RU2756476C2 - Molecular computing device based on essentially non-complementary single-stranded nucleic acids with low affinity to each other (options) - Google Patents

Abstract

FIELD: molecular computing.

SUBSTANCE: group of inventions relates to the field of molecular computing devices. A molecular computing device (options), a method for performing calculations using a device, a pharmaceutical formulation containing devices, as well as the use of a pharmaceutical formulation for regulating gene expression are proposed. The molecular computing device includes a set of single-stranded nucleic acids, wherein the numerical measure of input signals are measures related to input molecules, the numerical measure of output signals are measures related to output molecules, input signals are transformed into output signals as a result of interactions between input molecules and output molecules. In the first option, single-stranded nucleic acids have low affinity of interaction with each other. In the second option, the total concentration of each single-stranded nucleic acid exceeds a value of the dissociation constant of complexes formed by single-stranded nucleic acid and other single-stranded nucleic acids of a set of complexes by no more than 100,000 times. In the third option, interactions between single-stranded nucleic acids are reversible binding of not completely complementary between single-stranded nucleic acids, wherein such acids are used that for any two single-stranded nucleic acids, the length of the longest connected section of their mutual complementarity does not exceed 10 bases.

EFFECT: inventions provide fast calculation, solving a wide range of problems, as well as operating with oligonucleotides of various lengths.

49 cl, 2 tbl, 23 ex, 13 dwg

Description

Description of the invention

The technical field to which the invention relates

The invention relates to the field of molecular computing devices, namely systems capable of processing and transforming signals presented in the form of various quantitative characteristics of sets (sets) of molecules, according to various mathematical, algebraic, or logical laws. Indexes of the heading of the current edition of the International Patent Classification (IPC) to which the invention belongs: C12Q 1/68, G01N 33/50, H01L 51/00.

State of the art

Molecular computing devices (MVDs) can be used both for computational purposes and for various biomedical applications: diagnostics and therapy of diseases, as well as for the implementation of the brain-computer interface. For example, these systems can be used for complex multifactorial control over gene expression (for example, DNA computing systems) in terms of correcting various genetic diseases, targeted drug delivery to target cells, and the like.

At the moment, computing systems based on biomolecules are just beginning to develop. Other fields, such as medicine, require different kinds of computing systems. For example, systems would be extremely useful, consisting of a computing module that would register the presence or absence of various signals from the environment, as well as an operating module, which, depending on the result of the computing module's operation, could perform one or another biologically meaningful effect. , for example, kill a cancer cell in the body. Such systems require the transfer of information between the computing and operating modules. It is clear that the larger the spectrum of possible signals of the computing system and the larger the spectrum of the effects of the operating module, the better. Such effects do not have to be due to the presence of any molecules - it can be electromagnetic radiation (including light and low-frequency field), a change in pH, temperature, etc.

The known method (patent application WO 2011116151 (A2) dated September 22, 2011), in which a set of enzymes is used to carry out a biocatalyzed reaction, which is a logical Boolean function from the obtained "input" signals of biomarkers. In this case, a binary output signal is generated, and a signal equal to one means the presence of a disease or injury.

The disadvantages of this known method are that:

1) Only substrates, cofactors or products of action of the enzymes used can be used as input signals. At the same time, the number of combinations when using enzymes is very limited.

2) Enzymes are used as the basis of the logical element, the variety of which is very limited.

The known method (US Patent 7,745,594 B2, issued June 29, 2010), in which the logical element is a set of oligonucleotides, in which logical operations are performed on the input oligonucleotides due to branch migration of DNA as part of the logical element. Different combinations of oligonucleotides of different structures are used to implement various logical functions.

The disadvantages of this known method are that:

1) The signal transmission time is extremely long due to the low rate of displacement by one oligonucleotide of another by the strand displacement mechanism, in which the displacing oligonucleotide (olig) 1 binds to the free end (toehold) of the DNA duplex formed by olig 2 and olig 3 for due to its site complementary to olig 1, gradually displacing olig 3 from the bond with olig 2.

2) Only sufficiently long oligonucleotides, usually more than 15 bases, can be compatible with the method of calculations.

3) Applicability of the method for solving problems of exclusively Boolean algebra.

Thus, the required technical result consists in creating a molecular computing device capable of performing fast calculations (within a few minutes, and preferably in less than 1 minute); solve a wider range of problems, including not only Boolean (digital) algebra problems, but also various "analog" algebraic problems with continuously changing values of variables, as well as operate with oligonucleotides of various lengths, including short oligonucleotides less than 10 bases.

Brief description of the invention

To achieve the specified technical result, a molecular computing device based on the unexpectedly discovered by us new mechanism for storing information in nucleic acids (DNA / RNA) has been proposed.

After the great discovery of the structure of the most reliable biological storage of information, DNA, it is difficult to imagine any other more reliable storage of biomolecular information. Due to the high specificity, selectivity and affinity of two complementary DNA strands, the information is packed securely with a built-in backup, which allows you to save, read and copy information with fantastic accuracy to avoid errors.

It is securely packed, precisely readable and copied information based on strict correspondence (complementarity) of two DNA strands due to ATHC base-pairing and codons. A lot of studies have been devoted to this - various repair systems, error tracking, etc. have been found. The present invention is based on another method of storing information in DNA and other molecules discovered by us, which can be implemented with equal efficiency not on DNA / RNA, but on absolutely any other molecules, since based on low specific and low affinity interactions.

Namely, a molecular computing device is proposed that includes at least a set of single-stranded nucleic acids, and for which: the numerical measure of the input signals are the measures associated with the input molecules; signals are transformed into output signals as a result of interactions between the input molecules, said single-stranded nucleic acids of said set, and output molecules, characterized in that said single-stranded nucleic acids of said set have a low affinity of interaction with each other such that at least 0.1% of the amount of each of them exists in free molecular form, and does not enter into complexes with other molecules.

In addition, a molecular computing device that includes at least a set of single-stranded nucleic acids, and for which: the numerical measure of the input signals are the measures associated with the input molecules, the numerical measure of the output signals are the measures associated with the output molecules, the input signals are transformed into output signals as a result of interactions between the input molecules, said single-stranded nucleic acids of said set, and output molecules, and the complexes formed by single-stranded nucleic acids of said set are in equilibrium (or come into equilibrium) with the free molecular form of said single-stranded nucleic acids so that is not less than 0.1% of the total concentration of each single-stranded nucleic acid of the above set is in free molecular form.

In addition, a molecular computing device is proposed that includes at least a set of single-stranded nucleic acids, and for which: the numerical measure of the input signals are the measures associated with the input molecules; signals are transformed into output signals as a result of interactions between input molecules, said single-stranded nucleic acids of said set, and output molecules, and the total concentration of each said single-stranded nucleic acid exceeds the value of the dissociation constant of the complexes formed by this single-stranded nucleic acid and other single-stranded nucleic acids of said set no more than 100,000 times.

In addition, a molecular computing device is proposed that includes at least a set of single-stranded nucleic acids, and for which the numerical measure of the input signals are the measures associated with the input molecules, the numerical measure of the output signals are the measures associated with the output molecules, the input signals are transformed into output signals as a result of interactions between input molecules, said single-stranded nucleic acids of said set, and output molecules, characterized in that said interactions between single-stranded nucleic acids of said set are reversible binding of incompletely complementary ones between single-stranded nucleic acids of said set, and such, that for any two single-stranded nucleic acids of the aforementioned set, the length of the most extended connected region of their mutual complementarity does not exceed 10 bases.

In addition, a method for performing computations using the above described molecular computing devices.

In addition, a method of performing computations using the above-described molecular computing devices, including the following steps: selecting said set of single-stranded nucleic acids, mixing said set with said input molecules and said output molecules if they are not part of said set, incubating the mixture to ensure interactions between input molecules, said single-stranded nucleic acids of said set and said output molecules, and transformation of input signals into outputs as a result of said interactions, registration of said output signal.

In addition, the proposed pharmaceutical formulations, including the described VLMs for the regulation of gene expression.

Dictionary

To facilitate understanding of the essence of this invention, here (earlier and later in this text) the following terms are used:

Free molecular form is a state of a molecule in which it is not bound (does not form a complex / is not associated) with other molecules. This concept can be viewed in a statistical context, i.e. observed behind an ensemble of identical molecules of a given type, for example, all ACTGTAC oligonucleotides. It is clear that these molecules are in an equilibrium process of formation-decomposition of a complex with other oligonucleotides. Moreover, if there is a second oligonucleotide interacting with the first, then, most likely, each ACTGTAC molecule from time to time will periodically be complexed with the second nucleotide. In this sense, free form refers to the percentage of ACTGTAC molecules in an unbound state.

Weak (low) affinity is such a mutual affinity of two molecules at which the molecules are in such a concentration that a significant part of these molecules is in free molecular form (in our case, at least 0.01% of molecules, preferably 0.02%, preferably 0.05%, preferably 0.1%, more preferably 0.2%, more preferably 0.5%, more preferably 1%, more preferably 2%, more preferably 5%, more preferably 10%, more preferably 20%). As shown below, the number of molecules in free molecular form is correlated with the concentrations of molecules and the constants of affinity for other molecules according to the law of mass action. To clarify the general connection between the affinity constant of two molecules A, B and some of these molecules in free molecular form, we present the following reasoning. According to the law of mass action in equilibrium:

In this case, let us assume for simplicity that the total (or the initial concentration of molecules A and B upon mixing) is the same and equal to [A] 0. Let us designate the equilibrium concentration of the AB complex as [AB] eq. Then the equilibrium concentration [A] eq = [B] eq = ^: [A] 0- [AB] eq. Solving the equations, we get that the following part A will be in free molecular form:

Note that oligonucleotides often form dimers and numerous associates with themselves. Therefore, the translation of the value of [A in the complex,%] (which, for example, are given in the Examples of implementation of the invention) into the dissociation constant should also take into account such multidimensions formed only by molecules A or only by molecules B. However, in the first approximation (to determine the order of the constant), you can use the above formula and table.

As seen in FIG. 1, even the most non-affinity polyA-polyT oligonucleotides with a length of 20 bases, in the case of complementarity, reach a Kd of about 10-16 M. Accordingly, at standard concentrations used in the field of DNA calculations (10 ^-8 -10 ^-6 M), the ratio Kd [A ] 0 = 10 ^-8 -10 ^-10 , respectively, less than 0.001-0.01% of the molecules will be present in free molecular form. More affinity complementary variants of the same length, for example, polyG-polyC-reach Kd of about 10 ^-28 M. Accordingly, in order for at least 0.001% of molecules to remain in free molecular form, the concentration of molecules [A] 0 must be less than 10 ^-18 M, those. 6,000 molecules per milliliter. Surprisingly, even at such a concentration, not a single molecule will remain in molecular form in 1 milliliter of solution (6000 * 0.001% = 0.06)!

Parasitic affinity is such a mutual affinity of two molecules in which the molecules are in such a concentration that a small part of these molecules is in free molecular form (in our case, not more than 20%, preferably 10%, preferably 5%, preferably 2%, preferably 1%, more preferably 0.5%, more preferably 0.2%, more preferably 0.1%, more preferably 0.05%, more preferably 0.02%, more preferably 0.01%, more preferably 0.005%, more preferably 0.002%, more preferably 0.001%). It is clear that the concentration of molecules in a free molecular form can be recalculated in the ratio of the dissociation constants of complexes of a molecule with other molecules of the system according to the law of mass action as shown above.

Non-interaction of molecules - in this case, it is understood that these molecules have a parasitic affinity for each other.

Substantially non-complementary: under the essential non-complementarity of two nucleic acids is meant a chaotic (sporadic, etc.) mismatch (in terms of complementarity) of nucleotides throughout their sequences. Moreover, the percentage of such non-complementary bases is significant in relation to the total length of the nucleic acid - 2%, preferably 5%, more preferably 10% or more. Moreover, the longer the oligonucleotides, the higher the percentage should be. It is understood that this is significantly different from the partial non-complementarity in strand displacement systems using the dangling ends shown in FIG. 1, i.e. when a long complementary sequence is flanked by a long non-complementary sequence. Also, due to the large percentage of non-complementary bases, this is significantly different from traditional single nucleotide polymorphisms SNP (single nucleotide polymorphism).

The output threshold is the signal value that corresponds to the boundary between the output value = 1 and the output value = 0. It is used to convert an analog signal to binary digital. Since molecular systems are analog devices (that is, the signal can continuously change from small to large), a threshold is used to classify the output signal as a Boolean True or False. For different applications, different strategies can be used as such a threshold value. As the threshold value in all the examples given, the average value between the maximum and minimum output signals among all the input combinations achieved is used.

Formulations can be understood as substances, substances, molecules, supramolecular agents, nanoparticles, microparticles, nanoagents, microagents, formulations, dosage forms, etc., which are used both for therapy and diagnosis of various diseases or conditions of the body or patient, incl. include labels, contrast agents, controlled release components, paramagnetic and magnetic agents, cytotoxic agents, and the like.

"Measure" - by "measure associated with a molecule" means any numerical or other value that can be considered the input of a computing device associated with the molecule itself or its parameters. Such a numerical value of the input can directly equal the numerical value of a parameter (for example, concentration), or correlate with it according to a certain rule - for example, when a certain threshold value of concentration is exceeded, the input is considered = 1, otherwise the input = 0. Such parameters can be the nucleic acid sequence, its length, concentration, mass, parameters of its complexes with other molecules, etc.

Equilibrium with free molecular form means equilibrium in the thermodynamic sense.

As used herein, the term "pharmaceutical" refers to a composition that is useful in treating a disease or symptom of a disease.

The term "treatment" or "therapy" (treating, treatment, therapy) refers to any indication of success in the treatment or improvement of a disease, injury, pathology or condition, including any objective or subjective parameters such as reduction; remission; reducing symptoms or slowing down the deterioration of the patient's condition, pathology or patient's condition; slowing down the rate of degeneration; what makes the final stage of the disease less painful; improving the physical or mental well-being of the patient. Treatment or improvement of symptoms can be based on objective or subjective parameters; including results of physical examination, neuropsychiatric examinations, and / or psychiatric evaluation. For example, certain methods presented here can successfully treat cancer by reducing the incidence of cancer and inducing cancer remission. The term "treatment" and its continuation includes the prevention of injury, pathology, condition or disease.

"Disease" or "condition" refers to the state of health of a patient or subject to be treated with various drugs, dispersions, or other methods provided herein.

"Patient" or "subject in need thereof" refers to a living organism suffering from or (possibly) prone to a disease or condition that can be treated by administering a drug, pharmaceutical composition, or agent as defined herein. Non-limiting examples include humans, other mammals, bulls, rats, mice, dogs, monkeys, goats, sheep, cows, deer, and other non-mammalian animals. In particular, the patient can be human.

It should be noted that the given constants of interaction of various oligonucleotides within the framework of the Description and Examples are calculated for interactions at a temperature of 25 degrees Celsius.

It should be noted that throughout the application, alternatives are written in Markush groups, for example, each position of a therapeutic agent may contain more than one candidate therapeutic agent. In particular, it is assumed that each member of the Markush group should be considered separately, thus containing a different embodiment, and the Markush group should not be read as a whole.

Implementation of the invention. Detailed description of the embodiments of the invention.

The characteristics and advantages of the molecular computing device according to the present invention will be further illustrated in the following detailed description. While the manufacture and use of various embodiments of the present invention are discussed in detail below, it should be understood that the present invention provides many applicable inventive concepts that may be embodied in a wide variety of specific contexts. The specific embodiments discussed herein merely illustrate various ways of making and using the invention and do not limit the scope of the invention. All content provided herein (earlier and later) is incorporated herein by reference in its entirety for all purposes.

The discovery of the double-stranded structure of DNA and the genetic code turned biologists' understanding of the storage of hereditary information upside down. The high specificity and affinity of the interaction of two DNA strands with each other is an amazing mechanism invented by nature to transfer data from one organism to another. Encoding genetic information using a linear sequence of ATGC letters in two chains, in which the letter A on one chain strictly corresponds to T on the other, and the letter G to the letter C, is an extremely convenient mechanism that makes it possible to copy information extremely efficiently, as well as reliably. store, correcting random errors at the expense of two versions of the same information in a single indissoluble biomolecular complex.

Nevertheless, looking at all the mysteries of DNA (for example, the presence of non-coding DNA / RNA, including small non-coding oligonucleotides, an unexpected upregulation of genes by small RNAs), the question arises - with all the power of DNA - and does not DNA / RNA store information as or else - by some mechanism, which is not yet known and not discovered, for which all these incomprehensible entities would be needed?

This mystery encourages various researchers to look for alternative ways of storing and processing information in nucleic acids. Thus, various systems have been proposed based on the phenomena of strand displacement, DNAzymes, etc. In addition, there is an active search for non-nucleic molecular computing systems for working with information.

However, existing methods consider the transfer of information from between molecules to within the Paradigm of highly specific interactions, within which living systems are presented as a set of molecules that transmit useful information through highly selective interactions. In particular, the aforementioned DNA computing systems are also based on the complementarity of two DNA strands to ensure stand displacement reactions and specificity of DNAzymes (as shown in Fig. 1 below). Moreover, the so-called molecular pathways are distinguished, along which the signal propagates from one molecule to another.

We discovered a fundamentally different and extremely efficient mechanism for storing and processing information in molecular systems. In the discovered mechanism, molecules transmit information in reverse, using weak affine interactions with each other.

In principle, this information processing mechanism can be implemented by any molecule, but it is especially effective when implemented using nucleic acids.

We noticed a unique DNA / RNA feature, the beauty of which went unnoticed for a long time in the shadow of the DNA double helix. Take the single-stranded oligonucleotide X. Typically, the complementary oligonucleotide has the highest affinity of all other strands of the same length. But if we take all possible oligonucleotides (with the same length as for X) and sort them by their affinity for X, then we get a tightly packed “affinity continuum”.

FIG. 2 shows the density of this continuum for 10 base DNA, calculated using the NUPACK DNA double strand affinity calculation algorithm (unpack.org). It turns out that even for such short DNAs, for any chosen value of the affinity constant K, you can choose an oligonucleotide that will have an affinity constant for X equal to K + - 10% (with the exception of 25 minimum / maximum affinity values).

Moreover, FIG. 3 shows that an equally dense packing of oligonucleotides by affinities has a distribution with respect to two oligonucleotides at once - X and its complementary X , or to X and arbitrary Y. with the desired affinity for multiple oligonucleotides at once.

This feature of DNA / RNA is the key to our discovered new mechanism for storing information in molecular systems.

Let's look at what happens in a mixture of short single-stranded DNA molecules, if we deliberately remove all complementary strands from there (that is, remove from consideration in general all “specific” interactions - that is, do not use a “specific” ligand for each receptor ). In this case, oligonucleotides will remain in the mixture with weak affinity and reversibly binding to each other according to the law of mass action. At the same time, various complexes of one chain with all the others will coexist simultaneously, and there will probably be fewer of them than free molecules. Such interactions can easily be found on carrier proteins such as BSA and small molecules that compete for binding to the hydrophobic pocket of the protein.

The difference of such a system from the traditional paradigm of storing information in specific (complementary) interactions is shown in Fig. 4 (compare with Fig. 1)

Mathematically, such a system will be described by a system of equations for the interaction of molecules of the ensemble. In the simplest case, assuming that only two-component associates can be formed, and considering thermodynamically equilibrium systems, we obtain the system of equations shown in Fig. 4.

The present invention demonstrates the possibility of creating molecular computing devices (MVDs) operating on the basis of this non-linear system of equations. IDL data store and process information outside the standard paradigm of highly specific interactions.

In the present invention, the proposed molecular computing devices store, process information using an ensemble of molecules, between which there are many weak low-affinity and weakly specific (nonspecific) interactions, and signal transmission from input molecules to output molecules is transmitted due to changes in the concentrations (equilibrium or nonequilibrium) of free components of the ensemble and their complexes.

MVPs that implement Boolean algebra

According to this invention, the creation of IDLs that implement components for solving problems of Boolean algebra (which is the basis for the functioning of electronic computers) is based on the following rules.

1) YES logic gate. An implementation diagram is shown in FIG. 5 and experimentally shown in Examples 1-6. The MVL consists of an input oligonucleotide I, an output oligonucleotide S, and a single oligonucleotide Q that handles signal transmission between input and output. We will consider the amount of S in a free molecular form (i.e., not in a state of complex with other oligonucleotides) as a measure of the yield. Input value = 1 if I is added to the system, and input = 0 if not added.

To create an IDL, oligonucleotides I, S, Q are selected so that Q has weak affinity for I and Q; a S and I interacted (see the definition of this term in the dictionary) with each other.

According to the invention, the concentrations of I, Q, S are selected in the region of their interaction constants (to provide the required levels of complexation).

In this case, the system operates as follows. For example, let oligos S and Q interact in such a way that, in the absence of other oligos, 90% of oligos S would be connected with Q. Then if we add I to their system, it will begin to pull off part of Q, which in turn will increase the number free S (will release from the complex with Q). In this case, if the constants are approximately the same, then a significant delay can be achieved when the concentration of I exceeds S. In other words, the efficiency of redistribution of Q - can be changed by the choice of constants, as well as by the relationship between the concentrations of oligonucleotides.

2) Logic gate NO (NOT logic gate). An implementation diagram is shown in FIG. 6 and experimentally shown in Example 7. The MVL consists of an input oligonucleotide I, an output oligonucleotide S, and two oligonucleotides Q and M, which process signal transmission between input and output. We will consider the amount of S in a free molecular form (i.e., not in a state of complex with other oligonucleotides) as a measure of the yield. Input value = 1 if I is added to the system, and input = 0 if not added.

To create an IDL, oligonucleotides I, S, Q, M are selected so that Q has weak affinity for I and for Q; M - weak affinity for Q and I; and the other couples did not interact (see the definition of this term in the dictionary) with each other.

According to the invention, the concentrations of I, M, Q, S are selected in the region of their interaction constants (to provide the required levels of complexation).

In this case, the system functions similarly to that described above. Without I, Q is associated more with M than with S. When I is added to their system, it will begin to draw back a part of M, which in turn increases the availability of Q for binding to S. The efficiency of Q redistribution can be changed by choosing constants, as well as the relationship between the concentrations of oligonucleotides.

3) OR logic gate. An implementation diagram is shown in FIG. 7 and experimentally shown in Example 8. The MVL consists of input oligonucleotides In (n = 1, 2, 3 ...), output oligonucleotide S, and a single oligonucleotide Q, which handles signal transmission between input and output. We will consider the amount of S in a free molecular form (i.e., not in a state of complex with other oligonucleotides) as a measure of the yield. Input value In = 1 if In is added to the system, and input = 0 if not added.

To create an IDL, choose oligonucleotides I, S, Q so that Q has weak affinity for In and for Q; a S and I did not interact (see the definition of this term in the dictionary) with each other.

According to the invention, the concentrations of I, Q, S are selected in the region of their interaction constants (to provide the required levels of complexation).

In this case, the system operates as follows. Then if we add a system of any of In, then it will begin to pull off a part of Q, which in turn will increase the amount of free S (release it from the complex with Q). The efficiency of Q redistribution - can be changed by the choice of constants, as well as by the relationship between the concentrations of oligonucleotides.

4) AND logic gate. The implementation diagram is shown in Fig. 8 and experimentally shown in Example 9. The MVL consists of input oligonucleotides In (n = 1, 2, 3 ...), output oligonucleotide S, and oligonucleotides Qn, which processes signal transmission between input and output. We will consider the amount of S in a free molecular form (i.e., not in a state of complex with other oligonucleotides) as a measure of the yield. Input value In = 1 if In is added to the system, and input = 0 if not added.

For the creation of IDM, the oligonucleotides In, S, Qn are selected so that each Qn has a weak affinity for In and for Q; and other couples interacted (see the definition of this term in the dictionary) with each other.

According to the invention, the concentrations of In, Qn, S are selected in the region of their interaction constants (to provide the required levels of complexation).

In this case, the system operates as follows. To release S from complexes with all Qn, it is necessary to add all In (otherwise, Qx, for which no Ix will bind to S). The efficiency of redistribution can be changed by the choice of constants, as well as by the relationship between the concentrations of oligonucleotides.

The combination of principles for constructing these gates constitutes a functionally complete set of logic functions (i.e., a complete basis). Those. it can be used to implement complex boolean functions.

Implementation of complex systems based on Boolean algebra

Memory cell and square root calculation:

Examples 10-11 demonstrate systems that implement a memory location (with the ability to store and start reading) and calculating the root of a 4-bit binary number. FIG. 9. gives a scheme for constructing the corresponding MVEs that implement complex Boolean algebra.

It should be emphasized that Example 11 demonstrates the advantages of the present invention over previously known molecular computing systems. Namely, in 2010, Qian and Winfree implemented an IDL capable of solving the same problem (calculating the root of a 4-bit number), but they needed to use 137 oligonucleotides to transmit / process a signal between inputs and outputs according to the strand displacement principle, and the calculations took over 10 hours. Then, we achieved a decrease in the number of oligonucleotides in the system to 37 oligonucleotides and calculations in 1 hour by adding an enzyme to the system.

The MVL according to the present invention makes it possible to calculate the root in 5 minutes and using only 17 oligonucleotides (4 of which are inputs).

Gene expression control beyond the traditional paradigm of DNA interactions:

This invention allows you to create an MVP capable of influencing gene expression. Moreover, it makes it possible to create such MVLs, the functioning of which go beyond the traditional (standard, usual) paradigm of DNA interactions, and in general beyond the modern concepts of the effect of small nucleic acids on gene expression.

Currently, a large number of mechanisms are known for the influence of small nucleic acids on gene expression. For example, the antisense mechanism of nucleic acids is known, which can bind, for example, to messenger RNA and, through such competitive binding, reduce the expression of this gene. In addition, silencing RNAs are known, which involve a complex enzymatic cascade for the degradation of messenger RNA. However, it is known that, in certain cases, an attempt to suppress the expression of a particular gene leads to suppression of other so-called off-target genes. It is attempted to predict such side effects by comparing the sequence of a small nucleic acid (which should produce suppression of the desired gene) and other sequences in the genome that have sufficient affinity for this small nucleic acid.

We show that with the help of this invention it is possible to create an MVL that allows a particular small nucleic acid to influence the target sequence of a particular gene, having with it:

1) not a single complementary base without taking into account shifts (i.e. absolute non-complementarity)

2) non-exceeding maximum of complementarity, taking into account all possible relative shifts:

1 shift option: 1 AGGC tetraplet; 2 shift options: 1 GC doublet; 2 shift option: 1 doublet CU.

This behavior completely leaves the traditional paradigm of storing information in DNA, and in general the interactions in living systems that are currently being considered by DNA.

Example 19 shows an example of such an experimentally designed molecular computing device that implements the logical DA function to increase gene expression (output) upon the appearance of an input nucleotide for the following sequences:

5'-AGGCGCCCUGAC-3 '- target sequence in GFP mRNA:

3'-AGUCGUCCGAGU-5 '(5'-UGAGCCUGCUGA-3') - Input oligonucleotide.

It can be seen that, in view of the lack of complementarity, this input oligonucleotide cannot directly regulate gene expression according to none of the nucleic acid interactions known within the traditional paradigm - neither by the antisense regulation mechanism (antisense), nor by the siRNA mechanism (including by virtue of its short length), etc.

However, Example 19 shows efficient upregulation of gene expression when this input olig is added to the system. This is due to the fact that in the system of Example 19, the input oligonucleotide acts not on the target region of the gene itself, but on another short RNA, which it pulls from the mRNA by the mechanism of the DA logic gate, demonstrated in Example 6. This invention for the first time discloses the mechanism of upregulation of gene expression small RNA. Moreover, it can be seen that this is easily achieved in the presence in the system of several small RNAs with weak mutual complementarity, as well as the complementarity of some of them to mRNA. It is obvious that all the VLMs according to this invention can be applied to the regulation of gene expression, simply by using mRNA, not a fluorescently labeled model, but directly as a signal oligonucleotide. Thus, it is obvious that IDL data can be used for therapy and diagnosis of diseases, since represent a completely new and extremely multifunctional tool for the regulation of genetic information.

In addition, a similar mechanism can be realized if the input molecule is (the complex of input molecules will consist of) mRNA.

500-bit Logic AND gate

This invention enables molecular computing devices that are capable of:

1) be more efficient in storing and processing information than the traditional concept of storing information in the primary DNA sequence,

2) compete and even surpass electronic devices.

Let the following problem be given. There is a specific gene that has a specific sequence of 20 bases, which can be influenced in some way by using single-stranded oligonucleotides of the same size (for example, the antisense oligonucleotides mentioned above, silencing RNA, etc.).

Then, within the framework of the DNA complementarity paradigm, this sequence of 20 bases in length allows 4 ²⁰ ≈10 ¹² opportunities to influence it, i.e. there are 10 ¹² oligonucleotides that can either bind or not bind to a given sequence (and suppress / or not suppress gene expression - as described above). Example X shows the IDP is able to realize ≈10 2 ^{608 183} opportunities to influence the gene in the same formulation. This is significantly more than the number of elementary particles in the visible universe according to any currently published estimate.

Namely, Example 12 shows an MFP that implements a 500-bit AND logic gate. In this case, in the case of using a target sequence of a certain gene as an oligonucleotide S, the output of this logical gate, namely, the amount of oligonucleotide S free from complexes, will characterize the expression of the gene (as described above).

It should be noted that Figure 11 (below) proves the possibility of creating a 608-bit logic gate I. Indeed, if we consider the operation of 100-, 200-, 300, and 500-bit gates, it can be seen that the extrapolation of the maximum output values for inputs of the type "single input = 0 "and threshold values (average between inputs" all inputs = 0 "," all inputs = 1 ") is achieved with a 608-bit AND gate.

It should be noted that, at the moment, electronic computers with such a bit rate have not been implemented, which indicates the colossal computational capabilities of the proposed principle of constructing IDLs and, in general, this previously unknown phenomenon / principle of storing information in nucleic acids.

Elementary algebra

This invention allows you to create an IDP capable of processing data and for solving various algebraic equations for continuously changing values of a variable. This invention is the first and currently the only option for creating an IDP capable of solving such mathematical problems.

Examples 13-18 show various MVPs solving mathematical equations with an error of less than 2%.

Various aspects of the invention

It should be noted that the discovered phenomena and these inventions are based on the fundamental law of mass action, which determines the passage of various reversible reactions, and is in no way limited to nucleic acids (DNA / RNA).

We have demonstrated the implementation of such information processing using nucleic acids as an example. However, we do not use any "special" properties of DNA (DNA-specific properties), except that this is the only type of molecules between which at the moment the affinity of two-component systems is predicted quite accurately (based on the primary structure), and synthesis of a large the amount of substances and this does not seem to be a problem. The implementation of such systems on other molecules will mathematically be just as efficient.

In principle, this information processing / transmission mechanism can be implemented by any molecules, but if it is implemented with the help of DNA, processing can use the ability of olig to bind not only with its complementary (well, or with its variants containing monobasic SNP polymorphisms - single nucleotide polymorphisms) , but with the ability to weakly associate with many other olig with a sequence only vaguely reminiscent of the complementary (see Fig. 4).

While modern biology is enthusiastically mastering high-affinity interactions, our invention realizes and demonstrates the power of low-affinity (low-affinity) interactions. This invention is a completely new concept in biomolecular computation, and surpasses all currently known in speed and power.

This invention can be used to combine electronic systems with biological ones. In addition, since the shown mechanism, which is based on the most basic and basic laws of chemical reactions, can be used by nature, and she had millions of years to perfect this mechanism. Therefore, the possibility of combining IDLs with natural systems on the same mechanism gives undeniable advantages to these IDLs.

Information storage is possible not only in the concentrations of free oligonucleotides (or other molecules) and / or complexes. But it is also possible to use a) the rate of concentration change, especially if you need to operate quickly - such as memory, etc., b) we worked in a binary system, but you can also create MVDs that work 1) analog, 2) react to the smallest changes and 3) storing (processing) information using complex functional dependencies that will be able to store very large amounts of data.

In the above examples, data storage and processing was carried out using popular complexes or free oligonucleotides. In addition, the storage of information is possible at the rate of concentration change, which makes it possible to achieve an even higher rate of data processing. In addition, in the above examples with Boolean algebra, the interpretation of the outputs as true or false (0 or 1) is given. However, these examples can be considered not as a digital version of data processing, but also as analog. Likewise, Nature uses continuous analog processes and threshold-based ones, for example, neuron firing.

Compared to other molecular computational methods that allow complex calculations (i.e., they are not limited to several Boolean functions and are able to solve more than one specific problem), the unique advantage of our invention is the almost complete absence of any restrictions on the types and properties of molecules that compatible with our approach. There are no restrictions on the ratio of input, output and processing substances. In the case of DNA or RNA, these can be oligonucleotides of different sizes (for example, in contrast to the concepts based on DNAzymes), they can be very short (for example, in contrast to the concept of strand displacement), they may not be nucleic acid at all acids (for example, as opposed to concepts based on DNA winters).

In addition, as the example with the solution of mathematical problems of elementary algebra shows, it can be seen that the choice of oligonucleotides can be easily adjusted for almost any input and output oligonucleotides. This invention provides wide freedom in the selection of both input and output and processing oligonucleotides. Moreover, any IDL can be organized for almost any set of input and output oligonucleotides due to a wide, finely tuned system of processing oligonucleotides, as shown in the example with problems of elementary algebra.

Special uses of IDPs

It is already clear that small RNA / DNA is an important natural tool, for a small number of which they have found meaning (for example, siRNA). There are various single-stranded oligonucleotides that perform different functions: from the regulation of intracellular processes to the provision of intercellular communication using exosomes.

Our invention completely changes the perspective for their analysis. We show that it is not enough to bioinformatically identify complementary sequences; it is important and necessary to look at all potential interactions of small (and ideally also large) nucleic acids. Because The “first frontier” of complementary oligonucleotides can be controlled by a significantly larger amount of RNA, as in the demonstrated VLFs.

The biomedical significance of this phenomenon can be extremely important for the diagnosis and treatment of a large number of diseases. This phenomenon (and our invention) must and can be taken into account and used in gene therapy (including short RNAs). In addition, the invention can be used for fine multifactorial adjustment of such a therapy. Its use will provide additional control over the specificity and effectiveness of therapy, as well as minimize the risks of off-target modifications in the genome.

We discovered a mechanism for storing information in DNA and, in a more general case, for any low-affinity interactions between molecules, and IDLs invented on its basis can be used and applied in various fast processes (for example, short-term memory), or long-term processes (for example, aging or evolutionary systems ).

The existence of this phenomenon significantly complicates the analysis of the interactome, i.e. many intermolecular interactions, which in the traditional paradigm are considered as molecular pathways based on high-affinity and specific interactions. Although, in this case, it may be worth talking about a separate concept of "affinome", which considers, on the contrary, a lot of low-affinity interactions that affect various processes in living systems. The analysis of such an "affinoma" may be required to understand the full picture and may reveal the secrets of many diseases that were previously inaccessible and could not be addressed in the paradigm of high-affinity and highly specific drugs and analyzes.

Various aspects of the embodiment of the invention

Thus, the present invention provides a molecular computing device (MVD) (as well as methods for performing calculations using the described MVD), including at least a set of single-stranded nucleic acids, and for which: the numerical measure of the input signals are measures related with input molecules (including input oligonucleotides, input proteins, mRNA, etc., including those included or not included in the mentioned set), the numerical measure of output signals is the measures associated with output molecules (including including output oligonucleotides, output proteins, mRNA, etc., including those included or not included in the said set), the input signals are transformed into output signals as a result of interactions between the input molecules, the mentioned single-stranded nucleic acids of the said set, and output molecules, characterized in that said single-stranded nucleic acids of said set have a low affinity of interaction between oh such that at least 0.1% of the amount of each of them exists in free molecular form, and is not included in complexes with other molecules.

In addition, a molecular computing device that includes at least a set of single-stranded nucleic acids, and for which: the numerical measure of the input signals are the measures associated with the input molecules, the numerical measure of the output signals are the measures associated with the output molecules, the input signals are transformed into output signals as a result of interactions between the input molecules, said single-stranded nucleic acids of said set, and the output molecules.

In addition, a molecular computing device, characterized in that said single-stranded nucleic acids of said set have a low affinity of interaction with each other such that not less than 0.001% (preferably 0.003%, preferably 0.01%, preferably 0.03%, preferably 0.1%, preferably 0.3% , more preferably 0.5%, more preferably 1%, more preferably 2%, more preferably 3%, more preferably 5%, more preferably 10%, more preferably 15%, more preferably 20%, more preferably 30%, more preferably 50%) the amount (concentration, weight) of each of them exists in free molecular form, and does not enter into complexes with other molecules.

In addition, a molecular computing device, characterized in that the said single-stranded nucleic acids of the said set have a low affinity of interaction with each other, such that at least 0.1% of the amount of each of them exists in free molecular form, and is not included in complexes with other molecules.

In addition, a molecular computing device in which the complexes formed by single-stranded nucleic acids of said set are in equilibrium (thermodynamic) with the free molecular form of said single-stranded nucleic acids so that not less than 0.1% of the total (total) concentration of each single-stranded nucleic acid of said set is in free molecular form.

In addition, a molecular computing device, characterized in that for each pair of said single-stranded nucleic acids of said set, their mutual affinity is so low that in the absence of other single-stranded nucleic acids of said set, at least 0.1% of the total concentration of each single-stranded nucleic acid of said pair is in free molecular form.

In addition, a molecular computing device, characterized in that the mentioned single-stranded nucleic acids of the mentioned set are so low-affinity to each other that at least 0.1% of the amount of each of them exists in free molecular form, and is not included in complexes with other molecules.

In addition, a molecular computing device, characterized in that the said single-stranded nucleic acids of the said set have a low affinity of interaction with each other, such that at least 0.1% of the amount of each of them exists in free molecular form.

In addition, a molecular computing device, characterized in that the mentioned single-stranded nucleic acids of the said set have such a low affinity of interaction with each other that at least 0.1% of the amount of each of them exists in free molecular form, and not in complexes with other molecules.

In addition, a molecular computing device, characterized in that the said single-stranded nucleic acids of the said set have such a low pairwise affinity of interaction that at least 0.1% of the amount of each of them exists in free molecular form, and not in complexes with other molecules.

In addition, a molecular computing device, characterized in that for each pair of said single-stranded nucleic acids of said set, their mutual affinity is so low that in the absence of other single-stranded nucleic acids of said set, at least 0.1% of the total concentration of each single-stranded nucleic acid of said pair is in free molecular form.

In addition, a molecular computing device that includes at least a set of single-stranded nucleic acids, and which is capable of: accepting input signals, the numerical measure of which are the measures associated with the input molecules added to the said device, generate output signals, the numerical measure of which is are measures associated with output molecules to transform input signals into output signals as a result of interactions between output molecules, if they are not part of said set, input molecules, and said single-stranded nucleic acids of said set.

In addition, a molecular computing device, characterized in that for each pair of said single-stranded nucleic acids of said set, their mutual affinity is so low that in the absence of other single-stranded nucleic acids of said set, at least 0.1% of the total concentration of each single-stranded nucleic acid of said pair is in free molecular form.

In addition, a molecular computing device, characterized in that the complexes formed by single-stranded nucleic acids of said set are in equilibrium with the free molecular form of said single-stranded nucleic acids so that at least 0.1% of the total concentration of each single-stranded nucleic acid of said set is in free molecular form.

In addition, a molecular computing device, characterized in that for each pair of said single-stranded nucleic acids of said set, their mutual affinity is so low that in the absence of other single-stranded nucleic acids of said set, at least 0.1% of the total concentration of each single-stranded nucleic acid of said pair is in free molecular form.

In addition, a molecular computing device, characterized in that for each pair of said single-stranded nucleic acids of said set, their mutual affinity is so low that in the absence of other single-stranded nucleic acids of said set, at least 0.1% of the total concentration of each single-stranded nucleic acid of said pair is in free molecular form.

In addition, a molecular computing device, characterized in that for each pair of said single-stranded nucleic acids of the said set, their mutual affinity is so low that, in the absence of other single-stranded nucleic acids of the said set, not less than 0.001% (preferably 0.003%), preferably 0.01 %, more preferably 0.03%, more preferably 0.1%, more preferably 0.3%, more preferably 0.5%, more preferably 1%, more preferably 2%, more preferably 3%, more preferably 5%, more preferably 10%, more preferably 15%), more preferably 20%, more preferably 30% , preferably 50%) of the total concentration of each single-stranded nucleic acid of said pair is in free molecular form.

In addition, a molecular computing device, characterized in that the mentioned single-stranded nucleic acids of the mentioned set are so low-affinity with each other that at least 0.1% of each of them exists in free molecular form, and is not included in complexes with other molecules.

In addition, a molecular computing device, characterized in that said single-stranded nucleic acids of said set are so low-affinity to each other that they are not less than 0.001%) (preferably 0.003%, preferably 0.01%, preferably 0.03%, preferably 0.1%, preferably 0.3%, preferably 0.5%, more preferably 1%, more preferably 2%, more preferably 3%, more preferably 5%, more preferably 10%, more preferably 15%, more preferably 20%, more preferably 30%, more preferably 50%) each of them exists in free molecular form and not is a part of complexes with other molecules.

In addition, an MVL, in which said output molecules are single-stranded nucleic acids.

In addition, an MVL, in which the said output molecules are additional single-stranded nucleic acids not included in the specified set.

In addition, an MVL, in which the said output molecules are single-stranded nucleic acids of the said set.

In addition, the MVU, in which the mentioned output molecules are mRNA.

In addition, the MVP, in which the said measure of the output signal is related to the level of expression of the gene encoded by the said mRNA.

In addition, an MVL, in which the mentioned output molecules are proteins.

In addition, an MVL, in which said input molecules are single-stranded nucleic acids.

In addition, an MVL, in which said input molecules are single-stranded nucleic acids of said set.

In addition, the MVU, in which the mentioned input molecules are mRNA.

In addition, an MVL, in which the mentioned input molecules are proteins.

In addition, an IDL in which said nucleic acids of said set are DNA.

In addition, an IDL, in which said nucleic acids of said set are RNA.

In addition, an MVL, in which said nucleic acids of said set are oligonucleotides.

In addition, the IDL, in which the numerical measure of the input signal is related to the total concentration of the input molecule.

In addition, the MVL, in which the numerical measure of the input signal is related to the total mass of the input molecule.

In addition, the MVT, in which the numerical measure of the output signal is related to the total concentration of the output molecule in free molecular form.

In addition, the MVL, in which the numerical measure of the output signal is related to the mass of the output molecule in free molecular form.

In addition, the MVC, in which the numerical measure of the output signal is related to the concentration of the complex formed by the output molecule with the nucleic acids of said set.

In addition, the MVC, in which the numerical measure of the output signal is related to the concentration of complexes formed by the output molecules with the nucleic acids of the above set.

In addition, an IDL, in which said measure of the output signal is the level of expression of a gene encoded by a single-stranded nucleic acid of said set, which is an output molecule.

In addition, the MVD, in which the said numerical measure of the output signal is classified as a boolean value "0/1" as a result of comparison with the threshold value: if the signal is less than the threshold value, then the output is 0, if it is greater than the threshold value, then the output is 1.

In addition, the MVA, in which the transformation of input signals into output signals occurs as a result of a shift in the equilibrium of complexation between the input molecules, the mentioned single-stranded nucleic acids of the mentioned set and the output molecules, as a result of which the mentioned numerical measure of the output signals changes.

In addition, the IDL, in which at least 0.1% of the total (total) concentration of each of the mentioned single-stranded nucleic acid of the said set is in free molecular form.

In addition, the IDL, in which at least 1% of the total concentration of each said single-stranded nucleic acid of the said set is in free molecular form.

In addition, the IDL, in which at least 5% of the total concentration of each said single-stranded nucleic acid of the said set is in free molecular form.

In addition, the MVL, which includes at least a set of single-stranded nucleic acids, and for which: the numerical measure of the input signals are the measures associated with the input molecules, the numerical measure of the output signals are the measures associated with the output molecules, the input signals are transformed into output signals as a result of interactions between the input molecules, said single-stranded nucleic acids of said set, and output molecules, moreover, the total concentration of each said single-stranded nucleic acid exceeds the value of the dissociation constant of the complexes formed by this single-stranded nucleic acid and other single-stranded nucleic acids of said set, not more than, than 100,000 times.

In addition, the MVL, which includes at least a set of single-stranded nucleic acids, and for which: the numerical measure of the input signals are the measures associated with the input molecules, the numerical measure of the output signals are the measures associated with the output molecules, the input signals are transformed into output signals as a result of interactions between input molecules, said single-stranded nucleic acids of said set, and output molecules, characterized in that said interactions between single-stranded nucleic acids of said set are reversible binding not completely complementary between single-stranded nucleic acids of said set, and such, for any two single-stranded nucleic acids of the above set, the length of the most extended connected region of their mutual complementarity does not exceed 10 bases.

In addition, a method for performing computations using the above described molecular computing devices.

In addition, a method of performing computations using the above-described molecular computing devices, including the following steps: selecting said set of single-stranded nucleic acids, mixing said set with said input molecules and said output molecules if they are not part of said set, incubating the mixture to ensure interactions between input molecules, said single-stranded nucleic acids of said set and said output molecules, and transformation of input signals into outputs as a result of said interactions, registration of said output signal.

In addition, a molecular computing device including at least a set of single-stranded nucleic acids, in which the numerical measure of the input signal is a measure associated with at least one single-stranded nucleic acid of the set, the numerical measure of the output signal is a measure associated with at least one single-stranded nucleic acid of said set, the input signal is transformed from into an output signal as a result of interactions between single-stranded nucleic acids of said set, and the complexes formed by single-stranded nucleic acids of said set are in equilibrium with the free molecular form of said single-stranded nucleic acids so that is not less than 0.1% of the total concentration of each single-stranded nucleic acid of the above set is in free molecular form.

Moreover, a molecular computing device in which said nucleic acids are DNA.

Moreover, a molecular computing device in which said nucleic acids are RNA.

Moreover, a molecular computing device in which said nucleic acids are DNA or RNA.

In addition, a molecular computing device in which said nucleic acids are oligonucleotides.

Moreover, a molecular computing device in which said numerical measure of the input signal is the total concentration of said single-stranded nucleic acid.

In addition, a molecular computing device in which said numerical measure of an input signal is the amount of said single stranded nucleic acid.

Furthermore, a molecular computing device in which said numerical measure of the output signal is the concentration of said single-stranded nucleic acid in free molecular form.

Furthermore, a molecular computing device in which said numerical measure of the output signal is the concentration of complexes formed by said single-stranded nucleic acid with other nucleic acids of said set.

Furthermore, a molecular computing device in which said numerical measure of the output signal is the concentration of complexes formed by said single-stranded nucleic acid with certain nucleic acids of said set.

Furthermore, a molecular computing device in which said numerical measure of the output signal is the amount of said single-stranded nucleic acid in free molecular form.

Furthermore, a molecular computing device in which said numerical measure of the output signal is the number of complexes formed by said single-stranded nucleic acid with other nucleic acids of said set.

Furthermore, a molecular computing device in which said numerical measure of the output signal is the number of complexes formed by said single-stranded nucleic acid with certain nucleic acids of said set.

In addition, a molecular computing device in which the transformation of an input signal into an output signal occurs due to a shift in the complexation equilibrium between said single-stranded nucleic acids of said set, as a result of which said numerical measure of the output signal associated with the concentration of at least one single-stranded nucleic acid of said set changes. set.

In addition, a molecular computing device in which at least 0.2% of the total concentration of each of the mentioned single-stranded nucleic acid of the said set is in free molecular form.

In addition, a molecular computing device in which at least 0.5% of the total concentration of each of the mentioned single-stranded nucleic acid of the said set is in free molecular form.

In addition, a molecular computing device in which at least 1% of the total concentration of each said single-stranded nucleic acid of said set is in free molecular form.

In addition, a molecular computing device in which at least 2% of the total concentration of each said single-stranded nucleic acid of said set is in free molecular form.

In addition, a molecular computing device in which at least 5% of the total concentration of each said single-stranded nucleic acid of said set is in free molecular form.

In addition, a molecular computing device in which at least 10% of the total concentration of each said single-stranded nucleic acid of said set is in free molecular form.

In addition, a molecular computing device in which at least 20% of the total concentration of each said single-stranded nucleic acid of said set is in free molecular form.

In addition, a molecular computing device that transforms a binary input signal into an output signal according to the law of Boolean algebra.

In addition, a molecular computing device that transforms a continuous input signal into a continuous output signal according to the rule of elementary algebra.

In addition, a molecular computing device in which said measure of the output signal, which is a measure associated with at least one single-stranded nucleic acid of said set, is associated with the expression level of the gene of that nucleic acid of said set.

Furthermore, a molecular computing device in which said measure of the output signal is the expression level of a gene encoded by a single stranded nucleic acid of said set.

In addition, a molecular computing device in which an input signal is converted into an output signal according to the rule of logical DA, and which consists of (includes - comprises): an output oligonucleotide (i.e. an output molecule that is an oligonucleotide, included or not included in the mentioned set), an input oligonucleotide (i.e. an input molecule, which is an oligonucleotide, included or not included in the above set) - and its presence corresponds to a logical input equal to 1, and its absence corresponds to a logical input equal to 0, a processing oligonucleotide, and the processing oligonucleotide binds with the output oligonucleotide in the absence of other oligonucleotides so that not less than 0.1% and not more than 50% of the processing oligonucleotide and not less than 0.1% and not more than 50% of the output oligonucleotide is in free molecular form, for the input oligonucleotide: the processing oligonucleotide binds to it in the absence of other oligonucleotides so that n f less than 0.1% and not more than 50% of the processing oligonucleotide and not less than 0.1% and not more than 50% of the said input oligonucleotide is in free molecular form, for the input oligonucleotide: the output oligonucleotide binds to it in the absence of other oligonucleotides so that not less than 90% processing oligonucleotide and at least 90% of said input oligonucleotide is in free molecular form, while the result of the calculation is read after adding input oligonucleotides to a solution containing output and processing oligonucleotides as the amount of output oligonucleotide in free molecular form.

In addition, a molecular computing device for converting an input signal into an output signal according to the rule of logical DA, and which consists of: an output oligonucleotide, an input oligonucleotide, and its presence corresponds to a logical input equal to 1, and its absence corresponds to a logical input equal to 0, a processing oligonucleotide selected so that the processing oligonucleotide is able to bind to the output oligonucleotide in the absence of other oligonucleotides so that not less than 0.1% and not more than 50% of the processing oligonucleotide and not less than 0.1% and not more than 50% of the output oligonucleotide remains in free molecular form, the processing oligonucleotide is able to bind to input oligonucleotide in the absence of other oligonucleotides so that not less than 0.1% and not more than 50% of the processing oligonucleotide and not less than 0.1% and not more than 50% of the said input oligonucleotide remains in free molecular form, the output oligonucleotide is able to bind with the input oligonucleotide in the absence of other oligonucleotides so that at least 90% of the processing oligonucleotide and at least 90% of the said input oligonucleotide remains in free molecular form, and in which the result of the calculation corresponds to the amount of the output oligonucleotide in free molecular form after equilibration (or after incubation) after adding the input oligonucleotides to the solution containing the output and processing oligonucleotides.

In addition, a molecular computing device in which an input signal is converted into an output signal according to the logical NOT rule, and which consists of (includes - comprises): an output oligonucleotide, an input oligonucleotide (and its presence corresponds to a logical input equal to 1, and its absence corresponds to a logical input equal to 0), processing oligonucleotide A, processing oligonucleotide B, moreover, processing oligonucleotide A binds to the output oligonucleotide in the absence of other oligonucleotides so that not less than 0.1% and not more than 50% of the processing oligonucleotide and not less than 0.1% and not more than 50% of the output oligonucleotide is in free molecular form, processing oligonucleotide B binds to processing oligonucleotide A in the absence of other oligonucleotides so that no less than 0.1% and no more than 50% of processing oligonucleotide A and no less than 0.1% and no more than 50% of processing oligonucleotide B is in free molecule ular form, for the input oligonucleotide: the processing oligonucleotide B binds to it in the absence of other oligonucleotides so that not less than 0.1% and not more than 50% of the processing oligonucleotide B and not less than 0.1% and not more than 50% of the said input oligonucleotide is in free molecular form , for the input oligonucleotide: the output oligonucleotide binds to it in the absence of other oligonucleotides so that at least 90% of the processing oligonucleotide and at least 90% of the said input oligonucleotide is in free molecular form, for the input oligonucleotide: the processing oligonucleotide A binds to it in the absence of others oligonucleotides so that at least 90% of the processing oligonucleotide A and at least 90% of the said input oligonucleotide is in free molecular form, for the processing oligonucleotide B: the output oligonucleotide binds to it in the absence of other oligonucleotides so that at least 90% of the processing oligon ucleotide B and at least 90% of said output oligonucleotide is in free molecular form, and the result of the calculation is read after adding input oligonucleotides to a solution containing output and processing nucleotides as the amount of output oligonucleotide in free molecular form.

In addition, a molecular computing device for converting an input signal into an output signal according to the logical NOT rule, and which consists of: an output oligonucleotide, an input oligonucleotide, and its presence corresponds to a logical input equal to 1, and its absence corresponds to a logical input equal to 0, processing oligonucleotide A, processing oligonucleotide B selected so that the processing oligonucleotide A is able to bind to the output oligonucleotide in the absence of other oligonucleotides so that no less than 0.1% and no more than 50% of the processing oligonucleotide and no less than 0.1% and no more than 50% of the output oligonucleotide is in free molecular form, processing oligonucleotide B is able to bind to processing oligonucleotide A in the absence of other oligonucleotides so that no less than 0.1% and no more than 50% of processing oligonucleotide A and no less than 0.1% and no more than 50% of processing oligonucleotide B is in free molecular form, the processing oligonucleotide B binds to the input oligonucleotide in the absence of other oligonucleotides so that not less than 0.1% and not more than 50% of the processing oligonucleotide B and not less than 0.1% and not more than 50% of the said input oligonucleotide is in free molecular form, the output oligonucleotide is binds to the input oligonucleotide in the absence of other oligonucleotides so that at least 90% of the processing oligonucleotide and at least 90% of the said input oligonucleotide is in free molecular form, for the input oligonucleotide: processing oligonucleotide A binds to it in the absence of other oligonucleotides so that at least 90% of the processing oligonucleotide A and at least 90% of the said input oligonucleotide is in free molecular form, for the processing oligonucleotide B: the output oligonucleotide binds to it in the absence of other oligonucleotides so that at least 90% of the processing oligonucleotide B and at least 90% of said output oligonucleotide is in free molecular form, and the result of the calculation is read after adding input oligonucleotides to a solution containing output and processing nucleotides as the amount of output oligonucleotide in free molecular form.

In addition, a molecular computing device in which an input signal is converted into an output signal according to the logical OR rule, and which consists of (includes - comprises): an output oligonucleotide, input oligonucleotides (the presence of each of which corresponds to a logical input equal to 1, and the absence corresponds to a logical input equal to 0) of the processing oligonucleotide, moreover, the processing oligonucleotide binds to the output oligonucleotide in the absence of other oligonucleotides so that not less than 0.1% and not more than 50% of the processing oligonucleotide and not less than 0.1% and not more than 50% of the output oligonucleotide is in free molecular form, for each input oligonucleotide: the processing oligonucleotide binds to it in the absence of other oligonucleotides so that not less than 0.1% and not more than 50% of the processing oligonucleotide and not less than 0.1% and not more than 50% of the said input oligonucleotide is in free molecular form , d For each input oligonucleotide: the output oligonucleotide binds to it in the absence of other oligonucleotides so that at least 90% of the processing oligonucleotide and at least 90% of the said input oligonucleotide is in free molecular form, for each input oligonucleotide: each of the other input oligonucleotides binds to it in the absence of other oligonucleotides so that at least 90% of each of these two oligonucleotides is in free molecular form, while the result of the calculation is read after adding the input oligonucleotides to the solution containing the output and processing nucleotides, as the amount of the output oligonucleotide in free molecular form.

In addition, a molecular computing device in which an input signal is converted into an output signal according to the logical AND rule, and which consists of (includes - comprises): an output oligonucleotide, input oligonucleotides {Ix = I1,12 ...} (the presence of each of which corresponds to a logical input equal to 1, and the absence corresponds to a logical input equal to 0), processing oligonucleotides {Qx = Q1, Q2 ...}, each of which corresponds to one and only one input oligonucleotide, moreover, each processing oligonucleotide binds to the output oligonucleotide in the absence of other oligonucleotides so that not less than 0.1% and not more than 50% of the processing oligonucleotide and not less than 0.1% and not more than 50% of the output oligonucleotide is in free molecular form, for each input oligonucleotide: the corresponding processing oligonucleotide binds to it in the absence of other oligonucleotides, so, which is not less than 0.1% and not more than 50% of the processing o oligonucleotide and not less than 0.1% and not more than 50% of the said input oligonucleotide is in free molecular form, for each input oligonucleotide: the output oligonucleotide binds to it in the absence of other oligonucleotides so that not less than 90% of the processing oligonucleotide and not less than 90% of the mentioned input oligonucleotide is in free molecular form, for each input oligonucleotide: each of the other input oligonucleotides binds to it in the absence of other oligonucleotides so that at least 90% of each of these two oligonucleotides is in free molecular form, while the result of the calculation is read after adding input oligonucleotides to the solution containing the output and processing nucleotides as the amount of the output oligonucleotide in free molecular form.

In addition, a molecular computing device in which the input signal is converted into an output signal according to the function of Boolean algebra F, and which functions due to a combination of the above molecular computing devices that implement the corresponding functions YES, NOT, AND, OR, in which the output oligonucleotides of some molecular computing devices of said combination to input oligonucleotides of other molecular computing devices of said combination according to the expansion of function F in terms of the full system of functions YES, NOT, AND, OR.

In addition, a molecular computing device, in which all said oligonucleotides of said combination of molecular computing devices are mixed in one solution, and the result of the calculation is read after adding to said solution input oligonucleotides that correspond to the arguments of said function F, as the amount of output oligonucleotide that corresponds to the external functions for function F, in free molecular form.

In addition, a molecular computing device in which input signals are converted into an output signal according to a function with continuously changing arguments.

In addition, a molecular computing device in which an input signal X is converted into an output signal Y according to the function Y = F (x) with a continuously changing argument X, and which consists of (includes - comprises): an output oligonucleotide, an input oligonucleotide, and its total concentration corresponds to the value X of the processing oligonucleotides, and the processing oligonucleotides bind to the output oligonucleotide in the absence of other oligonucleotides so that not less than 0.1% and not more than 90% of the output oligonucleotide is in free molecular form, the processing oligonucleotides bind to the input oligonucleotide in the absence other oligonucleotides so that not less than 0.1% and not more than 90% of the input oligonucleotide is in free molecular form, while the result of the calculation of Y is associated with the amount of the output oligonucleotide in free molecular form.

In addition, a molecular computing device including at least a set of single-stranded nucleic acids, in which the numerical measure of the input signal is a measure associated with at least one single-stranded nucleic acid of the set, the numerical measure of the output signal is a measure associated with at least one single-stranded nucleic acid of said set, the input signal is transformed from into the output signal as a result of interactions between single-stranded nucleic acids of said set, moreover, the total concentration of each said single-stranded nucleic acid exceeds the value of the dissociation constant of the complexes formed by this single-stranded nucleic acid and other single-stranded nucleic acids of the above set no more than 100,000 times (preferably no more than 30,000, preferably no more than 10,000, preferably no more than 3,000, preferably no more than 1 000, preferably no more than 300, preferably no more than 100, preferably no more than 30 times.)

In addition, a molecular computing device including at least a set of single-stranded nucleic acids, in which the numerical measure of the input signal is a measure associated with at least one single-stranded nucleic acid of the set, the numerical measure of the output signal is a measure associated with by at least one single-stranded nucleic acid of said set, the input signal is transformed from into the output signal as a result of reversible non-complementary complexation interactions between single-stranded nucleic acids of said set, wherein (i.e., different in that) the total concentration of each said single-stranded nucleic acid exceeds the value of the dissociation constant of the complexes formed by this single-stranded nucleic acid and other single-stranded nucleic acids of the above set is not more than 100,000 times.

In addition, a molecular computing device that computes in no longer than 1 hour, preferably no longer than 30 minutes, preferably no longer than 10 minutes, preferably no longer than 5 minutes, more preferably no longer than 1 minute, more preferably no longer than 10 seconds.

In addition, a molecular computing device including at least a set of single-stranded nucleic acids, in which the numerical measure of the input signal is a measure associated with at least one single-stranded nucleic acid of the set, the numerical measure of the output signal is a measure associated with at least one single-stranded nucleic acid of said set, the input signal is transformed from into an output signal as a result of interactions between single-stranded nucleic acids of said set, characterized in that said interactions between single-stranded nucleic acids of said set are reversible binding of incompletely complementary ones between single-stranded nucleic acids of said set set, and such, for any two single-stranded nucleic acids of the above set, the length of the most extended connected region of their mutual complementarity does not exceed 10, preferably 9, preferably more readily 8, more preferably 7, more preferably 6, more preferably 5, more preferably 4 bases. A connected region of mutual complementarity of two oligonucleotides means a region of the nucleotide chain of one nucleic acid in which all bases in a row are complementary to the nucleotide chain of the second nucleic acid (i.e., there are no non-complementary bases throughout the entire region). If the areas are small, then a weak affinity of the given complexes is achieved, which allows information to be transferred due to the redistribution of nucleic acids (due to the displacement of some molecules by others) in the complexes without the need for the phenomenon of strand displacement, in which displacement occurs due to toe-hold (hanging free ends ) regions (as shown in Fig. 1).

In addition, methods of performing computations using said MVPs, including the following steps: selecting said set of single-stranded nucleic acids, mixing said set with said input molecules and said output molecules (if they are not part of said set), incubating the mixture to ensure transformation inputs to outputs as a result of interactions between input molecules, said single-stranded nucleic acids of said set and said output molecules, and detection of an output signal (as mentioned above related to output molecules).

In addition to the single-stranded nucleic acids included in said kits, the molecular computing devices of the present invention may include other molecules that interact with these elements of the kit and affect the functioning of the molecular computing device. So, in Example 23, the MVL is shown, which include the protein lysozyme, the interaction of which with a set of single-stranded nucleic acids leads to the implementation of the Boolean function of DA. Moreover, such additional (functional) molecules can both correspond to the rules of low-affinity interaction with the elements of the mentioned sets of nucleic acids, and be high-affinity to certain nucleic acids of the sets. Thus, example 23 shows a variant of a molecular computing device that uses fluorescently labeled lysozyme, the interaction of which with the single-stranded DNA aptamer is reduced due to the presence of fluorescent labels. In this case, the functioning of the MVL is similar to the functioning of the MVL, implemented in example 1. On the other hand, lysozyme can also be used as an input signal, which is high affinity for the aptamer mentioned, but at the same time allows the MVL to function due to the described redistribution of oligonucleotides of the set of MVL between complexes and free molecular forms.

In addition, in certain cases, a molecular computing device may include, in addition to said set of single-stranded nucleic acids having low affinity among themselves, various nucleic acids that have high affinity for oligonucleotides of said set. So, for example, completely complementary oligonucleotides to the oligonucleotides of the above set can be used as inputs of a molecular computing device. On the one hand, this can improve the dynamic range of the output signals, on the other hand, it makes it difficult to minimize the parasitic affinity in the set.

The above molecules can be used as input molecules and output. In fact, for each given MVC, the affinity “matrix” is important (ie which molecules have higher affinity than the parasitic one). At the same time, it is possible to "unfold" each MVD by changing the designation of "input" and "output" molecules, taking into account that it is necessary to change the mutual (relative) concentrations (as a rule, the concentration of input molecules should be higher than the output ones). In this case, for example, with such a "reversal" of the MVU from Example 23 - the lysozyme protein will become the input molecule, and the oligonucleotide, which performed the function of the output protein in Example 23, will become the output molecule. It is also possible to "unfold" (reverse, invert) the MVL in Example 20. In this case, the mRNA will become the input of the MVL, and the small RNA, which previously performed the function of the input, will become the output molecule.

In addition, Example 23 shows an example of the use of an aptamer as one of the nucleic acids of a set of VCMs in the VLM, so it is clear that in addition to lysozyme, input and output molecules can use any molecules with which aptamers can interact - other proteins, small molecules, ions, etc. etc.

In addition, it should be noted that a set of input molecules can contain both one molecule (function YES, NO) and many (functions AND, OR).

The same reasoning is true for a set of output molecules: it can include either one molecule (DA function) or a set (memory cell).

In addition, said numerical measure of the input signal can be related to the total concentration of the individual said input molecules, i.e. for example, in the case of the AND function, the numerical measure of each of the several inputs of the AND function (0 or 1) is associated (is, is equal to) the total concentration of one of the input molecules (for example, an oligonucleotide). However, in some cases, it makes sense to use the sum (or difference, or other numerical characteristic) of the total concentrations of several input molecules as the value of one input of a function with multiple inputs.

Examples of

The embodiments of the invention are varied. Here are various examples. The following examples are given to illustrate the present invention and do not limit its application.

Solving Boolean Algebra Problems:

Example 1) Molecular computing device (MVD), which implements the logical function of DA with DNA oligonucleotides 10 bases in length. The binary input of the system is the presence of oligonucleotide I (addition to the solution I = 1; non-addition = 0). A binary measure of the result of the calculation (yield) is the concentration of the oligonucleotide S in the form free from the complex with the oligonucleotide Q. The calculation result is read by measuring the fluorescence of the solution, taking into account the fact that S is labeled with a fluorescent Cy3 label, and Q is a querncher (quenching) BHQ2 molecule, i.e. upon binding of S to Q, the Cy3 fluorescence is quenched, which is detected by a fluorometer. IDL is a solution:

92 μl 100 mM Tris-HCl buffer; 1M NaCl;

+1 μl 100 μM oligonucleotide S = 5'-Cy3-GCAGTATTCG-3 'in water;

+ 2 μl 100 μM oligonucleotide Q = 5'-CGAATGCTGC-BHQ2-3 'in water.

The calculation is carried out by adding to the IDL:

Input I = 1) 5 μl 100 μM oligonucleotide I = 5'-GCAGCATTCG-3 'in water.

Input I = 0) 5 μl water.

The fluorescence signal of the Cy3 label is detected.

Results:

i) INPUT I = 1 - Signal 100 ± 4 RFU (relative fluorescence units),

i) INPUT I = 0 - Signal 17 ± 2 RFU (relative fluorescence units).

The affinity of oligonucleotides of a given MVU is presented as the concentration of a complex of two oligonucleotides (in μM) when they are mixed at a total concentration of 1 μM (calculated using NUPACK at 25 ° C). If necessary, these values can be easily converted to dissociation constant, as shown in the description above:

Parasitic affinity level: 0.05 (which corresponds to Kd ~ 10 ^-5 M if oligonucleotides do not form multidimensional units by themselves).

Example 2) Molecular Computing Device (MVD) implementing

logical function of DA with DNA oligonucleotides 7 bases long. The experimental setup is the same as in Example 1.

IDL is a solution:

69 μl of 100 μM Tris-HCl buffer; 1M NaCl;

+1 μl 100 μM oligonucleotide S = 5'-Cy3-GGCGGGG-3 'in water;

+10 μl 100 μM oligonucleotide Q = 5'-CCCCGGA-BHQ2-3 'in water.

The calculation is carried out by adding to the IDL:

Input I = 1) 20 μl 100 μM oligonucleotide I = 5'-TCCGGGG-3 'in water.

Input I = 0) 20 μl water.

The fluorescence signal of the Cy3 label is detected.

Results:

i) INPUT I = 1 - Signal 100 ± 7 RFU (relative fluorescence units),

i) INPUT I = 0 - Signal 68 ± 2 RFU (relative fluorescence units).

The affinity of oligonucleotides of a given MVU is presented as the concentration of a complex of two oligonucleotides (in μM) when they are mixed at a total concentration of 1 μM (calculated using NUPACK at 25 ° C). If necessary, these values can be easily converted to dissociation constant, as shown in the description above:

Parasitic affinity level: 0.2 (which corresponds to Kd = 6E-6 M if oligonucleotides do not form multidimensional units by themselves).

Example 3) Molecular computing device (MVD), implementing the logical function of DA with DNA oligonucleotides 15 bases in length.

The experimental setup is the same as in Example 1.

IDL is a solution:

92 μl 100 mM Tris-HCl buffer; 1M NaCl;

+1 μl 100 μM oligonucleotide S = 5'-Cy3-TTGAGTTGGCTATGA-3 'in water;

+ 2 μl 100 μM oligonucleotide Q = 5'-CCATATCCGACTCAC-BHQ2-3 'in water.

The calculation is carried out by adding to the IDL:

Input I = 1) 5 μl 100 μM oligonucleotide I = 5'-GGGAGTCGAATGTGG-3 'in water.

Input I = 0) 5 μl water.

The fluorescence signal of the Cy3 label is detected.

Results:

i) INPUT I = 1 - Signal 100 ± 3 RFU (relative fluorescence units),

i) INPUT I = 0 - Signal 22 ± 2 RFU (relative fluorescence units).

The affinity of oligonucleotides of a given MVU is presented as the concentration of a complex of two oligonucleotides (in μM) when they are mixed at a total concentration of 1 μM (calculated using NUPACK at 25 ° C). If necessary, these values can be easily converted to dissociation constant, as shown in the description above:

Parasitic affinity level: 0.05 (which corresponds to Kd ~ 10 ^-5 M if oligonucleotides do not form multidimensional units by themselves).

Example 4) Molecular computing device (MVD) that implements the logical function of DA with DNA oligonucleotides 15 bases in length in the range of nanomolar concentrations. The experimental setup is the same as in Example 1.

IDL is a solution:

92 μl 100 mM Tris-HCl buffer; 1M NaCl;

+1 μl 100 nM oligonucleotide S = 5'-Cy5-CCATATCCGACTCAC-3 'in water;

+ 2 μl 100 nM oligonucleotide Q = 5'-CTCAGTCGGATATGT-BHQ3-3 'in water.

The calculation is carried out by adding to the IDL:

Input I = 1) 5 μl of 100 nM oligonucleotide I = 5'-TCACATCCGACTGAG-3 'in water.

Input I = 0) 5 μl water.

The fluorescence signal of the Cy5 label is detected.

Results:

i) INPUT I = 1 - Signal 100 ± 15 RFU (relative fluorescence units),

i) INPUT I = 0 - Signal 42 ± 20 RFU (relative fluorescence units).

The affinity of oligonucleotides of a given MVU is presented as the concentration of the complex

two oligonucleotides (in μM) when mixed at a total concentration of 1 μM (calculated using NUPACK at 25 ° C). If necessary, these values can be easily converted to dissociation constant, as shown in the description above:

Parasitic affinity level: 0.05 (which corresponds to Kd ~ 10 ^-5 M if oligonucleotides do not form multidimensional units by themselves).

Example 5) Molecular computing device (MVD), implementing the logical function of DA with DNA oligonucleotides 20 bases in length. The experimental setup is the same as in Example 1.

IDL is a solution:

92 μl 100 μM Tris-HCl buffer; 1M NaCl;

+1 μl 100 μM oligonucleotide S = 5'-Cy3-AACTGTGACCACTTGTCAAG-3 'in water;

+2 μl 100 μM oligonucleotide Q = 5'-CTTGACACGTGGGCAAATGA-BHQ2-3 'in water.

The calculation is carried out by adding to the IDL:

Input I = 1) 5 μl 100 μM oligonucleotide I = 5'-CTTGACAAGTGGTCACAGTT-3 'in water.

Input I = 0) 5 μl water.

The fluorescence signal of the Cy3 label is detected.

Results:

i) INPUT I = 1 - Signal 100 ± 5 RFU (relative fluorescence units),

i) INPUT I = 0 - Signal 28 ± 4 RFU (relative fluorescence units).

The affinity of oligonucleotides of a given MVU is presented as the concentration of the complex

two oligonucleotides (in μM) when mixed at a total concentration of 1 μM (calculated using NUPACK at 25 ° C). If necessary, these values can be easily converted to dissociation constant, as shown in the description above:

Parasitic affinity level: 0.05 (which corresponds to Kd ~ 10 ^-5 M if oligonucleotides do not form multidimensional units by themselves).

Example 6) Molecular computing device (MVD), which implements the logical function of DA with RNA oligonucleotides. The experimental setup is the same as in Example 1.

IDL is a solution:

92 μl 100 mM Tris-HCl buffer; 1M NaCl;

+1 μl 100 μM oligonucleotide S = 5'-Cy3-AGGCGCCCUGAC-3 'in water;

+ 2 μl 100 μM oligonucleotide Q = 5'-UUCAGGGGCUCU-BHQ2-3 'in water.

The calculation is carried out by adding to the IDL:

Input I = 1) 5 μl 100 μM oligonucleotide I = 5'-UGAGCCUGCUGA-3 'in water.

Input I = 0) 5 μl water.

The fluorescence signal of the Cy3 label is detected.

Results:

i) INPUT I = 1 - Signal 100 ± 2 RFU (relative fluorescence units),

i) INPUT I = 0 - Signal 33 ± 3 RFU (relative fluorescence units).

The affinity of oligonucleotides of a given MVU is presented as the concentration of a complex of two oligonucleotides (in μM) when they are mixed at a total concentration of 1 μM (calculated using NUPACK at 25 ° C). If necessary, these values can be easily converted to dissociation constant, as shown in the description above:

Parasitic affinity level: 0.05 (which corresponds to Kd ~ 10 ^-5 M if oligonucleotides do not form multidimensional units by themselves).

Example 7) Molecular computing device (MVD) implementing the logical function NO with DNA oligonucleotides 15 bases in length.

The experimental setup is the same as in Example 1.

IDL is a solution:

83 μl 100 mM Tris-HCl buffer; 1M NaCl;

+1 μl 100 μM oligonucleotide S = 5'-Cy3-TTGAGTTGGCTATGA-3 'in water;

+ 2 μl 100 μM oligonucleotide Q = 5'-CCATATCCGACTCAC-BHQ2-3 'in water.

+ 4 μl 100 μM oligonucleotide M = 5'-GGGAGTCGAATGTGG-3 'in water.

The calculation is carried out by adding to the IDL:

Input I = 1) 10 μl 100 μM oligonucleotide I = 5'-CCAGATTAAACTCCC-3 'in water.

Input I = 0) 10 μl water.

The fluorescence signal of the Cy3 label is detected.

Results:

i) INPUT I = 1 - Signal 28 ± 4 RFU (relative fluorescence units),

i) INPUT I = 0 - Signal 100 ± 4 RFU (relative fluorescence units).

The affinity of oligonucleotides of a given MVU is presented as the concentration of the complex

two oligonucleotides (in μM) when mixed at a total concentration of 1 μM (calculated using NUPACK at 25 ° C). If necessary, these values can be easily converted into dissociation constant as shown in the description above:

Parasitic affinity level: 0.05 (which corresponds to Kd ~ 10 ^-5 M if oligonucleotides do not form multidimensional units by themselves).

Example 8) Molecular computing device (MVD) implementing a 3-bit logical OR function. The binary input of the system is the presence of oligonucleotides I1, I2, I3 (addition to the solution = 1; non-addition = 0). A binary measure of the calculation result (yield) is the concentration of oligonucleotide S in a complex-free form with quencher (quenching) oligonucleotide Q. The calculation result is read by measuring the fluorescence of the solution, taking into account that S is labeled with a fluorescent Cy3 tag, and oligonucleotide Q is labeled with a querncher BHQ2 molecule, i.e. upon binding of S to Q, the Cy3 fluorescence is quenched, which is detected by a fluorometer.

IDL is a solution:

83 μl 100 mM Tris-HCl buffer; 1M NaCl;

+1 μl 100 μM oligonucleotide S = 5'-Cy3-GCAGTATTCG-3 'in water;

+1 μl 100 μM oligonucleotide Q = 5'-CGAAAACTGC-BHQ2-3 'in water.

The calculation is carried out by adding 15 μL of various combinations of inputs to the MVC:

Input I1 = 1) 5 μL of 100 μM oligonucleotide I1 = 5'-TCAGTTTTCA-3 'in water.

Input I2 = 1) 5 μl 100 μM oligonucleotide I2 = 5'-GTAGTTTTCG-3 'in water.

Input I3 = 1) 5 μL of 100 μM oligonucleotide I3 = 5'-ACAGTTTTCC-3 'in water.

Input In = 0) 5 μl water - for n = 1, 2, 3.

The fluorescence signal of the Cy3 label is detected.

Results:

i) INPUT: I1 = 0; I2 = 0; I3 = 0 - Signal: 10 ± 1 RFU (relative fluorescence units).

i) INPUT: I1 = 0; I2 = 0; I3 = 1 - Signal: 40 ± 2 RFU (relative fluorescence units).

i) INPUT: I1 = 0; I2 = 1; I3 = 0 - Signal: 41 ± 1 RFU (relative fluorescence units).

i) INPUT: 1I = 0; 12 = 1; 13 = 1 - Signal: 46 ± 1 RFU (relative fluorescence units).

i) INPUT: I1 = 1; I2 = 0; I3 = 0 - Signal: 39 ± 1 RFU (relative fluorescence units).

i) INPUT: I1 = 1; I2 = 0; I3 = 1 - Signal: 46 ± 1 RFU (relative fluorescence units).

i) INPUT: I1 = 1; I2 = 1; I3 = 0 - Signal: 47 ± 1 RFU (relative fluorescence units).

i) INPUT: I1 = 1; I2 = 1; I3 = 0 - Signal: 50 ± 1 RFU (relative fluorescence units).

The threshold value is the average between the maximum and minimum, i.e.

(10 + 50) / 2 = 30. Therefore, the OR function is implemented.

The affinity of oligonucleotides of a given MVU is presented as the concentration of a complex of two oligonucleotides (in μM) when they are mixed at a total concentration of 1 μM (calculated using NUPACK at 25 ° C). If necessary, these values can be easily converted into dissociation constant as shown in the description above:

Parasitic affinity level: 0.05 (which corresponds to Kd ~ 10 ^-5 M if oligonucleotides do not form multidimensional units by themselves).

Example 9) Molecular computing device (MVD) that implements a 3-bit logical function I. The binary input of the system is the presence of oligonucleotides I1, I2, I3 (addition to solution = 1; non-addition = 0). A binary measure of the calculation result (yield) is the concentration of oligonucleotide S in the form free from the complex with quencher oligonucleotides Qn (n = 1, 2, 3). The calculation result is read by measuring the fluorescence of the solution, taking into account that S is labeled with a fluorescent Cy3 label, and Qn oligonucleotides - with a querncher BHQ2 molecule, i.e. upon binding of S to Qn, the Cy3 fluorescence is quenched, which is detected by a fluorometer.

IDL is a solution:

78 μl of 100 mM Tris-HCl buffer; 1M NaCl;

+1 μl 100 μM oligonucleotide S = 5'-Cy3-GCAGTATTCG-3 'in water;

+ 2 μl 100 μM oligonucleotide Q1 = 5'-CGAAAACTGC-BHQ2-3 'in water.

+ 2 μl 100 μM oligonucleotide Q2 = 5'-CGAATGCTGC-BHQ2-3 'in water.

+ 2 μl 100 μM oligonucleotide Q3 = 5'-CGAATAGTGC-BHQ2-3 'in water.

The calculation is carried out by adding 15 μL of various combinations of inputs to the MVC:

Input I1 = 1) 5 μL of 100 μM oligonucleotide I1 = 5'-GCAGTTTTCG-3 'in water.

Input I2 = 1) 5 μL of 100 μM oligonucleotide I2 = 5'-GCAGCATTCG-3 'in water.

Input I3 = 1) 5 μl 100 μM oligonucleotide I3 = 5'-GCACTATTCG-3 'in water.

Input In = 0) 5 μl water - for n = 1, 2, 3.

The fluorescence signal of the Cy3 label is detected.

Results:

i) INPUT: I1 = 0; I2 = 0; I3 = 0 - Signal: 10 ± 0.2 RFU (relative fluorescence units).

i) INPUT: I1 = 0; I2 = 0; I3 = 1 - Signal: 14 ± 0.6 RFU (relative fluorescence units).

i) INPUT: I1 = 0; I2 = 1; I3 = 0 - Signal: 13 ± 1 RFU (relative fluorescence units).

i) INPUT: I1 = 0; I2 = 1; I3 = 1 - Signal: 21 ± 0.3 RFU (relative fluorescence units).

i) INPUT: I1 = 1; I2 = 0; I3 = 0 - Signal: 18 ± 0.6 RFU (relative fluorescence units).

i) INPUT: I1 = 1; I2 = 0; I3 = 1 - Signal: 33 ± 1 RFU (relative fluorescence units).

i) INPUT: I1 = 1; I2 = 1; I3 = 0 - Signal: 28 ± 1 RFU (relative fluorescence units).

i) INPUT: I1 = 1; I2 = 1; I3 = 0 - Signal: 67 ± 0.5 RFU (relative fluorescence units).

The threshold value is the average between the maximum and minimum, i.e.

(10 + 67) /2=38.5. Therefore, the AND function is implemented.

The affinity of oligonucleotides of a given MVU is presented as the concentration of a complex of two oligonucleotides (in μM) when they are mixed at a total concentration of 1 μM (calculated using NUPACK at 25 ° C). If necessary, these values can be easily converted into dissociation constant as shown in the description above:

Parasitic affinity level: 0.3 (which corresponds to Kd ~ 2 * 10 ^-6 M, if oligonucleotides do not form multidimensional ones by themselves).

Example 10) Molecular computing device (MVD) implementing a 3-bit memory cell with a storage and reading function. The IDL diagram is shown in FIG. 9. The number to be stored using the IDC is represented as a 3-bit binary number. Each digit of the number is associated with an oligonucleotide In (n = 1, 2, 3, addition to solution = 1; non-addition = 0). Then, this number is fed to the input of the IDL by adding it to the solution. Then, to read the result of the calculation, a “reading” oligonucleotide is fed to the input of the IDL, which causes an increase in the fluorescence of 3 labels of different spectrum according to the number recorded in the memory of the IDL (if I1 = 1, the fluorescence of the Cy3 label increases / appears, if I12 = 1, it increases / the fluorescence of the Cy5 label appears, if I3 = 1 - the fluorescence of the FAM label increases / appears). This MVU functions as 3 parallel logical AND functions with one common input.

MVU is a solution: 61 μl of 100 mM Tris-HCl buffer; 1M NaCl;

+1 μl 100 μM oligonucleotide S1 = 5'-Cy3-AACTGTGACCACTTGTCAAG-3 'injected;

+1 μl 100 μM oligonucleotide S2 = 5'-Cy5-CGCTGAGCGCACCGCTCGAC-3 'in water;

+1 μl 100 μM oligonucleotide S3 = 5'-FAM-CACTGTGTCAACATCTCACC-3 'in water;

+4 μl 100 μM oligonucleotide Q1 = 5'-GGTGAGGAGTGGGCTCAGTG-BHQ2-3 'in water.

+1 μl 100 μM oligonucleotide Q2 = 5'-CTTGACCCTTAGTCACAGTT-BHQ2-3 'in water.

+4 μl 100 μM oligonucleotide Q3 = 5'-GATTCTGTTTACGCTAACTA-BHQ2-3 'in water.

+1 μl 100 μM oligonucleotide QR = 5'-CGTCAGATGTTAACAAAGTG-BHQ2-3 'in water.

The number is written into the memory cell of the MVC by adding 15 μl of various combinations of inputs to the MVC:

Input I1) = 1: +20 μl 100 μM oligonucleotide I1 = 5'-CTTTGTGACTAAGTGTCAAG-3 'in water; = 0: +20 μl water.

Input I2) = 1: +11 μl 100 μM oligonucleotide I2 = 5'-TAGTTAGCGTTAGCAAAATT-3 'in water; = 0: +11 μl water.

Input I2) = 1: +14 μl 100 μM oligonucleotide I3 = 5'-CACTTTGTAACCATGTGACG-3 'in water; = 0: +14 μl water.

Reading is carried out by adding 20 μl of the "reading" oligonucleotide to the MVU

R = 5'-CTGTCCCGTCACTACTCACC-3 'in water.

The fluorescence signal of the FAM, Cy3, Cy5 labels is detected.

The results obtained, confirming the correct operation of this MVD, are shown in Fig. nine.

The affinity of oligonucleotides of a given MVU is presented as the concentration of the complex

two oligonucleotides (in μM) when mixed at a total concentration of 1 μM (calculated using NUPACK at 25 ° C). If necessary, these values can be easily converted to dissociation constant, as shown in the description above:

Parasitic affinity level: 0.1 (which corresponds to Kd ~ 8 * 10 ^-6 M if oligonucleotides do not form multidimensional ones by themselves).

Example 11) A molecular computing device (MVD) that implements the calculation of the square root of a 4-bit binary number. Each digit of the number is associated with an oligonucleotide In (n = 1, 2, 3, 4, addition to the solution = 1; non-addition = 0), i.e. the number is written as {I1-I2-I3-I4}. Then, this number is fed to the input of the IDC by adding to the solutions to calculate each of the two digits of the response. The answer, i.e., the base of the square root (integer) is calculated as a 2-bit binary number of the form XY, in which the X, Y digits are calculated according to Boolean algebra:

X = I1 OR I2; Y = HE {[I2 OR NOT (I14)] AND [I2 OR NOT (I3)] AND [NOT (I2) OR NOT (I1)}

A binary measure of the result of the calculation of X is the concentration of the S1 oligonucleotide in a complex-free form with the Q1 quencher oligonucleotide. The calculation result is read by measuring the fluorescence of the solution, taking into account the fact that S is labeled with a fluorescent Cy3 label, and the oligonucleotide with a Q-querncher molecule BHQ2, i.e. upon binding of S to Q, the Cy3 fluorescence is quenched, which is detected by a fluorometer.

A binary measure of the result of the calculation of Y is the concentration of the oligonucleotide S2 in the form free from the complex with the quencher oligonucleotide Q2. The calculation result is read by measuring the fluorescence of the solution, taking into account that S is labeled with a fluorescent Cy5 label, and the oligonucleotide with a Q-querncher molecule BHQ3 (i.e., when S binds to Q, the fluorescence of Cy5 is quenched, which is detected by a fluorometer.

The IDL is two solutions (for calculating X and Y).

Solution for calculating X:

77 μl of 100 mM Tris-HCl buffer; 1M NaCl;

+1 μl 100 μM oligonucleotide SX = 5'-Cy5-TTGTGATTGGAAACT-3 'in water;

+ 2 μl 100 μM oligonucleotide QX = 5'-AGGTTCCATGCACAA-BHQ3-3 'in water.

Solution for calculating Y:

63 μl 100 mM Tris-HCl buffer; 1M NaCl;

If input = 0, then 5 μl of water is added instead of the oligonucleotide.

The calculated results obtained 5 minutes after the addition of the inputs are shown in FIG. 10 and demonstrate the correct operation of this IDP. Note that after 60 minutes, the output values practically do not change, so the solution calculated within 5 minutes (or faster) may be final.

The affinity of oligonucleotides of a given MVU is presented as the concentration of a complex of two oligonucleotides (in μM) when they are mixed at a total concentration of 1 μM (calculated using NUPACK at 25 ° C). If necessary, these values can be easily converted to dissociation constant, as shown in the description above:

Parasitic affinity level: 0.05 (which corresponds to Kd ~ 10 ^-5 M if oligonucleotides do not form multidimensional units by themselves).

Another variant of the sequences of oligonucleotides for MVU # 2 for calculating the square root:

The affinity of oligonucleotides of a given MVU is presented as the concentration of a complex of two oligonucleotides (in μM) when they are mixed at a total concentration of 1 μM (calculated using NUPACK at 25 ° C). If necessary, these values can be easily converted to dissociation constant, as shown in the description above:

Parasitic affinity level: 0.1 (which corresponds to Kd ~ 8 * 10 ^-6 M if oligonucleotides do not form multidimensional ones by themselves).

Example 12) Molecular computing device (MVD) implementing a 500-bit logical AND function in a NUPACK simulation. The binary input of the system is the presence of In oligonucleotides (n = 1-500, addition to the system = 1; non-addition = 0). A binary measure of the calculation result (yield) is the concentration of oligonucleotide S in free form. The MVL is a collection of oligonucleotides in Table 1. The calculation of the 500-bit logical function AND is carried out by adding to the MVL various combinations of the inputs indicated in Table 1.

At the same time, the following criteria were used to select oligonucleotides:

[S] = 1 μM

50 μM <[Q] <100 μM, 50 μM <[I] <120 μM

and the following affinities:

0.4 μM <Kd [QS] <1 μM, 0.4 nM <Kd [IQ] <3 nM.

In fact, in the set presented in Table 1,

50.5 μM <= [Q] <= 89.8 μM, 52.7 μM <= [I] <= 112 μM

0.4 μM <Kd [QS] <0.898 μM, 0.415 nM <= Kd [IQ] <= 2.63 nM.

Due to the enormous amount of data, Table 1 presents only the concentrations of oligonucleotides, all other parameters can be calculated using the publicly available NUPACK algorithm (unpack.org).

The affinities between non-interacting oligonucleotides (parasitic affinity) should have been Kd> 18 μM.

The NUPACK algorithm calculates the amount of free S.

The value of work without adding inputs (all inputs = 0) = 4.10E-05 (share of S in free

molecular form).

FIG. 11 shows the results of the operation of the IDL when calculating the following input combinations:

A) all inputs = 1

B) all inputs except In = 1; In = 0. (for all n = 1..500).

FIG. 11 shows that the output for group C = 0 for all n. Therefore, for all other combinations of inputs (where more than one input = 0) there will be even less, i.e. also = 0. Therefore, the IDL does indeed implement logical AND.

Elementary Algebra

Example 13) Molecular computing device (MVD) calculating the value of the function y = Ax for a continuously varying value of x in the NUPACK simulation.

The value of x is: x = log ([I] / [X0), where [I] is the concentration of the input oligonucleotide I in units of mol / L, [X0] = 10 ^∧ -6 mol / L is an arbitrarily chosen reference shift concentration along X ...

The value of y is: y = ([S] - [Y0]) / [C0], where [S] is the concentration of free output oligonucleotide S in units of mol / l, [Y0] = 5 * 10 ^∧ -11 mol / l and [С0] = 10 ^∧ -11 mol / l - arbitrarily chosen reference concentration of the shift along Y and the normalization factor, respectively.

IDL is a collection of oligonucleotides: in Table 2.

Calculation of the value of the function y = x (for -2 <x <2) is calculated by adding to the system the corresponding amount of input oligonucleotide I in the range: 10 ^-8 M <[I] <10 ^-4 M, where [I] is the concentration of the input oligonucleotide I.

Figure 12 shows the theoretical value of the function (solid line) and the value of the calculated MVL, and at all points calculated and shown on the graph, the error in calculating the value of the MVL function does not exceed 0.96% (in terms of concentration S).

Example 14) Molecular computing device (MVD) calculating the value of a function y = Ax ^∧ 2 for a continuously varying value of x in the NUPACK simulation.

The value of x is: x = log ([I] / [X0), where [I] is the concentration of the input oligonucleotide I in units of mol / L, [X0] = 10 ^∧ -6 mol / L is an arbitrarily chosen reference shift concentration along X ...

The value of y is: y = ([S] - [Y0]) / [C0], where [S] is the concentration of free output oligonucleotide S in units of mol / l, [Y0] = 5 * 10 ^∧ -11 mol / l and [С0] = 10 ^∧ -11 mol / l - arbitrarily chosen reference concentration of the shift along Y and the normalization factor, respectively.

The MVL is a set of oligonucleotides: in Table 2. Calculation of the value of the function y = 0.25 * x ^∧ 2 (for -2 <x <2) is calculated by adding to the system the corresponding amount of input oligonucleotide I within: 10 ^-8 M <[I ] <10 ^-4 M, where [I] is the concentration of the input oligonucleotide I.

Figure 12 shows the theoretical value of the function (solid line) and the value of the calculated MVC, and at all points calculated and shown on the graph, the error in calculating the value of the MVC function does not exceed 0.82% (in terms of concentration S).

Example 15) Molecular computing device (MVD) calculating the value of a function y = Ax ^∧ 3 for a continuously varying value of x in the NUPACK simulation.

The value of x is: x = log ([I] / [X0), where [I] is the concentration of the input oligonucleotide I in units of mol / L, [X0] = 10 ^∧ -6 mol / L is an arbitrarily chosen reference shift concentration along X ...

The value of y is: y = ([S] - [Y0]) / [C0], where [S] is the concentration of free output oligonucleotide S in units of mol / l, [Y0] = 5 * 10 ^∧ -11 mol / l and [С0] = 10 ^∧ -11 mol / l - arbitrarily chosen reference concentration of the shift along Y and the normalization factor, respectively.

The MVL is a set of oligonucleotides: in Table 2. Calculation of the value of the function y = 0.125 * x ^∧ 3 (for -2 <x <2) is calculated by adding to the system the corresponding amount of input oligonucleotide I within the range: 10 ^-8 M <[I ] <10 ^-4 M, where [I] is the concentration of the input oligonucleotide I.

Figure 12 shows the theoretical value of the function (solid line) and the value of the calculated MVL, and at all points calculated and shown on the graph, the error in calculating the value of the MVL function does not exceed 0.88% (in terms of concentration S).

Example 16) Molecular computing device (MVD) calculating the value of the function y = sqrt (Ax) for a continuously varying value of x in the NUPACK simulation.

The value of x is: x = log ([I] / [X0), where [I] is the concentration of the input oligonucleotide I in units of mol / L, [X0] = 10 ^∧ -6 mol / L is an arbitrarily chosen reference shift concentration along X ...

The value of y is: y = ([S] - [Y0]) / [C0], where [S] is the concentration of free output oligonucleotide S in units of mol / l, [Y0] = 5 * 10 ^∧ -11 mol / l and [С0] = 10 ^∧ -11 mol / l - arbitrarily chosen reference concentration of the shift along Y and the normalization factor, respectively.

The MVL is a set of oligonucleotides: in Table 2. Calculation of the value of the function y = sqrt (x / 2) is calculated by adding to the system the corresponding amount of input oligonucleotide I in the range: 10 ^-8 M <[I] <10 ^-4 M, where [I] is the concentration of the input oligonucleotide I.

Figure 12 shows the theoretical value of the function (solid line) and the value of the calculated MVL, and at all points calculated and shown on the graph, the error in calculating the value of the MVL function does not exceed 0.88% (in terms of concentration S).

Example 17) Molecular computing device (MVD) calculating the value of the function y = sin (Ax) for a continuously varying value of x in the NUPACK simulation.

The value of x is: x = log ([I] / [X0), where [I] is the concentration of the input oligonucleotide I in units of mol / L, [X0] = 10 ^∧ -6 mol / L is an arbitrarily chosen reference shift concentration along X ...

The value of y is: y = ([S] - [Y0]) / [C0], where [S] is the concentration of free output oligonucleotide S in units of mol / l, [Y0] = 5 * 10 ^∧ -11 mol / l and [С0] = 10 ^∧ -11 mol / l - arbitrarily chosen reference concentration of the shift along Y and the normalization factor, respectively.

The MVL is a set of oligonucleotides: in Table 2. Calculation of the value of the function y = sin (πx / 2) is calculated by adding to the system the corresponding amount of input oligonucleotide I in the range: 10 ^-8 M <[I] <10 ^-4 M, where [I] is the concentration of the input oligonucleotide I.

Figure 12 shows the theoretical value of the function (solid line) and the value of the calculated MVL, and at all points calculated and shown on the graph, the error in calculating the value of the MVL function does not exceed 0.93% (in terms of concentration S).

Example 18) Molecular computing device (MVD) solving an equation of the form Bx ^∧ 2 = sin (Ax) for a continuously varying value of x in a NUPACK simulation. Create MVL No. 1 according to Example H and MVL No. 2 according to Example H. The amount of input oligonucleotide I is varied in both mentioned MVCs within the concentration range: 10 ^-8 M <[I] <10 ^-4 M, the yields of both MVCs are recorded and compared. The amount of input oligonucleotide I, at which both IDLs produced the same (within 1% difference) yield, is matched to the solution x of the specified equation. FIG. 13 shows the solution for the equation -0.25 * x ^∧ 2 = sin (πx / 2): x1 = -1.6 (1 = 2.5 * 10 ^∧ -8M) (the error from the correct answer x = -1.57 is 1.8%), x1 = 0.05 (I = 1.13 * 10 ^∧ -6M), the correct answer is x = 0.

MVP, which regulates gene expression

Example 19) A molecular computing device (MVD) that implements the DA logic function to control gene expression.

The binary input of the system is the presence of RNA oligonucleotide I (addition to the solution I = 1; non-addition = 0). A binary measure of the calculation result (yield) is the amount of synthesized protein in the extracellular expression solution. This example demonstrates the performance of the IDM to control GFP expression, so the output signal is recorded by GFP fluorescence. Targeted mRNA sequence S = AGGCGCCCUGAC.

IDL is a solution:

20 μl of a mixture of the components of the 1-Step Human Coupled IVT Kit - DNA (Thermo Scientific ™, USA, catalog number 88881) with a plasmid containing the GFP gene (supplied in the kit) according to the manufacturer's recommendations (but with a 2-fold dilution with water) ;

+1 μl of 50 μM oligonucleotide Q = 5'-UUCAGGGGCUCU-3 'in water;

The calculation is carried out by adding to the IDL:

Input I = 1) 1 μl of 500 μM oligonucleotide I = 5'-UGAGCCUGCUGA-3 'in water.

Input I = 0) 1 μl water.

Incubate for 6 hours at +30 degrees C and detect the fluorescence of the synthesized GFP.

Results:

i) INPUT I = 1 - Signal 100 ± 20 RFU (relative fluorescence units),

i) INPUT I = 0 - Signal 22 ± 10 RFU (relative fluorescence units).

Thus, they achieve influence on the expression of the gene (namely, increase its expression) due to the presence in the mixture of an oligonucleotide having complementarity in the target sequence in only two not adjacent bases.

Expression was also increased when using the following combinations of Q, I:

The same target mRNA sequence S = AGGCGCCCUGAC;

Q = GACAGGACGCCU

I = CGGCGUCCAGUU: maximum length of sequential complementary bases to the plasmid - 5 nucleotides (CAGUU), to mRNA - 5 nucleotides (UCCAG).

Expression was also increased when using the following combinations of Q, I:

Target mRNA sequence S = TACGGCTTCTACCAC;

Q = GUGGUCGAAUCCGUA

I = UAGAGAUUCGACAGC: maximum length of sequential complementary bases to the plasmid - 5 nucleotides (ACAGC), to mRNA - 4 nucleotides (CAGC).

Expression was also increased when using the following combinations of Q, I:

Target mRNA sequence S = TACGGCTTCTACCAC;

Q = CGGUCUCUAUAUGCG

I = CGUAUAGAGAGACUU: maximum length of sequential complementary bases to the plasmid - 5 nucleotides (GACUU), to mRNA - 4 nucleotides (ACUU).

Example 20) Molecular computing device (MVD) implementing the logical function NO to control gene expression.

The binary input of the system is the presence of RNA oligonucleotide I (addition to the solution I = 1; non-addition = 0). A binary measure of the calculation result (yield) is the amount of synthesized protein in the extracellular expression solution. This example demonstrates the performance of the IDM to control GFP expression, so the output signal is recorded by GFP fluorescence.

IDL is a solution:

20 μl of a mixture of the components of the 1-Step Human Coupled IVT Kit - DNA (Thermo Scientific ™, USA, catalog number 88881) with a plasmid containing the GFP gene (supplied in the kit) according to the manufacturer's recommendations (but with a 2-fold dilution with water) ;

+1 μl of 50 μM oligonucleotide Q = 5'-UUCAGGGGCUCU-3 'in water;

+ 5 μl of 50 μM oligonucleotide M = 5'-AGAGACCCUUAA-3 'in water;

The calculation is carried out by adding to the IDL:

Input I = 1) 2 μl of 500 μM oligonucleotide I = 5'-UUAACGGUCUCU-3 'in water.

Input I = 0) 1 μl water.

Incubate for 6 hours at +30 degrees C and detect the fluorescence of the synthesized GFP.

Results:

i) INPUT I = 1 - Signal 60 ± 3 RFU (relative fluorescence units),

i) INPUT I = 0 - Signal 100 ± 2 RFU (relative fluorescence units).

Thus, they achieve influence on the expression of the gene (namely, to reduce its expression) due to the presence in the mixture of an oligonucleotide having complementarity in the target sequence in only two not adjacent bases.

The affinity of oligonucleotides of a given MVU is presented as the concentration of the complex

two oligonucleotides (in μM) when mixed at a total concentration of 1 μM (calculated using NUPACK at 25 ° C). If necessary, these values can be easily converted into a dissociation constant as shown in the description above (as S for calculations, the target mRNA sequence of the -AGGCGCCCTGAC gene is used):

Parasitic affinity level: 0.05 (which corresponds to Kd ~ 10 ^-5 M if oligonucleotides do not form multidimensional units by themselves).

Example 21) A molecular computing device (MVD) that implements a logical AND function to control gene expression.

The binary input of the system is the presence of the RNA oligonucleotide In (n = 1, 2; addition to the solution In = 1; non-addition = 0). A binary measure of the calculation result (yield) is the amount of synthesized protein in the extracellular expression solution. This example demonstrates the performance of the IDM to control GFP expression, so the output signal is recorded by GFP fluorescence. IDL is a solution:

20 μl of a mixture of the components of the 1-Step Human Coupled IVT Kit - DNA (Thermo Scientific ™, USA, catalog number 88881) with a plasmid containing the GFP gene (supplied in the kit) according to the manufacturer's recommendations (but with a 2-fold dilution with water) ;

+1 μl 25 μM oligonucleotide Q1 = 5'-UUCAGGGGCUCU-3 'in water;

+1 μl 25 μM oligonucleotide Q2 = 5'-UCCAGGUCGCCU-3 'in water;

The calculation is carried out by adding to the IDL:

Input I1 = 1 I2 = 1) 1 μL of 500 μM oligonucleotide I1 = 5'-UGAGCCUGCUGA-3 'in water +1 μL of 500 μM oligonucleotide I2 = 5'-AUGCGACCUCGG-3' in water. Input I1 = 0 I2 = 1) 1 μl water +1 μl 500 μM oligonucleotide I1 in water. Input I1 = 1 I2 = 0) 1 μl 500 μM oligonucleotide I2 in water +1 μl water. Input I1 = 0 I2 = 0) 2 μl water.

Incubate for 6 hours at +30 degrees C and detect the fluorescence of the synthesized GFP. Results:

i) INPUT I1 = 0 I2 = 0 - Signal 42 ± 2 RFU (relative fluorescence units),

i) INPUT I1 = 0 I2 = 1 - Signal 65 ± 4 RFU (relative fluorescence units).

i) INPUT I1 = 1 I2 = 0 - Signal 59 ± 3 RFU (relative fluorescence units),

i) INPUT I1 = 1 I2 = 1 - Signal 100 ± 3 RFU (relative fluorescence units).

Boundary (true / false) = (100 + 42) = 71, so the IDL actually drives gene expression according to logical AND.

The affinity of oligonucleotides of a given MVU is presented as the concentration of a complex of two oligonucleotides (in μM) when they are mixed at a total concentration of 1 μM (calculated using NUPACK at 25 ° C). If necessary, these values can be easily converted into a dissociation constant, as shown in the description above (as S for calculations, the target mRNA sequence of the gene is used - AGGCGCCCGGAC):

Parasitic affinity level: 0.05 (which corresponds to Kd ~ 10 ^-5 M if oligonucleotides do not form multidimensional units by themselves).

Example 22) Molecular computing device (MVD) that implements a logical OR function to control gene expression.

The binary input of the system is the presence of the RNA oligonucleotide In (n = 1, 2; addition to the solution In = 1; non-addition = 0). A binary measure of the calculation result (yield) is the amount of synthesized protein in the extracellular expression solution. This example demonstrates the performance of the IDM to control GFP expression, so the output signal is recorded by GFP fluorescence.

IDL is a solution:

20 μl of a mixture of the components of the 1-Step Human Coupled IVT Kit - DNA (Thermo Scientific ™, USA, catalog number 88881) with a plasmid containing the GFP gene (supplied in the kit) according to the manufacturer's recommendations (but with a 2-fold dilution with water) ;

+1 μl of 50 μM oligonucleotide Q1 = 5'-UUCAGGGGCUCU-3 'in water;

The calculation is carried out by adding to the IDL:

Input I1 = 1 I2 = 1) 1 μL of 500 μM oligonucleotide I1 = 5'-UGAGCCUGCUGA-3 'in water +1 μL of 500 μM oligonucleotide I2 = 5'-AGAGCCAGUGAC-3' in water. Input I1 = 0 I2 = 1) 1 μl water +1 μl 500 μM oligonucleotide I1 in water. Input I1 = 1 I2 = 0) 1 μl 500 μM oligonucleotide I2 in water +1 μl water. Input I1 = 0 I2 = 0) 2 μl water.

Incubate for 6 hours at +30 degrees C and detect the fluorescence of the synthesized GFP.

Results:

i) INPUT I1 = 0 I2 = 0 - Signal 24 ± 4 RFU (relative fluorescence units),

i) INPUT I1 = 0 I2 = 1 - Signal 71 ± 4 RFU (relative fluorescence units),

i) INPUT I1 = 1 I2 = 0 - Signal 75 ± 3 RFU (relative fluorescence units).

i) INPUT I1 = 1 I2 = 1 - Signal 100 ± 4 RFU (relative fluorescence units).

The border (true / false) = (100 + 24) = 62, so the IDL does indeed control gene expression according to a logical OR.

The affinity of oligonucleotides of a given MVU is presented as the concentration of a complex of two oligonucleotides (in μM) when they are mixed at a total concentration of 1 μM (calculated using NUPACK at 25 ° C). If necessary, these values can be easily converted into the dissociation constant as shown in the description above (as S for calculations, the target mRNA sequence of the gene is used - AGGCGCCCГGAC):

Parasitic affinity level: 0.05 (which corresponds to Kd ~ 10 ^-5 M if oligonucleotides do not form multidimensional units by themselves).

Example 23) Molecular computing device (MVD), which implements the logical function of DA, including a DNA aptamer, as well as a protein. The binary input of the system is the presence of oligonucleotide I (addition to the solution I = 1; non-addition = 0). A binary measure of the calculation result (yield) is the concentration of the model protein lysozyme in the free from the complex with the quencher oligonucleotide - the DNA aptamer to lysozyme. The calculation result is read by measuring the fluorescence of the solution, taking into account the fact that lysozyme is labeled with a fluorescent label Cy3, and aptamer Q is labeled with a querncher molecule BHQ2, i.e. upon binding of Q to a protein, the Cy3 fluorescence is quenched, which is detected by a fluorometer. A 60-nucleotide aptamer is used as an aptamer (see Iran DT, et al. (2010) Selection and characterization of DNA aptamers for egg white lysozyme. Molecules 15: 1127 - 1140; Zou M et al. (2012) The homogeneous fluorescence anisotropic sensing of salivary lysozyme using the 6-carboxyfluorescein-labeled DNA aptamer. Biosens. Bioelectron. 32: 148-154.). The rest of the nucleotides are created affine to a part of the aptamer: GCGGCTCACAAAACCATT.

IDL is a solution:

92 μl buffer 25 mM Tris-HCl, 200 mM glycine, 5 mM K2HPO4, 300 mM NaCl, pH 8.3;

+1 μl of Cy3-labeled lysozyme (initial concentration 10 μg / ml)

+1 μl 100 μM aptamer Q labeled at the 5 'end of BHQ2 (in water);

The calculation is carried out by adding to the IDL:

Input I = 1) 6 μl of 500 μM oligonucleotide I = 5'-AATGATTCTGGGTGCCGC -3 'in water.

Input I = 0) 6 μl water.

The fluorescence signal of the Cy3 label is detected.

Results:

i) INPUT I = 1 - Signal 100 ± 4 RFU (relative fluorescence units),

i) INPUT I = 0 - Signal 67 ± 8 RFU (relative fluorescence units).

The affinity of oligonucleotides of a given MVU is presented as the concentration of a complex of two oligonucleotides (in μM) when they are mixed at a total concentration of 1 μM (calculated using NUPACK at 25 ° С): I-Q = 0.86. If necessary, this value can be easily converted into a dissociation constant, as shown in the description above.

The operation of the proposed method is illustrated by the drawings of FIG. 1-13. and tables 1-2.

Fig 1. The traditional paradigm of complementary interactions of nucleic acids. Above - the affinity of complementary DNA molecules depending on the length of the oligonucleotides (calculated using the NUPACK algorithm). Below - the principle of transmission and processing of information in complementary interactions of nucleic acids (within the traditional paradigm of highly specific interactions).

FIG. 2. Density of the "continuum" of dissociation constants for DNA with a length of 10 bases, calculated using the algorithm for calculating the affinity of two DNA strands NUPACK (unpack.org). The histogram shows the distribution of the interaction constants of all 10-membered oligonucleotides with a randomly selected GCTTATGACG oligonucleotide.

FIG. 3. Distribution by affinities of 10-membered oligonucleotides in relation to a pair of oligonucleotides: 1 - below) GCTTATGACG and its complementary CGTCATAAGC, or 2) GCTTATGACG and arbitrary TAGCGCAGTA. It can be seen that the affinities form a "multidimensional continuum", among which an oligonucleotide with the desired affinity for many oligonucleotides can be selected at once.

FIG. 4. The mechanism and principle of transmission and processing of information through essentially non-complementary interactions of nucleic acids (within the framework of this invention). The system of essentially non-complementary interacting oligonucleotides is described by a system of equations for the interaction of molecules of the ensemble. In the simplest case, when only two-component associates can be formed, and also considering thermodynamically equilibrium systems, we get the system of equations shown in the Figure.

FIG. 5. Scheme of the MVU, which implements the logic gate YES. Arrows indicate oligonucleotides that interact with each other to a greater extent than others (in other pairs). The absence of arrows between oligonucleotides indicates that the affinity between them is no stronger than the parasitic one.

FIG. 6. The scheme of the MVU, which implements the logic gate NO. Arrows indicate oligonucleotides that interact with each other to a greater extent than others (in other pairs). The absence of arrows between oligonucleotides indicates that the affinity between them is no stronger than the parasitic one. In the case of a fluorescent readout, the signal oligonucleotide is labeled with a fluorophore indicated by the empty diamond; the black rhombus denotes a quencher (quenching fluorescence during the formation of a QS complex).

FIG. 7. The scheme of the MVU, which implements the logical gate OR. Arrows indicate oligonucleotides that interact with each other to a greater extent than others (in other pairs). The absence of arrows between oligonucleotides indicates that the affinity between them is no stronger than the parasitic one. In the case of a fluorescent readout, the signal oligonucleotide is labeled with a fluorophore indicated by the empty diamond; the black rhombus denotes a quencher (quenching fluorescence during the formation of a QS complex).

FIG. 8. Scheme of the IDL that implements the logical gate I. The arrows show oligonucleotides interacting with each other to a greater extent than the rest (in other pairs). The absence of arrows between oligonucleotides indicates that the affinity between them is no stronger than the parasitic one. In the case of a fluorescent readout, the signal oligonucleotide is labeled with a fluorophore indicated by the empty diamond; the black rhombus denotes a quencher (quenching fluorescence during the formation of a QS complex).

FIG. 9. Scheme and results of the operation of the MVU, which implements a 3-bit memory cell with the function of storing and reading. Arrows indicate oligonucleotides that interact with each other to a greater extent than others (in other pairs). The absence of arrows between oligonucleotides indicates that the affinity between them is no stronger than the parasitic one. In the case of a fluorescent readout, the signal oligonucleotide is labeled with a fluorophore indicated by the empty diamond; the black rhombus denotes a quencher (quenching fluorescence during the formation of a QS complex).

FIG. 10. Scheme and results of the work of the MVU, which calculates the square root of a 4-bit number. Arrows indicate oligonucleotides that interact with each other to a greater extent than others (in other pairs). The absence of arrows between oligonucleotides indicates that the affinity between them is no stronger than the parasitic one. In the case of a fluorescent readout, the signal oligonucleotide is labeled with a fluorophore indicated by the empty diamond; the black rhombus denotes a quencher (quenching fluorescence during the formation of a QS complex).

FIG. 11. Results of the MVG implementing the logic gate 500-bit logic gate AND (top) and the assessment of the gate performance based on the analysis of the operation of 100-, 200-, 300-, and 500-bit logic gates I. The output signal is the fraction of S in the free molecular form.

FIG. 12. The results of calculating the MVU, solving problems of elementary algebra.

FIG. 13. The results of calculating the IDL, the solving equation -0.25 * x ^∧ 2 = sin (πx / 2).

Table 1. List of oligonucleotides that are part of the MVU that implements the 500-bit logical gate I.

Table 2. List of oligonucleotides that are part of the IDU, solving problems of elementary algebra.

Molecular computing device based on substantially non-complementary single-stranded nucleic acids with low affinity to each other (variants)

Molecular computing device based on essentially non-complementary single-stranded nucleic acids with low affinity to each other (variants)

Molecular computing device based on substantially non-complementary single-stranded nucleic acids with low affinity to each other (variants)

Molecular computing device based on substantially non-complementary single-stranded nucleic acids with low affinity to each other (variants)

Molecular computing device based on substantially non-complementary single-stranded nucleic acids with low affinity to each other (variants)

Molecular computing device based on substantially non-complementary single-stranded nucleic acids with low affinity to each other (variants)

Claims (117)

1. Molecular computing device including at least a set of single-stranded nucleic acids and for which: the numerical measure of the input signals are the measures associated with the input molecules, the numerical measure of the output signals are the measures associated with the output molecules, input signals are transformed into output signals as a result of interactions between input molecules, said single-stranded nucleic acids of said set and output molecules, characterized in that the said single-stranded nucleic acids of the said set have a low affinity of interaction with each other, such that at least 0.1% of the amount of each of them exists in free molecular form, and is not included in complexes with other molecules. 2. The molecular computing device of claim 1, wherein said output molecules are single stranded nucleic acids. 3. The molecular computing device of claim 1, wherein said output molecules are single stranded nucleic acids of said set. 4. The molecular computing device of claim 1, wherein said output molecules are mRNA. 5. The molecular computing device of claim 4, wherein said measure of output is related to the level of gene expression encoded by said mRNA. 6. The molecular computing device of claim 1, wherein said output molecules are proteins. 7. The molecular computing device of claim 1, wherein said input molecules are single stranded nucleic acids. 8. The molecular computing device of claim 1, wherein said input molecules are single stranded nucleic acids of said set. 9. The molecular computing device of claim 1, wherein said input molecules are mRNA. 10. The molecular computing device of claim 1, wherein said input molecules are proteins. 11. The molecular computing device of claim 1, wherein said nucleic acids of said set are DNA. 12. The molecular computing device of claim 1, wherein said nucleic acids of said set are RNA. 13. The molecular computing device of claim 1, wherein said nucleic acids of said set are oligonucleotides. 14. The molecular computing device of claim 1, wherein the numerical measure of the input signal is related to the total concentration of the input molecule. 15. The molecular computing device of claim 1, wherein the numerical measure of the input signal is related to the total mass of the input molecule. 16. The molecular computing device of claim 1, wherein the numerical measure of the output signal is related to the total concentration of the output molecule in free molecular form. 17. The molecular computing device of claim 1, wherein the numerical measure of the output signal is related to the mass of the output molecule in free molecular form. 18. The molecular computing device of claim 1, wherein the numerical measure of the output signal is related to the concentration of the complex formed by the output molecule with the nucleic acids of said set. 19. The molecular computing device of claim 1, wherein the numerical measure of the output signal is related to the concentration of the complexes formed by the output molecules with the nucleic acids of said set. 20. The molecular computing device of claim 1, wherein said measure of output is the level of expression of a gene encoded by a single-stranded nucleic acid of said set, which is an output molecule. 21. The molecular computing device of claim 1, wherein said numerical measure of the output signal is classified as a boolean value "0/1" as a result of comparison with a threshold value: if the signal is less than the threshold value, then the output is 0, if it is greater than the threshold value, then the output is 1. 22. The molecular computing device of claim 1, wherein the transformation of the input signals into output signals occurs as a result of a shift in the complexation equilibrium between the input molecules, said single-stranded nucleic acids of said set and output molecules, as a result of which said numerical measure of the output signals changes. 23. Molecular computing device according to claim 1, characterized in that said single-stranded nucleic acids of said set have a low affinity of interaction with each other, such that at least 0.2% of the amount of each of them exists in free molecular form, and is not included in complexes with other molecules. 24. Molecular computing device according to claim 1, characterized in that said single-stranded nucleic acids of said set have a low affinity of interaction with each other, such that at least 1% of the amount of each of them exists in free molecular form, and is not included in complexes with other molecules. 25. Molecular computing device according to claim 1, characterized in that said single-stranded nucleic acids of said set have a low affinity of interaction with each other, such that at least 5% of the amount of each of them exists in free molecular form, and is not included in complexes with other molecules. 26. The molecular computing device according to claim 1, characterized in that it is configured to transform binary input signals into output signals according to a Boolean algebra function. 27. Molecular computing device according to claim 1, characterized in that it is configured to transform a continuous input signal into a continuous output signal according to the rule of elementary algebra. 28. Molecular computing device according to claim 1, characterized in that it is configured to convert the input signal into the output signal according to the logical YES rule and which consists of: output oligonucleotide, input oligonucleotide, and its presence corresponds to a logical input equal to 1, and its absence corresponds to a logical input equal to 0, processing oligonucleotide, and the processing oligonucleotide binds to the output oligonucleotide in the absence of other oligonucleotides so that no less than 0.1 and no more than 50% of the processing oligonucleotide and no less than 0.1 and no more than 50% of the output oligonucleotide is in free molecular form, for the input oligonucleotide: the processing oligonucleotide binds to it in the absence of other oligonucleotides so that no less than 0.1 and no more than 50% of the processing oligonucleotide and no less than 0.1 and no more than 50% of the said input oligonucleotide is in free molecular form, for the input oligonucleotide: the output oligonucleotide binds to it in the absence of other oligonucleotides so that at least 90% of the processing oligonucleotide and at least 90% of the said input oligonucleotide is in free molecular form, while the molecular computing device is configured to read the result of the calculation after adding the input oligonucleotides to the solution containing the output and processing nucleotides as the amount of the output oligonucleotide in free molecular form. 29. Molecular computing device according to claim 1, characterized in that it is configured to convert the input signal into the output according to the logical NOT rule and which consists of: output oligonucleotide, input oligonucleotide, and its presence corresponds to a logical input equal to 1, and its absence corresponds to a logical input equal to 0, processing oligonucleotide A, processing oligonucleotide B, and processing oligonucleotide A binds to the output oligonucleotide in the absence of other oligonucleotides so that no less than 0.1 and no more than 50% of the processing oligonucleotide and no less than 0.1 and no more than 50% of the output oligonucleotide is in free molecular form, processing oligonucleotide B binds to processing oligonucleotide A in the absence of other oligonucleotides so that no less than 0.1 and no more than 50% of processing oligonucleotide A and no less than 0.1 and no more than 50% of processing oligonucleotide B is in free molecular form, for the input oligonucleotide: the processing oligonucleotide B binds to it in the absence of other oligonucleotides so that no less than 0.1 and no more than 50% of the processing oligonucleotide B and no less than 0.1 and no more than 50% of the said input oligonucleotide is in free molecular form, for the input oligonucleotide: the output oligonucleotide binds to it in the absence of other oligonucleotides so that at least 90% of the processing oligonucleotide and at least 90% of the said input oligonucleotide are in free molecular form, for the input oligonucleotide: the processing oligonucleotide A binds to it in the absence of other oligonucleotides so that at least 90% of the processing oligonucleotide A and at least 90% of the said input oligonucleotide is in free molecular form, for processing oligonucleotide B: the output oligonucleotide binds to it in the absence of other oligonucleotides so that at least 90% of the processing oligonucleotide B and at least 90% of said output oligonucleotide is in free molecular form, while the molecular computing device is configured to read the result of the calculation after adding the input oligonucleotides to the solution containing the output and processing nucleotides as the amount of the output oligonucleotide in free molecular form. 30. Molecular computing device according to claim 1, characterized in that it is configured to convert the input signal into the output signal according to the logical OR rule and which consists of: output oligonucleotide, input oligonucleotides, the presence of each of which corresponds to a logical input equal to 1, and the absence corresponds to a logical input equal to 0, processing oligonucleotide, and the processing oligonucleotide binds to the output oligonucleotide in the absence of other oligonucleotides so that no less than 0.1 and no more than 50% of the processing oligonucleotide and no less than 0.1 and no more than 50% of the output oligonucleotide is in free molecular form, for each input oligonucleotide: the processing oligonucleotide binds to it in the absence of other oligonucleotides so that no less than 0.1 and no more than 50% of the processing oligonucleotide and no less than 0.1 and no more than 50% of the said input oligonucleotide is in free molecular form, for each input oligonucleotide: the output oligonucleotide binds to it in the absence of other oligonucleotides so that at least 90% of the processing oligonucleotide and at least 90% of said input oligonucleotide is in free molecular form, for each input oligonucleotide: each of the other input oligonucleotides binds to it in the absence of other oligonucleotides so that at least 90% of each of these two oligonucleotides is in free molecular form, while the molecular computing device is configured to read the result of the calculation after adding the input oligonucleotides to the solution containing the output and processing nucleotides as the amount of the output oligonucleotide in free molecular form. 31. Molecular computing device according to claim 1, characterized in that it is configured to convert the input signal into the output signal according to the logical AND rule and which consists of: output oligonucleotide, input oligonucleotides, the presence of each of which corresponds to a logical input equal to 1, and the absence corresponds to a logical input equal to 0, processing oligonucleotides, each of which corresponds to one and only one input oligonucleotide, and each processing oligonucleotide binds to the output oligonucleotide in the absence of other oligonucleotides so that no less than 0.1 and no more than 50% of the processing oligonucleotide and no less than 0.1 and no more than 50% of the output oligonucleotide is in free molecular form, for each input oligonucleotide: the corresponding processing oligonucleotide binds to it in the absence of other oligonucleotides so that no less than 0.1 and no more than 50% of the processing oligonucleotide and no less than 0.1 and no more than 50% of the said input oligonucleotide is in free molecular form, for each input oligonucleotide: the output oligonucleotide binds to it in the absence of other oligonucleotides so that at least 90% of the processing oligonucleotide and at least 90% of the said input oligonucleotide are in free molecular form, for each input oligonucleotide: each of the other input oligonucleotides binds to it in the absence of other oligonucleotides so that at least 90% of each of these two oligonucleotides is in free molecular form, while the molecular computing device is configured to read the result of the calculation after adding the input oligonucleotides to the solution containing the output and processing nucleotides as the amount of the output oligonucleotide in free molecular form. 32. Molecular computing device according to claim 1, characterized in that it is configured to convert the input signal into the output according to the function of Boolean algebra F and which functions by a combination of molecular computing devices according to claims. 28-31, implementing the corresponding functions YES, NOT, AND, OR, in which the output oligonucleotides of some molecular computing devices of the mentioned combination are associated with the input oligonucleotides of other molecular computing devices of the mentioned combination according to the expansion of the function F in terms of the complete system of functions YES, NOT, AND, OR. 33. A molecular computing device according to claim 32, characterized in that it is configured to mix all said oligonucleotides of said combination of molecular computing devices in one solution, wherein the molecular computing device is configured to read the result of the calculation after adding to said solution input oligonucleotides that correspond to the arguments of said function F, as the amount of output oligonucleotide that corresponds to an external function for function F, in free molecular form. 34. Molecular computing device according to claim 1, characterized in that it is configured to convert input signals into an output signal according to a function with continuously changing arguments. 35. Molecular computing device according to claim 1, characterized in that it is configured to convert the input signal X into the output signal Y according to the function Y = F (x) with a continuously changing argument X and which consists of: output oligonucleotide, input oligonucleotide, and its total concentration corresponds to the value of X, processing oligonucleotides, and processing oligonucleotides bind to the output oligonucleotide in the absence of other oligonucleotides so that no less than 0.1 and no more than 90% of the output oligonucleotide is in free molecular form, processing oligonucleotides bind to the input oligonucleotide in the absence of other oligonucleotides so that no less than 0.1 and no more than 90% of the input oligonucleotide is in free molecular form, while the result of the calculation of Y is associated with the amount of the output oligonucleotide in free molecular form. 36. Molecular computing device, including at least a set of single-stranded nucleic acids and for which: the numerical measure of the input signals are the measures associated with the input molecules, the numerical measure of the output signals are the measures associated with the output molecules, input signals are transformed into output signals as a result of interactions between input molecules, said single-stranded nucleic acids of said set and output molecules, moreover, the total concentration of each said single-stranded nucleic acid exceeds the value of the dissociation constant of the complexes formed by this single-stranded nucleic acid and other single-stranded nucleic acids of the said set by no more than 100,000 times. 37. The molecular computing device according to claim 36, wherein the total concentration of each said single-stranded nucleic acid exceeds the value of the dissociation constant of the complexes formed by this single-stranded nucleic acid and other single-stranded nucleic acids of said set by no more than 10,000 times. 38. The molecular computing device according to claim 36, wherein the total concentration of each said single-stranded nucleic acid exceeds the value of the dissociation constant of the complexes formed by this single-stranded nucleic acid and other single-stranded nucleic acids of said set by no more than 1000 times. 39. A molecular computing device according to claim 36, wherein the total concentration of each said single-stranded nucleic acid exceeds the value of the dissociation constant of the complexes formed by this single-stranded nucleic acid and other single-stranded nucleic acids of said set by no more than 100 times. 40. A molecular computing device according to claim 36, wherein the total concentration of each said single-stranded nucleic acid exceeds the value of the dissociation constant of the complexes formed by this single-stranded nucleic acid and other single-stranded nucleic acids of said set by no more than 10 times. 41. Molecular computing device including at least a set of single-stranded nucleic acids and for which: the numerical measure of the input signals are the measures associated with the input molecules, the numerical measure of the output signals are the measures associated with the output molecules, input signals are transformed into output signals as a result of interactions between input molecules, said single-stranded nucleic acids of said set and output molecules, characterized in that said interactions between single-stranded nucleic acids of said set are reversible binding of incompletely complementary ones between single-stranded nucleic acids of said set, moreover, such that for any two single-stranded nucleic acids of the mentioned set, the length of the most extended connected region of their mutual complementarity does not exceed 10 bases. 42. The molecular computing device of claim 41, wherein for any two single-stranded nucleic acids of said set, the length of the longest connected portion of their mutual complementarity does not exceed 9 bases. 43. A molecular computing device according to claim 41, wherein for any two single-stranded nucleic acids of said set, the length of the longest connected portion of their mutual complementarity does not exceed 8 bases. 44. The molecular computing device of claim 41, wherein for any two single-stranded nucleic acids of said set, the length of the longest connected portion of their mutual complementarity does not exceed 7 bases. 45. The molecular computing device of claim 41, wherein for any two single-stranded nucleic acids of said set, the length of the longest connected portion of their mutual complementarity does not exceed 6 bases. 46. A molecular computing device according to claim 41, wherein for any two single-stranded nucleic acids of said set, the length of the longest connected portion of their mutual complementarity does not exceed 5 bases. 47. A method for performing computations using a molecular computing device according to claims 1-46, including the following steps: selection of said set of single-stranded nucleic acids, mixing said set with said input molecules and said output molecules if they are not part of said set, incubating the mixture to ensure interactions between the input molecules, said single-stranded nucleic acids of said set and said output molecules, and transforming input signals into outputs as a result of said interactions, registering said output signal as a computation result. 48. A pharmaceutical formulation designed to regulate gene expression, comprising molecular computing devices according to claims 1, 36, 41. 49. Use of a pharmaceutical formulation according to claim 48 for regulating gene expression for therapeutic purposes.

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