from the kinetic data and from their dependence on experimental conditions or perturbations. In particular, variation of the temperature (T-jump or iT-Jump) allows addressing many biologically relevant processes or their crucial elementary steps that occur on time-scales of microseconds. In recent years, the advancement in nanofabrication technology enabled the production of nanoscale devices that allow the detection of single molecule in a solution above the micromolar range. Such devices have been used to carry out single molecule studies of biological processes as DNA polymerization or protein-protein interaction. These devices are able to single molecule analysis but are not designed to allow brief variations of temperature of the molecule for studying microsecond time-scale processes and/or to thermally activate them on a microsecond-scale

Although not limited thereto, the invention applies to DNA sequencing and especially to the method referred to as Single Molecule Real Time or SMRT, developed by the company Pacific Biosciences.

SMRT belongs to next generation sequencing (NGS), “massively parallel” or “deep” sequencing which are related terms that describe a DNA sequencing technology which has revolutionized genomic research. Using NGS, an entire human genome can be sequenced within a single day. In contrast, the previous Sanger sequencing technology, used to decipher the human genome, required over a decade to deliver the final draft.

With NGS the determination of the succession of bases A, C, G, T of DNA strands involves the replication of said strand by an enzyme, the polymerase. The bases sequentially incorporated by the enzyme during the synthesis process are detected and identified one by one (sequencing by synthesis).

Patent US 9279155 B2 from Pacific Biosciences describes a method for stepwise nucleic acid sequencing. Polymerase/template/primer complexes are immobilized on a substrate and exposed to a solution comprising a non-catalytic metal and nucleotides labeled with a detectable label on a portion of the nucleotide that is released upon incorporation. The cognate nucleotide is sequestered in the active site of the polymerase, unable to proceed to incorporation. After observing the sequestered cognate nucleotide, the complex is exposed to a catalytic metal, resulting in the incorporation of the bound cognate nucleotide and consequent release of the label resulting in a single-base extended primer.

Patent application EP1681356 A1 from Cornell Research foundation discloses a method for nucleotide incorporation reactions involving polymerases having altered nucleotide incorporation kinetics and that are linked to an energy transfer donor moiety, and nucleotide molecules linked with at least one energy transfer acceptor moiety. The donor and acceptor moieties undergo energy transfer when the polymerase and nucleotide are proximal to each other during nucleotide binding and/or nucleotide incorporation. As the donor and acceptor moieties undergo energy transfer, they generate an energy transfer signal which can be associated with nucleotide binding or incorporation. Detecting a time sequence of the generated signals, or the change in the signals, can be used to determine the order of the incorporated nucleotides, and can therefore be used to deduce the sequence of the target molecule.

More specifically, SMRT is a method for sequencing DNA by simultaneous, real-time observation of hundreds of thousands of polymerases each working on the synthesis of a single DNA molecule. Each polymerase is immobilized at a bottom of a well called the zero mode waveguide (ZMW) in the method implemented by Pacific Biosciences. Each polymerase synthesizes DNA in the presence of nucleotides labeled with a fluorophore whose binding will be cleaved by the polymerase upon incorporation of the nucleotide into the DNA. The width of the well is such that an excitation light at an excitation wavelength cannot propagate through it, but the energy can penetrate a short distance and excites the fluorophores located near the bottom of the well. Each of the four nucleotides is labeled with a different fluorophore (for example indicated in red, yellow, green and blue, respectively for G, C, T and A in) so that they have distinct emission spectra and can therefore be identified. Thus, although huge progress has been achieved in about fifteen years, the limits in terms of sequencing speed are far from being reached. Indeed, the high throughput of the current sequencers is mainly due to the massive sequencing multiplexing allowed by the micro technologies (from 10⁶ to 10¹⁰ DNA fragments sequenced in parallel depending on the technologies), but the polymerization rates are still slow: between 0.001 and 3 bases/s while most polymerases can work in vivo at much higher speeds, from several tens of bases per second to 1000 bases/s depending on the polymerase.

This “braking” of the polymerization rate finds its origin in the fact that the incorporation of the bases is a stochastic process, and that it is necessary, in the SMRT technology, to individually follow a multitude of polymerases working asynchronously.

For the SMRT method which is based on the replication of unique DNA molecules, the polymerases synthesize DNA "freely", but the average speed of incorporation of the bases by the polymerases remains well below the observation rate in order to limit sequencing errors. The polymerization rate is then voluntarily "braked" by adjusting the physicochemical parameters of the reaction medium and of the polymerase, in order to limit the base detection error rate: The incorporation of the bases by the polymerase is random in time, if its average polymerization rate is too great, the probability becomes important that, for example, two successive identical bases are incorporated too quickly to be distinguished and thus only one base can be counted instead of two. Or, if the incorporation signal is too short, it may not be detected by the imaging system. To limit this kind of sequencing errors, the average rate of incorporation of the bases must remain well below the “natural” polymerization rate of the polymerases. It is this constraint that today leads to a compromise of about 3 bases / s for high resolution imaging frequencies of the order of one hundred frames per second.

It is further known that kinetics of polymerization is temperature dependent. The temperature of the well may then be varied through application of a localized and timely controlled photothermic effect resulting from an exposure to a heating light.

The currently known SMRT method, as disclosed for example in document US 7 170050, implements a molecular experimentation device comprising an array of confinements. The confinements are simple wells formed in a cladding made of an opaque metal layer of 100 nm thick deposited on a transparent substrate. However, such wells are not suitable for a control of temperature. In particular, because of the thermal conductivity of the metal cladding, the photothermic effect would be delocalized and thermal power could hardly be evacuated to allow rapid cooling. This problem is much more important for a large number of wells of an array.

More generally, there remains a considerable need for chemical and biological analyses to provide for the ability to observe or drive single molecule reactions with a microsecond time- scale temperature control. There also exists a need for small, mass produced and disposable devices that can aid in these goals by providing optical and thermal confinements that are amenable to perform single-molecule analysis and heating.

The present invention aims at meeting these needs.

Summary of the invention

According to a first aspect, the invention proposes a molecular experimentation device for studying a thermally activated chemical reaction, the molecular experimentation device comprising a cladding including at least one confinement provided with a well configured to receive a reaction medium, wherein the confinement is configured to photothermally heat the reaction medium upon exposure to a heating light having a heating light wavelength, and wherein the cladding further includes an insulating member surrounding the confinement, the insulating member being configured to thermally insulate the confinement from a cladding remaining portion of the cladding.

According to such provisions, the molecular experimentation device has improved thermal properties enabling photothermal heating confined to the wells.

The confinement may be configured to have a first absorptance with respect to the heating light, and the cladding remaining portion may be configured to have a second absorptance with respect to the heating light, the first absorptance being greater than the second absorptance.

The first absorptance may be between 15 % and 100 %, preferably between 40 % and 100 %, and the second absorptance may be between 0 % and 10 %, preferably between 0 % and 5 %. When the heating light wavelength is in infrared field, the confinement may include at least one of Titanium (Ti) and Chromium (Cr) and the cladding remaining portion may include at least one of Gold (Au), Silver (Ag), copper (Cu) and Aluminium (Al).

The insulating member may comprise a groove interposed between the confinement and the cladding remaining portion.

The groove may be filled with a material having a low thermal conductivity, preferably less than 2 W.m^.K ¹.

The confinement may be cylindrical of circular cross section about a central axis and may have a transverse dimension Dc between 300 nm and 500 nm, the well may be arranged centrally and may have has a transverse dimension D_w between 50 nm and 150 nm, and the insulating member may have a transverse dimension D_IM between 50 nm and 150 nm.

The cladding may include an array of confinements comprising at least one row of confinements and a plurality of insulating members each surrounding one of the confinements.

The confinements of the row may be spaced apart of a distance between 2 x Dc and 50 x Dc. The array may comprise parallel rows of confinements.

The molecular experimentation device may comprise a substrate presenting a support surface on which the cladding rests, the cladding presenting a height, preferably between 50 nm and 150 nm, between the support surface and an opposite upper surface, the confinement extending along the height of the cladding, the substrate being transparent to light in visible field.

The well may be configured to define a heating volume of reaction medium to be heated less than 1 femtoliter.

According to a second aspect, the invention proposes a molecular experimentation apparatus for studying a thermally activated chemical reaction, the molecular experimentation apparatus comprising:

- a molecular experimentation device as defined previously,

- an optical system configured to emit a heating light at a heating light wavelength, preferably in the infrared field, especially greater than 800 nm, towards a confinement of the molecular experimentation device so as to photothermally heat a reaction medium received in a well of the confinement.

For a reaction medium involving an optical signal upon thermal activation of the chemical reaction, the optical system may further be configured to detect the optical signal upon thermal activation of the chemical reaction.

When the cladding includes an array of confinements, the optical system may be configured to selectively and sequentially emit the heating light towards the confinements of the array.

The optical system may be configured to scan the array of confinements.

The optical system may further be configured to emit an excitation light towards the confinement so as to induce the optical signal of the reaction medium.

The optical system may further be configured to measure a temperature of the confinement.

According to a third aspect, the invention proposes a molecular experimentation method implementing a molecular experimentation device as defined previously for studying a thermally activated chemical reaction, the molecular experimentation method comprising the steps of:

- placing a reaction medium into a well of a confinement of the molecular experimentation device,

- emitting a heating light at a heating light wavelength towards the confinement so as to photothermally heat the reaction medium and thermally activate the chemical reaction.

Before the step of emitting the heating light, the reaction medium may be maintained in a temperature range preventing occurrence of the chemical reaction.

During the step of emitting the heating light, heating light may be emitted so as to generate a temperature pulse.

The temperature pulse may be generated by irradiating the confinement of the molecular experimentation device by a heating light pulse.

The duration of temperature pulse may be less than 100 ms, preferably less than 1 ms. During the step of placing the reaction medium, a plurality of reaction mediums may be placed respectively into the confinements, at least two of the reaction mediums being different from each other.

For a reaction medium involving an optical signal upon the chemical reaction, the molecular experimentation method may further comprise a step of detecting the optical signal upon the chemical reaction.

The steps of emitting the heating light and detecting the optical signal may be performed in an iterative manner.

The molecular experimentation method may be specifically implemented for a nucleic acid sequencing, the molecular experimentation method may then comprise:

- at the step of placing the reaction medium, a solution containing a nucleic acid molecule, a polymerase and at least one type of nucleotide or nucleotide analog is placed into the well of the confinement, the nucleotide or nucleotide analog being marked with a marker adapted to emit the optical signal, and

- at the steps of emitting the heating light and detecting the optical signal, the heating light is emitted to perform polymerization as chemical reaction allowing extension of the nucleic acid molecule by the polymerase, wherein only one nucleotide or nucleotide analog is incorporated into the nucleic acid molecule, the optical signal emitted by the marker marking the nucleotide or the nucleotide analog being detected synchronously with the heating of the confinement.

At least one of an amplitude or duration of emission of heating light may be increased to thermally activate the chemical reaction.

The chemical reaction may involve enzymes, protein-protein interactions, nucleic acid-protein interactions, nucleic acid-nucleic acid interactions, polymer folding or unfolding, protein folding or unfolding.

Brief description of the figures

Other objects and advantages of the invention will emerge from the following disclosure of a particular embodiment of the invention given as non-limitative example, the disclosure being made in reference to the enclosed drawings in which: - figure 1 is a reaction diagram of the incorporation of a base by a polymerase, where E: polymerase, E': closed polymerase, Dn: DNA of n bases, N: nucleotide (base), M: Mg2 +, P phosphate (by-product). Steps : [1] capture of a nucleotide, [2] structural transition of the polymerase called "closing", [3] capture of Mg²⁺, [4] chemical step where a covalent bond is created between the nucleotide and the DNA, [5] release of Mg 2+, [6] structural transition of the polymerase called "opening". [7] release of by-products, in particular a phosphate,

- figure 2 illustrates a general principal of SMRT sequencing,

- figure 3 illustrates a molecular experimental device according to an embodiment of the invention, the molecular experimental device comprising a cladding with an array of confinements each provided with a well configured to receive a reaction medium, each confinement being configured to photothermally heat the reaction medium upon exposure to a heating light having a heating light wavelength,

- figure 4 is an enlarged view of one of the confinements of the molecular experimentation device of figure 3 illustrating an annular groove as insulating member surrounding the confinement to thermally insulate the confinement from a cladding remaining portion of the cladding,

- figure 5 illustrates an evolution of temperature within the well of one of the confinements of the molecular experimentation device of figure 3 upon emission of a IR laser flash of 20 ps on a row of confinements (pitch: 2 pm, diameter of the confinement: 400 nm, height of the confinement: 80 nm, insulating member: 100 nm, height of the cladding remaining portion:

80 nm, 2 pm-wide laser line),

- figure 6 illustrates an evolution of temperature within the well of one of the confinements of the molecular experimentation device of figure 3 upon scanning by a IR laser line of the array of confinements (pitch: 2 pm, diameter of the confinement: 400 nm, height of the confinement: 80 nm, insulating member: 100 nm, height of the cladding remaining portion:

80 nm, 4 pm-wide laser line, scanning 200 mm/s),

- figure 7 illustrates a distribution of temperature on the array upon irradiation by an IR laser line (pitch: 2 pm, diameter of the confinement: 400 nm, height of the confinement: 80 nm, insulating member: 100 nm, height of the cladding remaining portion: 80 nm, laser line of

2 pm large, flash: 20ps), - figure 8 schematically illustrates an embodiment of a method for manufacturing the molecular experimentation device of figure 3,

- figure 9 schematically illustrates another embodiment of a method for manufacturing the molecular experimentation device of figure 3,

- figure 10 schematically illustrates a further embodiment of a method for manufacturing the molecular experimentation device of figure 3.

Detailed description of the invention

As used herein “stop-and-go iterative manner” means a process with repetitive steps wherein each steps of the process can be controlled (stopped or started) by the operator. For instance, in the present invention, switching between non-permissive and permissive temperatures allows to control the incorporation of the nucleotide by the polymerase to the nascent complementary strand during polymerization.

As used herein, the term “nucleic acid” or “polynucleotide” encompasses any physical string of monomer units that can be corresponded to a string of nucleotides, including a polymer of nucleotides ( e.g ., a typical DNA or RNA polymer), PNAs, modified oligonucleotides (e.g, oligonucleotides comprising nucleotides that are not typical to biological RNA or DNA, such as 2'-0-methylated oligonucleotides), and the like. A nucleic acid can be e.g, single-stranded or double-stranded. Unless otherwise indicated, a particular nucleic acid sequence of this invention encompasses complementary sequences, in addition to the sequence explicitly indicated.

As used herein, the “polymerization rate” corresponds to the number of base pairs (or nucleotides), which is included, per second, in the growing nascent nucleic acid strand.

As used herein “a permissive temperature” is a temperature greater than the in vivo temperature where the polymerase in reaction is activated. This temperature will vary depending on the polymerase used, for instance for the Thermus aquaticus DNA polymerase (Taq polymerase) a permissive temperature is greater than 70°C (Brock TD, Freeze H. Thermus aquaticus gen. n. and sp. //., a Nonsporulating Extreme Thermophile. Journal of Bacteriology. 1969;98(l):289-297.)

As used herein “a non-permissive temperature” is a temperature where the polymerase in reaction is inhibited. In other words, a non-permissive temperature is a temperature, typically a temperature range preventing occurrence of the polymerization reaction. Maintaining the reaction mixture at a non-permissive temperature drastically decreases the polymerization rate down to few percent of the in vivo temperature polymerization rate, i.e. less than 15%, more preferably less than 10%, even more preferably less than 5%, even more preferably less than 1% and even more preferably less than 0.5% of the in vivo temperature polymerization rate (typically the polymerization rate at in vivo temperature for a given polymerase is usually known from the literature). This non-permissive will vary depending on the polymerase used, for instance for the Taq polymerase, a non-permissive temperature is less than 45°C (Innis et al. DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc. Natl. Acad. Sci. U. S. A. 85, 9436-40 (1988).).

As used herein a “flash” of temperature is a high increase of temperature for a very short amount of time, for instance, an increase of 50°C for 100 ps. Typically, a “high increase of temperature” as used can be an increase of at least, 1°C, 2°C, 5°C, 10°C, 20°C, 40°C, 50°C, 60°C, 70°C, 80°C, 160°C above the baseline temperature {i.e., the temperature of the reaction mixture before the flash of temperature, e.g. the non-permissive temperature). It is noted that the baseline temperature and typically the non-permissive temperature typically depends on the polymerase, which is used in the reaction. For example, considering a baseline temperature of the reaction mixture before the flash of temperature of about 25°C, when using a thermophililic polymerase, a high increase of temperature would be typically of at least 30°C and would be in particular comprised between 30°C and 300°C, notably between 30°C and 150°C and more particularly between 30°C and 90°C. See also for more details paragraph below relative to the “flash of temperature”.

As used herein “the repeating rate of the flashes” corresponds to the frequency of the flashes of temperature {e.g., the number of flashes per second).

As used herein the “incorporation of a nucleotide” means the incorporation of the nucleotide by a polymerase to the complementary strand of a target nucleic acid molecule during a polymerization and the release of by-products, for example a phosphate. Thermo-synchronisation of nucleic acid polymerization.

The present disclosure proposes a method for rapid synchronization of the incorporation of bases by polymerases. Such method aims at solving the problem of stochastic incorporation which currently limits the polymerization rate in sequencing by synthesis methods.

The method consists in subjecting a target nucleic acid molecule to a template-directed polymerization reaction to yield a nascent nucleic acid strand that is complementary to the target nucleic acid molecule under temperature conditions where the polymerization rate is very low and controlling the incorporation of the bases by said polymerases with successive flashes of temperature. The polymerases are temperature-sensitive, so by putting them under temperature conditions where the polymerization rate is very low and by applying a very short flash of temperature to the polymerase molecules, the inventors discovered that it is possible to trigger the incorporation of only one single base only into the complementary strand. That way, the successive incorporation of the bases by the polymerases can be controlled temporally and synchronized with their detection in order to eliminate the errors due to the random timing of incorporation of the bases. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid molecule by the catalytic activity of the polymerase at each step in the sequence of base additions.

This method, when applied to the SMRT method, makes it possible to synchronize the incorporation of the bases with the image acquisition and thus reach polymerization rates equal to this imaging rate without losing of information. A significantly higher polymerization rate than current methods can therefore be achieved.

The method allows controlling the polymerization based on the natural mechanism of the polymerases. For about fifteen years, a consensus has emerged on said mechanism. Although described in a simplified schematic fashion, the actual biochemical process of incorporation is relatively complex. The biochemical process can be described as a sequence of biochemical steps, wherein each biochemical step can be characterized as having a particular forward reaction rate and a reverse reaction rate that can be represented by a rate constant.

One representation of the incorporation biochemistry is provided in figure 1. As shown in figure 1, when starting from an already formed DNA-Enzyme complex, the biochemical steps of the mechanism consist of [1] capture of a nucleotide, [2] a structural transition (enzyme isomerization) of the polymerase called "closing", [3] capture of Mg²⁺, [4] a chemical step where a covalent bond is created between the nucleotide and the DNA, [5] release of Mg 2+, [6] a structural transition (enzyme isomerization) of the polymerase called "opening", [7] release of by-products, in particular a phosphate.

It is to be understood that the scheme shown in figure 1 does not provide a unique representation of the biochemical process. In some cases, this process can be described using fewer biochemical steps. For example, this process is sometimes represented without inclusion of the enzyme isomerization steps [2] and [6] or without biochemical step [3] and [5] related to the adsorption or desorption of the cofactor. Alternatively, the process can be represented by including additional biochemical steps such as DNA binding or translocation. Generally, biochemical steps which can be slow, and thus limit the rate of polymerization will tend to be included.

In the SMRT method, a fluorophore is bound to the phosphate. The phosphate release then allows the detection of the nucleotide incorporation.

It is well established that the kinetics of polymerization is thermally activated. For example, for Taq polymerase, the kinetics of incorporation varies by an order of magnitude of about 20 K of temperature variation. When the nucleotide concentration in the reaction mixture is sufficient enough for the nucleotide capture to not limit the kinetics of reaction, the kinetic limiting step may vary depending on the polymerases. It is generally either the chemical step [4] or the one involving the structural transitions of the polymerase [2] or [6] which kinetically limits the polymerization. In any case, for temperatures where the polymerization rate is decreased to less than 15% of the in vivo temperature polymerization rate (non- permissive temperature), after capture of a nucleotide, the enzymatic complex will be drastically slowed in the state before the kinetic limiting step (for instance [2], [4] or [6]). In said state the nucleotide and/or its by-products remain captured by the polymerase complex.

The disclosure proposes a method for controlling nucleic acid polymerization in a stop-and-go iterative manner comprising the following steps: a. providing a reaction mixture comprising i. at least one type of a plurality of nucleotide or nucleotide analog ii. a polymerase, iii. at least one target nucleic acid molecule wherein the reaction mixture is maintained at a non-permissive temperature, such that the polymerization rate is decreased to less than 15% of the in vivo temperature polymerization rate of said polymerase in said reaction mixture; and b. applying at least one flash of permissive temperature to the reaction mixture, such that only one nucleotide or nucleotide analog is incorporated by the polymerase into the nascent complementary strand of said target nucleic acid molecule and one phosphate is released; c. Optionally, repeating step b. at least one time.

In the method, the at least one flash of permissive temperature (step b. of applying a flash of permissive temperature) drastically increase the reaction rates of the kinetic limiting steps for a very short amount of time and thus will unlock said kinetic limiting step(s). This at least one flash of permissive temperature needs to be hot enough to unlock the kinetic limiting step(s) with certainty during its duration, and needs to be brief enough so that only one nucleotide is incorporated by the polymerase into the complementary strand of the target nucleic acid molecule and its by-products released, in particular a phosphate. The present method allows to control step by step the incorporation of the nucleotides: one flash of temperature equals one incorporated base.

The reaction mixture is provided in order to subject a target nucleic acid molecule to a template-directed polymerization reaction to yield a nascent nucleic acid strand that is complementary to the target nucleic acid molecule in the presence of at least one type of a plurality of nucleotides or nucleotide analogs and polymerases (one or more that can be the same or not).

As explained previously, maintaining the reaction mixture at a non-permissive temperature kinetically slows down the polymerization by blocking a step of structural transition ([2] or [6]) or the chemical step [4] In other words, at a non-permissive temperature the polymerase has already captured a nucleotide [1] but is unable to rapidly finish the incorporation of said nucleotide, release the by-products, particularly a phosphate and to allow the capture of the next nucleotide. This non-permissive temperature will vary depending on the polymerase used (For instance, for phi 29 see: Soengas et al. 1995 J. Mol. Biol. 253(4):517-529). Typically, a non-permissive temperature is from -5°C to 15°C, preferably from 0°C to 10°C, more preferably from 0°C to 4°C for mesophilic polymerase and from -5°C to 45°C, preferably from 0°C to 25°C for thermophilic or hyper-thermophilic polymerase. For instance, the non- permissive temperature may be such that the incorporation kinetics of the polymerase is less than 2 bases per second in reaction mixture containing 100 mM of nucleotides. Preferably, the reaction mixture comprises at least one type (bases A, T, C or G) of a plurality of nucleotides or nucleotide analogs. In one embodiment, the nucleotide or nucleotide analogs are labelled, preferably each type of nucleotide (bases A, T, C or G) is labeled with a different label. Said label may be selected from the group consisting of chromophores, fluorescent moieties, enzymes, antigens, heavy metals, dyes, phosphorescent groups, chemiluminescent moieties, scattering or fluorescent nanoparticles, and Raman signal generating moieties. The label can be attached to the nucleotide or nucleotide analog at a base, sugar moiety, alpha phosphate, beta phosphate, or gamma phosphate of said nucleotide or nucleotide analog. Preferably, the label is attached to the nucleotide or nucleotide analog at its phosphate. More preferably, the label is attached to the nucleotide or nucleotide analog at its terminal phosphate. The label is enzymatically cleaved from the nucleotide or nucleotide analogs during the incorporation of said nucleotide or nucleotide into the complementary strand.

The invention applies equally to sequencing all types of nucleic acids (DNA, RNA, DNA/RNA hybrids etc.) using a number of polymerizing enzymes (DNA polymerases, RNA polymerases, reverse transcriptases, mixtures, etc.). Therefore, appropriate nucleotide analogs serving as substrate molecules for the nucleic acid polymerizing enzyme can consist of members of the groups of dNTPs, NTPs, modified dNTPs or NTPs, peptide nucleotides, modified peptide nucleotides, or modified phosphate-sugar backbone nucleotides, polyphosphate nucleotides or modified nucleotides such as tetra, penta or hexa phosphate nucleotides or modified nucleotides.

As used herein, a "nucleic acid" shall mean any nucleic acid molecule, including, without limitation, DNA, RNA, and hybrids thereof. The nucleic acid bases that form nucleic acid molecules can be the bases A, C, G, T and U, as well as derivatives thereof. Derivatives of these bases are well-known in the art. The term should be understood to include, as equivalents, analogs of either DNA or RNA made from nucleotide analogs. The term as used herein also encompasses cDNA, that is complementary, or copy, DNA produced from an RNA template, for example by the action of a reverse transcriptase. It is well known that DNA (deoxyribonucleic acid) is a chain of nucleotides consisting of 4 types of nucleotides; A (adenine), T (thymine), C (cytosine), and G (guanine) and that RNA (ribonucleic acid) is a chain of nucleotides consisting of 4 types of nucleotides; A, U (uracil), G, and C. It is also known that all of these 5 types of nucleotides specifically bind to one another in combinations called complementary base pairing. That is, adenine (A) pairs with thymine (T) (in the case of RNA, however, adenine (A) pairs with uracil (U)), and cytosine (C) pairs with guanine (G), so that each of these base pairs forms a double strand. As used herein, "nucleic acid sequencing data", "nucleic acid sequencing information", "nucleic acid sequence", "genomic sequence", "genetic sequence", "fragment sequence", or "nucleic acid sequencing read" denotes any information or data that is indicative of the order of the nucleotide bases (e.g., adenine, guanine, cytosine, and thymine/uracil) in a molecule (e.g., a whole genome, a whole transcriptome, an exome, oligonucleotide, polynucleotide, fragment, etc.) of DNA or RNA.

The target nucleic acid molecule may be selected from the group consisting of double- stranded DNA, circular DNA, single-stranded DNA, single stranded DNA hairpins, DNA/RNA hybrids, RNA with a recognition site for binding of the polymerase, and RNA hairpins.

The method may further comprise a waiting step right after step b. Said waiting step is performed at a non-permissive temperature in order to kinetically slow down the polymerization after one flash of permissive temperature. The duration of the waiting step may be such that a nucleotide or nucleotide analog is captured by the polymerase (step [1]) but is not fully incorporated into the complementary strand and its by-products not released, in particular a phosphate. As such, after the incorporation of a nucleotide and release of its by products in step b, the waiting step allows time for the next nucleotide or nucleotide analog to be captured by the polymerase. Preferably, the duration of the waiting step is less than Is, preferably from 0ms to 100ms, more preferably from 0ms to 10ms. Preferably, the waiting step may consist in various sub-steps (rinsing, chemistry, etc.) depending on the detection technique used (for instance, presentation of another type of nucleotide for detection by H⁺ ion).

Step b. or step b. and c. may be repeated at least one time. Preferably, these steps are repeated until the complete complementary strand of the target nucleic acid molecule is polymerized.

In one embodiment, the repeating rate of the flashes is at least 5 times the polymerization rate at the non-permissive temperature, preferably between 10 and 1000 times, more preferably between 20 and 200 times.

As defined previously, a flash of temperature is a high increase of temperature for a very short amount of time. In one embodiment, the temperature increase during the flash of permissive temperature may be higher than 30°C, preferably from 30°C to 300°C, notably from 30°C to 150°C, in particular from 30°C to 90°C. Also, the temperature reached during the flash may be higher than 100°C.

The at least one flash of permissive temperature according to the present invention will unlock the steps limiting the kinetics of polymerization (see Fig.1) to achieve the incorporation of the captured nucleotide and optionally allow the capture of the next nucleotide between two flashes.

The at least one flash of permissive temperature is such that only one nucleotide or nucleotide analog is incorporated by the polymerase to the complementary strand of the target nucleic acid sequence. In one embodiment, the duration of the flash of permissive temperature is less than Is, preferably less than 10ms. In another embodiment, the duration of the flash of permissive temperature is about 10ns to about Is, preferably about lps to about 10ms, more preferably from 1 Ops to 2ms.

Typically, the permissive temperature is such that the forward reaction rate constant of the limiting step(s) is between 100 s ¹ and 1 000 000 s ¹.

Preferably, the permissive temperature is such that the forward reaction rate constant of the limiting step(s) is at least 10 times the forward reaction rate constant of the limiting step(s) at the non-permissive temperature, preferably between 10 and 100 000 times, more preferably between 100 and 100 000 times.

The method can be tailored according to the polymerase used. In one embodiment, the polymerase according to the method is selected from the group consisting of DNA polymerase, RNA polymerase, reverse transcriptase, and mixtures thereof. Preferably, the polymerase is selected from DNA polymerases. In the case of DNA polymerases, a variety of polymerases may be employed including for example, Strand displacing polymerases, such as Phi29 derived polymerases (e.g., those described in U.S. Pat. Nos. 5,001,050, and published U.S. Patent Application No. 2007-0196846) , Taq polymerases, KOD polymerases, Bst Polymerases, SD polymerase, Klenow, 9 No polymerase, T7 DNA polymerase, E. coli pol I, Bacillus Stearothermophilus pol I, DNA polymerases alpha, beta, epsilon and gamma, RB 69 polymerase, pol IV (DINB), poly (UmuD2C), and others. In some embodiment, genetically engineered polymerases can be used.

Preferably, the polymerase is a thermostable polymerase. In one embodiment, the polymerase is processive.

In another embodiment, the polymerase is non-processive.

In some embodiments, the polymerase is bound to the target nucleic acid molecule at an origin of replication, a nick or gap in a double stranded target nucleic acid, a secondary structure in a single-stranded target nucleic acid, a binding site created by an accessory protein, or a primed single stranded nucleic acid.

In some embodiments, the polymerase is selected (and potentially genetically engineered) such that the polymerization reaction has only one limiting step, preferably wherein the limiting step is the “opening step”.

The disclosure also proposes a use of a flash of permissive temperature for controlling nucleic acid polymerization, wherein a reaction mixture comprising at least one type of a plurality of nucleotide or nucleotide analog, a polymerase and at least one target nucleic acid molecule is maintained at a non-permissive temperature such that the polymerization rate is decreased to few percent of the in vivo temperature polymerization rate of said polymerase and said flash of permissive temperature allows only one nucleotide or nucleotide analog to be incorporated by the polymerase to the nascent complementary strand of said target nucleic acid molecule.

Sequencing process

The disclosure also proposes a method of synchronized genotyping or sequencing of a target nucleic acid molecule comprising the method for controlling nucleic acid polymerization as described previously and a further step b’ of identifying the nucleotide or nucleotide analog incorporated by the polymerase in step b and wherein step b and b’ are repeated at least one time, preferably until the nucleotide sequence information for the target nucleic acid molecule is determined.

By repeating the steps of the present method, the succession of bases that have been identified in step b’ constitutes the sequence of the synthesized DNA. Depending on the nature of the step limiting the polymerization kinetics and the nature of the identification, these steps can last and behave differently. In particular, the waiting step may be of zero duration (therefore not present). The identifying step b’ is performed in a synchronized manner with the flashes of temperature (step b). Step b’ may be performed right before or right after step b depending on the identification method (see below). In other words, step b’ may be performed after step a and before step b, or alternatively step b’ may be performed after step b and before step c. Preferably, step b’ is performed with a temporal overlap with step b and more preferably simultaneously with step b, for example in the case of a continuous detection.

The identifying step b’ can be performed by any existing method of base incorporation identification. The detection and identification may be performed optically, electrically, by detecting by-product released by the complex after the incorporation of a nucleotide analog into the nucleic acid molecule, preferably using label properties of nucleotides analog.

Preferably, in the SMRT method, the step b’ consist of a detection by fluorescence wherein each type of nucleotide is labeled with a different fluorophore which bond is cleaved upon incorporation of the nucleotide by the polymerase. For this type of detection, the limiting kinetic step of the polymerase is preferably the “opening” step ([6] - see Fig.1). Preferably for this type of detection, step b’ is performed right before step b.

When the detection of fluorescence is carried out optically, i.e. by a camera, the image acquisition can be synchronized with the heating steps so that the succession of acquired images corresponds to the succession of incorporated bases. It is thus sufficient to use a frame rate equal to the rate of the heating steps. This result in a significant reduction of data volume compared to conventional methods and allows to achieve higher sequencing rates, up to the imaging rate of high-resolution cameras (see Examples).

Preferably, one or more chemical or physical treatment are performed before repeating step b and b’. This requires the exchange of the reaction mixture and treatments such as rinsing and adding nucleotides or nucleotide analogues to the mixture.

Step b’ is preferably performed at a non-permissive temperature, preferably equal to the non- permissive temperature of the waiting phase (if present).

In one embodiment, a plurality of reaction mixtures is placed in a plurality of individual locations maintained at a non-permissive temperature and present on a solid support, preferably the reaction mixtures can vary depending on the individual location {i.e. comprising different target nucleic acid molecule). Alternatively, each individual location comprises identical reaction mixtures. The method of synchronized genotyping or sequencing according to the invention may be performed simultaneously for each individual location. Preferably, each individual location gets flash heated individually (step b).

Preferably, the flashes of temperature (step b) are performed sequentially on individual locations or groups of individual locations.

Flashes of Temperature

Three parameters characterize the flashes of temperature: temperature, duration and repeating rate. The evaluations that follow are based on probabilities calculations made by considering the physical phenomena involved (capture, structural transition, etc.) as being random in time.

According to the present disclosure, these parameters can be defined as follow:

• Repeating rate of the flashes: to ensure the sequential incorporation of nucleotides during a succession of flashes, it is necessary that at least one nucleotide can be captured between two flashes. Setting, as a criterion, that the probability of a nucleotide being captured during the time lapse (t) between two flashes must be higher than a given value p2, we can establish the following relationship based on Poisson’s statistics:

Where t_¾ is the average duration for an available DNA-polymerase complex to capture a nucleotide.

By choosing a time lapse between each flash greater than t_min = thus

ensure the presence of a nucleotide during a flash with a probability higher than p_2. Hence, by choosing p₂ close to 1, we then ensure further incorporation of nucleotides close to the repetition rate of the flashes.

T_b depends on the polymerase and the physicochemical conditions of the mixture, it is noticeable inversely proportional to the nucleotide concentration (c) in the mixture xb can be measured experimentally (John EID et al. Real-Time DNA Sequencing from Single Polymerase Molecules, Science Vol. 323, Issue 5910, pp. 133-138 (2009)) or deducted from kinetic studies on polymerase mechanisms. (Catherine M. Joyce, Techniques used to study the DNA polymerase reaction pathway, Biochimica et Biophysica Acta, 1804, (2010), 1032- 1040). In particular, t_¾ can be assessed from the measurement of the binding rate constant of nucleotide (k_bmd)_·

Within the boundaries of a low nucleotide’s concentration (<100mM), we have:

k_bind varies from one polymerase to another and also depends on the solution’s physiochemical conditions (nature and concentration of ions, temperature, pH...). Its value is typically between 3mM^_1 s ¹ and 30mM^_1 s ¹.

The table 1 below shows the corresponding values of maximum repeating rates (fmax = 1/tmin) of T_b. These repeating rates are calculated for a capture’s efficiency higher than 99% (p₂=0.99) or higher than 90% (p₂=0.9).

The table 1 displays nucleotide’s concentration values for k_bmd =10 mM ¹ s ¹, which is a typical value for the polymerase Phi29.

Table 1 • Duration of the flashes:

By setting as a criterion that the probability of one or more nucleotide being captured during a flash is not higher than a specific value (pi), for instance 0.01 if we want to obtain a probability of capture during the flash of 1% maximum, we can derive from a Poissonian statistics the following relationship:

Where r is the average duration for an available DNA-polymerase complex to capture a nucleotide during the temperature flash.

As it is necessary to capture a nucleotide during the flash for a multiple insertion to occur, we will ensure that the probability of a multiple insertion will be below the predetermined value pi if we choose for an impulsion duration shorter than:

r cannot be measured experimentally with common techniques due to the high temperatures used during the flashes. It could possibly be estimated by studying the polymerization by flash thanks to a device described further in the text. However, the average time t for a nucleotide to be captured is limited by the diffusion’s time of the nucleotides, which is itself little dependent on the temperature. Supposing that this limit is reached during high temperature flashes, we can then estimate r:

Where D is the coefficient of diffusion of the nucleotides and r a factor depending on the enzyme. For polymerases, the theory of kinetic diffusion allows to predict a result for D r of about 10-100 mM ¹ s ¹ (R. Samson et al. Diffusion-controlled reaction rate to a buried active site The Journal of Chemical Physics 68, 285 (1978)). Table 2 here below displays values of maximum duration (d_max) of the flashes to use to make sure that no multiple insertion occurs during the flash, depending on the value of t of the reaction medium. For information, the table shows the expected nucleotide’s concentration for Z =10-100 mM ¹ s ¹ .

Table 2 · Flash temperatures: By setting as a criterion that the probability of inserting a nucleotide previously captured with a flash of temperature of duration d must be higher than a given value p3, and considering an exponential probability law, we can establish the following relationship:

where d is the average time of incorporation of a nucleotide at the temperature T of the flash. The polymerization being thermally activated, considering an Arrhenius equation,

Ea d can be expressed by d = S₀eRT_^ where E_a is the activation energy of bases’ insertion and do is a pre-exponential factor depending of the polymerase. The temperature T to be reached to fulfill the criterion is then obtained as a function of the duration of the flashes d , Ea, do and P₃:

Ea and do are values depending on the polymerase and the physicochemical properties of the medium (e.g.: pH and salt concentration). They can be estimated from an experiment of thermo-kinetic polymerization (A. Longer et al. Scientific reports 5: 12066 (2015)). p shall be defined close to 1 to ensure an efficient insertion of the captured nucleotide.

It is to be noted that the proceeding of the present disclosure can be implemented by the skilled person as proposed below.

The many parameters that have been described above, i.e., the repeating rate of the flashes, the flash duration, the nucleotide concentration, as well as the permissive and temperature and non-permissive temperature can be adjusted experimentally to thermally control polymerization.

One strategy for adjusting these parameters is to evaluate the average base incorporation rate (number of bases incorporated per molecule or nucleic acid target/number of flashes). This rate represents the polymerization efficiency.

For example, this can be done using the technique described in Example 2, by implementing it on DNA molecules of known sequence.

Alternatively, it can also be done by recovering the DNA produced during the polymerization of a single stranded DNA (ssDNA) template of a known size and by analyzing it by electrophoresis or mass spectrometry to evaluate the average number of nucleotides added.

The formulas provided in the description above allow calculating the repeating rate, the duration and temperature of the flashes, when the thermodynamics of the polymerase under the physicochemical conditions employed (k_bmd, E_a, do, D_r) are known exactly. When these parameters are not known and/or cannot be easily calculated, the tables of the present disclosure provide typical values which can be used to set initial experimental conditions for the control of the polymerization according to the present invention. In particular, table 4 provides values adapted for a mesophilic polymerase, while table 3 provides values adapted for a thermophilic polymerase. The experimenter may choose these values as a starting point for developing the process and then proceed by successively adjusting the parameters according to the difference between the desired theoretical polymerization efficiency ( Ef_poi =p2.p3) and the average incorporation rate observed (T_x). Typically, T_x increases with the non-permissive temperature, the nucleotide concentration, the duration of the flashes or their temperature (i.e. the permissive temperature) and varies inversely with the repeating rate of the flashes.

A typical strategy is to use the conditions of nucleotide concentration recommended in tables 3 or 4 (according to the desired repeating rate of the flashes) and at a temperature of 5°C for mesophilic polymerases or 25°C for thermophilic polymerases. Initially, with a repetition frequency selected well below /_max (typically by a factor 3 or 4), T_x is evaluated for the corresponding temperature and flash duration as indicated in table 3 or 4.

If T_x = E f_poi , the temperature of the flashes can be progressively decreased, while keeping T_x constant.

If T_x < Bf_poiJT_x can be adjusted by first increasing the flash duration (up to a factor of 4 to 5) and then if necessary the flash temperature until the desired T_x is obtained.

It must be kept in mind that the polymerase can be damaged from a certain temperature inducing decrease of T_x, this threshold should therefore not be exceeded. Low T_x or decreasing T_x with increasing of the flash duration indicates the flash temperature is too high. It must further be noted that a well-suited thermoactivated polymerase should have an activity which is increased by typically a factor of about 10 when the temperature is increased by 20°C.

If T_x> E f_poi, the base temperature and/or nucleotide concentration should be lowered. In a second step, after having adjusted the parameters for a low repeating rate, it can be increased to the desired one (T_x can then only decrease) and then increase the non-permissive temperature and/or the nucleotide concentration to adjust T_x if necessary.

Depending on the application, one shall pick a p2 value close to 1 : for example, 0.99 for a precise monitoring of the polymerization, or 0.90 if it is simply about synchronizing a detection with the polymerization.

Applying a flash of temperature to the reaction mixture:

During the brief heating of a system, its temperature increases, then decreases by exchanging heat with its external environment. The duration of the temperature flash cannot be shorter than the cooling time. The cooling of a system by thermal conduction is typically done on a time scale proportional to the square of its diameter. In water environment, this time is typically one second for a millimeter-sized system (volume of the system of a few microliters). Cooling is done within tens of milliseconds for a system whose size is about 100 pm (system of a few nanoliters), within a few hundred microseconds for a system whose size is about 10 pm (system of a few pi coliters), within a few microseconds for micrometric-sized systems (systems of a few femtoliters). Thus, it is possible to generate temperature flashes as short as a few seconds in the few microliters of a millimetric-sized system or as short as a few microseconds in the few femtoliters of a microsystem. (Kubelka, J. Time-resolved methods in biophysics. Photochem. Photobiol. Sci. 8, 499-512 (2009)). Heating of a microsystem can be carried out by bringing energy in optical form by photo-thermal effect by irradiating the system with a laser pulse (M Olinkovsky, et al. Ultrafast cooling reveals microsecond-scale biomolecular dynamics. Nat Commun 5, 5737 (2014)) or by bringing energy in electric form by Joule heating effect by running a brief electrical current through the system (C. Y Jin et al. Localized Temperature and Chemical Reaction Control in Nanoscale Space by Nanowire Array. Nano Letters, 11(11), 4818 4825, (2011)).

According to the present disclosure, a flash of temperature can be applied toward one or more confmement(s) of a molecular device configured to implement the method as herein disclosed. The confinement or well may be configured to define a volume to be heated less which is than 1 femtoliter, notably which is comprised between 0.01 and 1 femtoliter.

The temperature pulse may thus typically be generated by emitting a heating light at a heating light wavelength toward the one or more confmement(s) (wherein the reaction medium as herein defined is placed) of a molecular device, so as to photothermally heat the reaction medium to reach the permissive temperature.

Typically, the heating wavelength comprised between 0,4pm and 20pm, for instance a wavelength of 1064 nm for Nd YAD laser.

In some embodiments, a device as illustrated below can be set. For instance, a temperature pulse may be generated by irradiating the confinement of the molecular experimentation device by a heating light pulse, such as a laser. Device

Figures 3 and 4 illustrate a molecular experimentation device 1 according to an embodiment of the invention.

The molecular experimentation device 1 enables studying a thermally induced chemical reaction. In the meaning of the invention, the chemical reaction may be of any kind and especially but not exclusively an association, a dissociation, a conformation change or other.

The molecular experimentation device 1 comprises a cladding 2 supported by a support surface 4 of a substrate 3, preferably transparent to light in visible field and lowly thermally conductive, such as made of silica fused or borofloat. Alternatively, the substrate may be a layer of such material of 1 pm to 10 pm in thickness deposited on a highly thermally conductive support, such as Saphir or Diamond and preferably transparent to visible light.

The cladding 2 includes one or several confinements 5 each provided with a well 6 configured to receive a reaction medium to be studied. In the illustrated embodiment, the cladding 2 includes an array of confinements 5 comprising parallel rows of confinements 5. Each confinement 5 extends along a height of the cladding 2 between the support surface 4 and an opposite upper surface.

Each confinement 5 is configured to photothermally heat the reaction medium upon exposure to a heating light having a heating light wavelength.

The confinement 5 is made of one or several layers each including one or several materials chosen so that it has a first absorptance with respect to the heating light. In particular, the first absorptance, measured by any conventional manner especially by an absorption spectrometer, may be between 15 % and 100 %, preferably between 40 % and 100 %. The confinement 5 is preferably made to form a layer highly absorbent to infrared field and opaque to visible light. The cladding 2 further includes an insulating member 7 surrounding each of the confinements 5. The insulating member 7 is configured to thermally insulate the confinement 5 from a cladding remaining portion 8 of the cladding 2. In the represented embodiment, the confinements 5 and the cladding remaining portions 8 extended in a same plane so that the insulating members 7 are annular of a cross section matching a contour of the confinements 5. In other embodiments, the insulating member 7 may be of any suitable configuration. For example, the substrate 3 may be provided with an array of pads protruding from the support surface 4, each pad supporting one of the confinements 5 and the cladding remaining portions 8 resting on the support surface 4. The confinements 5 and the cladding remaining portions 8 extend in separate planes, offset with respect to a direction perpendicular to the support surface 4, and the insulating members 7 are formed by spaces between adjacent confinements 5.

In the represented embodiment, the insulating member 7 comprises a groove interposed between the confinement 5 and the cladding remaining portion 8. The groove may be filled with a material having a low thermal conductivity, preferably less than 2 W.m^.K ¹. Alternatively, the groove may remain empty and a solution will form the thermal barrier.

The cladding remaining portion 8 is also made of one or several layers each including one or several materials chosen so that it has a second absorptance with respect to the heating light. The second absorbtance of the cladding remaining portion 8 is smaller than the first absorbtance of the confinement 5. The second absorptance, measured by any conventional manner especially by an absorption spectrometer, may be between 0 % and 10 %, preferably between 0 % and 5 %. The cladding remaining portion 8 is preferably made to lowly absorb infrared light.

In a particular non-limitative example specifically adapted to be implemented in the above mentioned method SMRT for DNA sequencing comprising in substance:

- placing a solution containing a nucleic acid molecule, a polymerase and at least one type of nucleotide or nucleotide analog is placed into the well 6 of the confinement 5, the nucleotide or nucleotide analog being marked with a marker adapted to emit an optical signal, and in an iterative manner:

- emitting the heating light so as to photothermally heat the reaction medium and thermally induce polymerization as chemical reaction allowing extension of the nucleic acid molecule by the polymerase, wherein only one nucleotide or nucleotide analog is incorporated into the nucleic acid molecule,

- detecting the optical signal emitted by the marker marking the nucleotide or the nucleotide analog in a synchronized manner upon incorporation of said nucleotide or the nucleotide analog into the nucleic acid molecule.

Indeed, in the method SMRT, polymerase is immobilized at a bottom of each well to synthesize DNA in the presence of nucleotides labeled with a fluorophore as marker whose binding will be cleaved by the polymerase upon incorporation of the nucleotide into the DNA. The fluorophore emits an optical signal, namely a fluorescence signal, upon excitation by an excitation light at an excitation wavelength. The width of the well is such that the excitation light cannot propagate through it, but the energy can penetrate a short distance and excites the fluorophores located near the bottom of the well. Each of the four nucleotides is labeled with a different fluorophore (for example indicated in red, yellow, green and blue, respectively for G, C, T and A in) so that they have distinct emission spectra and can therefore be identified. Concentration in solution of fluorophore is such that the fluorescence signal emanating from the well remains weak. When a nucleotide is captured in the excitation volume by a polymerase in order to be incorporated into the DNA, its residence time becomes long in the excitation volume thus producing a fluorescence signal until that fluorophore is released by the polymerase after incorporation of the nucleotide.

In particular, the excitation wavelength is chosen so that the excitation light is either reflected or absorbed by the cladding 2 so as to avoid any transmission of the excitation light by the cladding 2. For example the excitation light may have a wavelength between 300 nm and 700 nm and a power between 1 W/mm² and 1000 W/mm²

Preferably, a plurality of solutions is placed respectively into the wells 6 of the confinements 5, at least two of the solutions being different from each other.

At least one of an amplitude or duration of emission of heating light can be increased to thermally induce the polymerization.

In such particular non-limitative example, a molecular experimentation apparatus comprising the molecular experimentation device 1 and an optical system is then provided. The optical system is configured to emit the heating light at the heating light wavelength, preferably in the infrared field, especially greater than 800 nm, selectively and sequentially towards each confinement of the molecular experimentation device. The optical system is further configured to emit the excitation light and to detect the optical signal upon thermal activation of the chemical reaction. The optical system may also be configured to measure a temperature of the confinement.

In such particular non-limitative example, the confinement 5 is cylindrical of circular cross section about a central axis A and has a height between 50 nm and 150 nm and a transverse dimension Dc, measured perpendicularly to the central axis A, between 300 nm and 500 nm. The well 6 is configured to define a heating volume of reaction medium to be heated less than 1 femtoliter. In such particular non-limitative example, the well 6 is arranged centrally and has a transverse dimension D_w, measured perpendicularly to the central axis A, between 50 nm and 150 nm.

The insulating member 7 has a transverse dimension D_IM, measured perpendicularly to the central axis A, between 50 nm and 150 nm.

When the heating light wavelength is in infrared field, the confinement 5 may be made of one of Titanium (Ti) and Chromium (Cr) having a first absorptance of 55 % at 1064 nm. The cladding remaining portion 8 is made of one of Gold (Au), Silver (Ag), copper (Cu) and Aluminium (Al). For a heating laser having a heating light wavelength of 1064nm, the first absorptance of the confinement 5 in Titanium or Chromium is twenty times higher than the second absorptance of the cladding remaining portion 8 in silver or gold. Hence, even badly focalized, the absorption of the laser light is localized on the confinements 5 around the wells 6 which are thermally insulated from the remaining of the cladding 2, so that a confined heating of the wells 6 can be obtained.

The molecular experimentation device 1 defines an array of nanostructures each including the well 6 and the confinement 5. A pitch of the array may be between 2 x Dc and 50 x Dc. Alternatively, the cladding remaining portion 8 defining a reflective layer may be discontinuous, being present only around the insulating members 7 with a radius between 1 pm and 2 pm.

The array may comprise from one hundred to several tenths of millions of nanostructures.

Irradiation by heating laser can be performed either through the substrate or through the solution. In the latter case, the confinements 5 can be multilayer where the external layer close to the solution and which is directly exposed to the heating light is highly absorbing (Cr or Ti), the other layers having other properties. For example, the layer in contact with the substrate can be chosen in a material providing to the confinement 5 properties of plasmonic resonance (gold or silver) in order to exhale fluorescence of the fluorophores attached to the nucleotides. The dimensions of the confinement 5 can be adapted to optimise that king of resonance.

Digital simulations based on finite element calculation (software COMSOL) show that such conception enables suitable temperature pulses to be obtained. The heated volumes are of submicronic dimensions, where temperatures higher than 100 °C can be reached (see figures 5 and 7).

This molecular experimentation device 1 can be the main element of sequencer by synchronized synthesis. Excitation of fluorescence may be made by a plurality of laser beams obtained thanks to a diffractive element and focalised by an objective on the wells 6 of the array, the detection of fluorescence being made in a confocal manner by cameras.

Heating of the nanostructures, is preferably made sequentially, for example row by row, by irradiating sequentially the rows of the molecular experimentation device by an infrared laser. It can be easily implemented by scanning the external surface of the cladding 2 by laser structured in line (thanks to a diffractive element or a Powell lens) and focalised by an objective, either through the substrate using for example an objective of a fluorescence imaging device, or through the solution by means of an immersed objective. For example, for an array of 1000 x 1000 wells, of a pitch of 2 pm x 2 pm, the scanning on the array at a speed of 200 mm/s, of a laser line of 4 pm width and of which length covers the dimension of the array (2 mm) enables production, in 10 ms, in each well of the millions of wells of the array, temperature pulses of 20 ps (see figure 6).

The invention is however no limited to a method for DNA sequencing an may apply to any molecular experimentation method for studying a thermally activated chemical reaction, wherein:

- a reaction medium is placed into a well of a confinement of the molecular experimentation device,

- a heating light at a heating light wavelength is emitted towards the confinement so as to photothermally heat the reaction medium and thermally activate the chemical reaction.

The chemical reaction may involve enzymes, protein-protein interactions, nucleic acid-protein interactions, nucleic acid-nucleic acid interactions, polymer folding or unfolding, protein folding or unfolding, or any other suitable thermally induced chemical reaction.

Possibly, before the step of emitting the heating light, the reaction medium is maintained in a temperature range preventing occurrence of the chemical reaction.

As apparent from the above, during the step of emitting the heating light, heating light may be emitted so as to generate a temperature pulse. The temperature pulse may then be generated by irradiating the confinement of the molecular experimentation device by a heating light pulse. The duration of temperature pulse is preferably less than 100 ms, preferably less than 1 ms.

Figure 8 schematically illustrates an embodiment of a method for manufacturing the molecular experimentation device 1. The method comprises the following steps:

(a) resin deposition on the support surface of the substrate and insulation,

(b) revelation,

(c) Chromium deposition,

(d) lift off resulting in ring of chromium,

(e) resin deposition on the support surface of the substrate and the ring of chromium, and insulation,

(f) revelation,

(g) Silver deposition,

(h) lift off resulting in ring of Chromium surrounded at a distance thereof by a layer of Silver.

Figure 9 schematically illustrates another embodiment of a method for manufacturing the molecular experimentation device 1. The method comprises the following steps:

(a’) resin deposition on the support surface of the substrate and insulation,

(b’) revelation,

(c’) Silver deposition,

(d’) lift off resulting in layer and ring of Silver,

(e’) resin deposition on the support surface of the substrate and the layer and ring of Silver, and insulation,

(f ) revelation,

(g’) Chromium deposition,

(h’) lift off resulting in ring of Silver covered by Chromium surrounded at a distance thereof by a layer of Silver.

Figure 10 schematically illustrates a further embodiment of a method for manufacturing the molecular experimentation device 1. The method comprises the following steps:

(a”) resin deposition on the support surface of the substrate and insulation,

(b”) revelation,

(c”) Chromium deposition,

(d”) lift off resulting in layer and ring of chromium,

(e”) resin deposition on the support surface of the substrate and the ring of chromium, and insulation,

(f”) revelation,

(g”) Silver deposition,

(h”) lift off resulting in ring of Chromium surrounded at a distance thereof by a layer of Chromium covered by Silver.

Example 1: Control of DNA polymerization by Taq Polymerase:

We give here parameters of temperature flashes for a good control of the DNA polymerization by Taq polymerase.

To drive the incorporation of the bases, that is to say to have: a flash = an incorporated base, we aim at a 98% efficiency for the incorporation on each flash of temperature and a probability of multiple incorporation of nucleotides during a flash less than 1%, giving pi = 0.01 and p2 =ps = 0.99.

With these conditions,

The maximum flash duration is: d_max = In 0.01t

The minimum time between two flashes (minimum period of repetition of a control of the polymerization) is: t_min % — 4.6 r_b, which means:

1 a maximum repeating rate f_r max 4.6t

The minimum temperature for flashes of duration d_maxis:

k_bmd, Ea and do are evaluated using the thermocinetic data of polymerization found in the literature for Taq polymerase (Ea = 102 kJmoT¹, do = 8. 9 10¹⁹ s (A. Langer et al. Scientific reports 5: 12066 (2015)); k_bmd = 3.3 mM ¹ s (Xu, C. Conformational dynamics of thermus aquaticus DNA polymerase i during catalysis. Journal of Molecular Biology , 426(16), 2901- 2917, (2014)). Minimum temperature of flash T_min , duration d_max and repeating rate f_max are calculated as a function of oligonucleotide concentration as illustrated in table 3 below.

Table 3 Each line of the table 3 give a set of parameters to achieve an accurate control of the DNA polymerization by Taq polymerase, for instance flashes of 20 ps at 148 °C with a maximum repeating rate of 36Hz for a nucleotide concentration of roughly 50 pM each. These temperatures above the boiling point of water can be achieved by nanometric heating without resulting in boiling. In addition, in the absence of oxygen and on such short times, Taq polymerases or other thermophile polymerase do not have time to inactivate or denature.

For instance, to perform thermal control of DNA polymerization, Taq polymerases can be immobilized by N-terminal on the surface of the thin film of titanium (lOOnm) supported by a transparent support (a borosilicate coverslip) and put in contact with the reaction mixture comprising nucleotides at 25 pM each (dATP, dTTP, dGTP, dCTP) and DNA target. A simple way to perform flashes of temperature is to focus a laser beam (infrared wavelengths, notably 1064 nm for Nd-YAG laser) on an area of sub-micrometer extension of the titanium film to heat this area by photothermal effect. Illuminating with laser pulses of 40 ps at a repetition rate in 1 Hz- 100 Hz range allows to generate 40 ps temperature flashes in the area at the same repetition rate. Support and mixture are maintained close to 25°C during pulsed illumination by the laser. For pulse rate in 3-18 Hz range, adjusting the laser power to produce temperature flashes of 140°C makes it possible to control the addition of bases in the DNA target by the immobilized Taq polymerases. In the pulsed heated area, the polymerization rate is equal to the laser pulse rate while the extension of the DNA is inhibited where temperature remains low, few tens micrometers far away (<0.2 bases/s). Alternatively, temperature pulses can also be carried out electrically, by resistive heating. Using conventional microfabrication techniques, the thin titanium film (100 nm) can be deposited on the substrate to form a wire 500nm width and few hundred of micrometers long between two electrical connections of gold. In this case, applying electrical current pulses of 40 ps through the wire at a repetition rate in 3 Hz- 18 Hz range will generate 40 ps temperature flashes along the wire at the same repetition rate. Adjusting the current intensity to produce temperature flashes of 140 °C makes it possible to control the addition of bases in the DNA target by the Taq polymerases immobilized on the surface of the titanium wire.

Example 2: Synchronized Single Molecule Real Time Sequencing

The controlled polymerization method of the present invention can be applied to SMRT with the molecular experimentation device previously disclosed. SMRT is the method with the highest polymerization rate (3 bases / s). It is relatively low multiplexed (<10⁶), and currently allows to obtain 5 to 6 gigabases in three hours thanks to its capacity of sequencing long DNA fragment (on average 15-20 kbases).

SMRT is a method for sequencing DNA by simultaneous, real-time observation of hundreds of thousands of polymerases each working on the synthesis of a single DNA molecule. Each polymerase is immobilized at the bottom of a 60 to 150 nm wide well called the zero-mode waveguide (ZMW) in the method implemented by Pacific Biosciences (See Figure 2 A). The wells are simple wells in an opaque aluminum layer of lOOnm thick deposited on a transparent substrate. Each polymerase synthesizes DNA in the presence of nucleotides labeled with a fluorophore whose binding will be cleaved by the polymerase upon incorporation of the nucleotide into the DNA. The width of the wells is such that the light cannot propagate through it, but the energy can penetrate a short distance (lOnm) and excites the fluorophores located near the bottom of the well. Each of the four nucleotides is labeled with a different fluorophore (indicated in red, yellow, green and blue, respectively for G, C, T and A in) so that they have distinct emission spectra and can therefore be identified. Over time, for concentrations in solution of fluorescent molecules up to 10 mM, less than 0.1 molecule is on average present in the excitation volume of a well (about 20-100 zeptolitre, residence time <10ps) so that the fluorescence signal emanating from the wells remains weak.

When a nucleotide is captured in the excitation volume by a polymerase in order to be incorporated into the DNA (see Figure 2 B), its residence time becomes long in the excitation volume thus producing a fluorescence signal until that fluorophore is released by the polymerase after incorporation of the nucleotide (see Figure 2 B). The duration of the fluorescence signal is random and depends on the kinetics of incorporation of the polymerase. For instance, the polymerase “Phi29” produces pulses of about 100ms on average.

High resolution and low noise cameras record in real time the fluorescence signals emanating from hundreds of thousands of wells at a rate of about 100 frames per second, i.e 10 ms of exposure per image. Identification of the sequence is done from the analyses of the change in intensity and spectrum of the light emanating from the wells. 2/3 of the sequencing error rate (about 15%) of this technique comes from fluorescence pulses which are too short, less than 10ms, and thus not detected. Unlike other synthetic sequencing techniques, the error rate is independent of the length of the sequenced DNA, allowing this technique to sequence a very long DNA fragment (up to 60,000 bases).

The process of the present invention allows the temporal control of the incorporation of bases. By implementing it within the framework of the SMRT, and in contrast with this method, it is no longer necessary to film the wells of the network at high speed in order to capture temporally random and low-rate intensity changes due to nucleotide capture events. By synchronizing the frame with the heating, one single frame corresponds to the detection of one base of the sequence, and it is thus enough to use a frame rate equal to the rate of the heating steps. This results in a significant reduction of data volume compared to the Pacific Biosciences SMRT technique (30 frames per base on average). In addition, it allows to achieve higher sequencing rates, up to the imaging rate of high-resolution cameras.

The sequencing method of the present invention does not require a polymerization efficiency of 98% as in Example 1, it is possible to choose higher flash rates, corresponding to efficiencies of 90% or less. Insofar as this reduction in efficiency comes from the fact that a base could not be captured by the DNA / polymerase complex between two temperature flashes, no fluorophore will be detected during the detection phase of the process and there will be no impact on the sequencing error rate. The process of the invention can be implemented with the device disclosed herein. For instance, unique target DNA - phi29 polymerase complexes are immobilized at the bottom of the wells of a confinement array of the device and are contacted with a solution comprising the four types of nucleotides each labeled with a different fluorophore (Alexa532, Alexa 568, Alexa 635, Alexa 680) linked to the terminal phosphate of a penta-phosphate nucleotide. Like the system published by Lundquist, P. M. et al. (. Parallel confocal detection of single molecules in real time. Opt. Lett. 33, 1026-1028 (2008)), an optical system coupled with the device allows the excitation of the fluorophores by means of multiple laser beams (532nm and 635nm) focused on the wells, the detection of the fluorescence being achieved with cameras imaging the network in four spectral ranges. The optical system also allows to irradiate any line of the network of confinements, for example with a laser at 1064nm in order to heat the wells by photo-thermal effect. This can be achieved by a 1064nm laser beam that has been focused and structured in line on the surface of the device, this line being set to coincide with the well lines through a fast positioning system. For the phi29 polymerase, synchronized sequencing conditions are given in Table 4 below (89% polymerization efficiency with p2=0.9 and p3=0.99):

Table 4 From table 4 above, for example, in order to carry out the sequencing of immobilized target DNAs in the wells, the device and the solution containing 12.5 mM of each fluorescent labeled nucleotide are maintained at 4°C., whereas flashes of temperature at 80°C for 80 ps are generated in the wells. These flashes allow control of the polymerization up to a repeating rate of 50Hz, i.e. for periods of at least 20ms. Thanks to the positioning system of the laser line, these flashes are generated sequentially in the various lines of the network by positioning themselves for 80 ps on each line, one after the other by scanning the network, making it possible to multiplexly generate flashes in the network of confinements.

Several modes of synchronization between the temperature flashes, the exposure of the camera sensor and the irradiation of the wells by the fluorophore excitatory lasers (532 nm and 635 nm) can be implemented in order to achieve synchronized sequencing.

In a global synchronization mode of the frame, when one or more lines are heated by multiplexing (step b), the excitation of the fluorophores and the exposure of each frame (detection step b ') begin with the flash of the last line of heated confinements and end with the flash of the first heated line of the next period. This defines an exposure duration of the frame which must be set as greater than the minimum period of repetition of a control of the polymerization, i.e. 20ms for our example. The periodicity of heating the confinements then corresponds to the sum of the duration of the heating step and the duration of exposure. For example, by sequentially heating 200 lines of confinements, one by one, during 80ps {i.e. a step b during 200x0.08ms = 16ms), with an exposure and excitation duration of 20ms, the periodicity of the heating step is 36ms yielding a repeating rate of 27.8Hz and corresponding to an average sequencing rate of about 25 bases / s since the nucleotide incorporation efficiency is 89% (89% of 27.8 Hz). Alternatively, the excitation and the exposure (stage b ') begin after a waiting phase starting after the heating of the last line, the essential feature being that the sum of the waiting duration and the duration of excitation has to be greater than the minimum repetition period of a polymerization control, i.e. 20ms for the flash conditions we have chosen. For example, a waiting phase of 19ms and an excitation of 1ms.

Alternatively, the synchronization is done using the "rolling shutter" camera exposure mode which is particularly suitable for sequential heating of the confinement lines by scanning of the heating laser line on the network. Indeed, the "rolling shutter" exposure mode of the cameras has the consequence of gradually starting the exposure of the camera sensor, line by line in the manner of a scan, and finishing it in the same way so that each line has an identical duration of exposure. By setting up the progression of the heating laser line on the confinement network with the progression of the sensor exposure, it is possible to synchronize the heating of each line of confinements and its detection. This allows the heating of the lines and the detection to be simultaneous, and in these conditions the imaging periodicity can reach the minimum period of repetition of a control of the polymerization, i.e. 20ms for the flash conditions that we have chosen in this Example, corresponding to a sequencing rate of approximately 45 bases / s.

Claims

1. Molecular experimentation device (1) for studying a thermally activated chemical reaction, the molecular experimentation device (1) comprising a cladding (2) including at least one confinement (5) provided with a well (6) configured to receive a reaction medium, the molecular experimentation device (1) being characterized in that the confinement (5) is configured to photothermally heat the reaction medium upon exposure to a heating light having a heating light wavelength, and in that the cladding (2) further includes an insulating member (7) surrounding the confinement (5), the insulating member (7) being configured to thermally insulate the confinement (5) from a cladding remaining portion (8) of the cladding (2).

2. Molecular experimentation device (1) according to claim 1, wherein the confinement (5) is configured to have a first absorptance with respect to the heating light, and the cladding remaining portion (8) is configured to have a second absorptance with respect to the heating light, the first absorptance being greater than the second absorptance.

3. Molecular experimentation device (1) according to claim 2, wherein the first absorptance is between 15 % and 100 %, preferably between 40 % and 100 %, and the second absorptance is between 0 % and 10 %, preferably between 0 % and 5 %.

4. Molecular experimentation device (1) according to any of claims 2 and 3 when the heating light wavelength is in infrared field, wherein the confinement (5) includes at least one of Titanium (Ti) and Chromium (Cr) and the cladding remaining portion (8) includes at least one of Gold (Au), Silver (Ag), copper (Cu) and Aluminium (Al).

5. Molecular experimentation device (1) according to any of claims 1 to 4, wherein the insulating member (7) comprises a groove interposed between the confinement (5) and the cladding remaining portion (8).

6. Molecular experimentation device (1) according to any of claims 1 to 5, wherein the cladding (2) includes an array of confinements (5) comprising at least one row of confinements (5) and a plurality of insulating members (7) each surrounding one of the confinements (5).

7. Molecular experimentation device (1) according to any of claims 1 to 6, comprising a substrate (3) presenting a support surface (4) on which the cladding (2) rests, the cladding (2) presenting a height, preferably between 50 nm and 150 nm, between the support surface (4) and an opposite upper surface, the confinement (5) extending along the height of the cladding (2), the substrate (3) being transparent to light in visible field.

8. Molecular experimentation apparatus for studying a thermally activated chemical reaction, the molecular experimentation apparatus comprising:

- a molecular experimentation device (1) according to any of claims 1 to 7,

- an optical system configured to emit a heating light at a heating light wavelength, preferably in the infrared field, especially greater than 800 nm, towards a confinement of the molecular experimentation device so as to photothermally heat a reaction medium received in a well of the confinement.

9. Molecular experimentation apparatus according to claim 8 for a reaction medium involving an optical signal upon thermal activation of the chemical reaction, wherein the optical system is further configured to detect the optical signal upon thermal activation of the chemical reaction.

10. Molecular experimentation method implementing a molecular experimentation device (1) according to any of claims 1 to 7 for studying a thermally activated chemical reaction, the molecular experimentation method comprising the steps of:

- placing a reaction medium into a well (6) of a confinement (5) of the molecular experimentation device (1),

- emitting a heating light at a heating light wavelength towards the confinement (5) so as to photothermally heat the reaction medium and thermally activate the chemical reaction.

11. Molecular experimentation method according to claim 10, wherein before the step of emitting the heating light, the reaction medium is maintained in a temperature range preventing occurrence of the chemical reaction.

12. Molecular experimentation method according to any of claims 10 and 11, wherein during the step of emitting the heating light, the heating light is emitted so as to generate a temperature pulse.

13. Molecular experimentation method according to any of claims 10 to 12 for a reaction medium involving an optical signal upon the chemical reaction, further comprising a step of detecting the optical signal upon the chemical reaction.

14. Molecular experimentation method according to claim 13, wherein the steps of emitting the heating light and detecting the optical signal are performed in an iterative manner.

15. Molecular experimentation method according to claim 14 specifically implemented for a nucleic acid sequencing, wherein: - at the step of placing the reaction medium, a solution containing a nucleic acid molecule, a polymerase and at least one type of nucleotide or nucleotide analog is placed into the well (6) of the confinement (5), the nucleotide or nucleotide analog being marked with a marker adapted to emit the optical signal, and

- at the steps of emitting the heating light and detecting the optical signal, the heating light is emitted to perform polymerization as chemical reaction allowing extension of the nucleic acid molecule by the polymerase, wherein only one nucleotide or nucleotide analog is incorporated into the nucleic acid molecule, the optical signal emitted by the marker marking the nucleotide or the nucleotide analog being detected synchronously with the heating of the confinement.