JP2022532995A - Homopolymer encoded nucleic acid memory - Google Patents

Abstract

Nucleic acid memory strands, which encode digital data using sequences of homopolymeric tracts of repeated nucleotides, offer a cheaper and faster alternative to conventional digital DNA storage techniques. The use of homopolymer tracts allows lower fidelity high-throughput sequencing techniques, such as nanopore sequencing, to read the data encoded in the memory chain. Specialized synthetic techniques are capable of encoding large volumes of data despite the reduced data density obtained by homopolymeric tracts compared to conventional single nucleotide sequences. Allows synthesis of memory chains.

Description

Related Applications This application claims priority to US Patent Application No. 16 / 393,510 filed April 24, 2019, the contents of which are incorporated herein by reference.

INDUSTRIAL APPLICABILITY The present invention relates to a method and an apparatus for storing data in a nucleic acid memory chain containing a homopolymer tract.

Background DNA digital memory is the process of representing digital data using the base sequence of DNA and storing that data via DNA synthesis of the polynucleotide corresponding to the base sequence that encodes the data. DNA digital memory offers several advantages over traditional data storage methods, targeting tens of billions of dollars in the market. Traditional data storage methods, including flash memory and recording on magnetic tape, pose problems with physical spatial requirements, scarcity resource reliance, and data integrity. DNA digital memory provides significantly higher data storage densities with significantly lower energy requirements. Current methods rely on high fidelity sequencing techniques with little error tolerance to accurately read the data encoded in DNA. The required sequencing method is relatively slow and expensive to meet fidelity requirements. Examples of current DNA digital memory techniques are described in Church, et al. U.S. Pat. No. 9,384,320 (incorporated herein by reference). To increase the fidelity of sequencing, current methods, such as those described by Church, encode data using sequences that avoid features that make it difficult to read or write sequence iterations, etc. ..

The synthesizer of current DNA digital memory techniques further limits the adoption of this technique due to lack of speed, production of toxic by-products and high cost. Most de novo nucleobases are synthesized using solid phase phosphoramidite techniques, including sequential deprotection and synthesis of sequences constructed from phosphoramidite reagents corresponding to natural (or non-natural) nucleobases. Will be done. Inkjet synthesis on array-based formats allows for very low cost phosphoramidite synthesis, but the strands produced are limited to 100-200 base lengths and length to index the sequence. It is made on a scale below the femtomolar concentration, which requires post-synthesis amplification to provide sufficient material for subsequent readout, at the expense of some of the. Using conventional synthetic techniques, nucleic acids longer than 200 base pairs (bp) in length cause a high rate of disruption and side reactions. In addition, conventional synthetic techniques produce toxic by-products, and disposal of this waste limits the availability of nucleic acid synthesizers and increases the cost of oligo production. These annoying problems associated with synthesis and readout in DNA digital memory have otherwise limited application for promising techniques.

US Pat. No. 9,384,320

Abstract The present invention provides systems and methods for storing data using sequences of homopolymeric tracts that encode digital data. Representing each bit in a data sequence using a homopolymeric tract of repeated bases (eg, 2-10 nucleotides) allows the use of higher throughput and less expensive sequencing techniques. Nanopore sequencing, zero-mode waveguide (ZMW) single molecule sequencing and mass, as sequence reading only relies on distinguishing transitions between homopolymeric tracts and does not require reliable reading of each individual nucleotide. Sequencing techniques such as analysis can be used to increase speed and reduce costs.

Recording of data using the homopolymeric tracts described herein is most efficiently achieved using long chain (eg, 5-10 kb) nucleic acids. Although traditional synthetic techniques are length limited, for example, template-independent polynucleotide synthesis using nucleotidyltransferase synthesizes long chains at a reduced cost and with lower waste production. It is possible. Enzymatically synthesized ssDNA memory strands require only 50% of DNA synthesis compared to traditional phosphoramidite approaches, which are complex with ssDNA strands longer than about 100-200 nucleotides in length. This is because it requires costly ligation or PCR techniques and can only produce ssDNA from dsDNA intermediates. Efcavitch, et al., Which is incorporated herein by reference. See US Pat. No. 8,808,989. The data encoding can be radix 2, radix 3, radix 4 using standard nucleotides, or the data density is radix (base) 8, radix 10, radix 12 or higher. Any number of modified nucleotide analogs can be used to increase to generate a large radix encoding scheme.

Restrictions on modified nucleotide analogs say they can be incorporated using selected synthetic techniques (eg, terminal deoxynucleotidyltransferase (TdT)) and distinguished from each other using selected sequencing analysis. It's just that. In some embodiments, synthesis can be accomplished using the polymerase theta in the presence of Mn ²⁺ .

Consistent homopolymeric tract lengths are not essential for the systems and methods of the invention, as only transitions between individual tracts need to be recognized. Although the tract length can be varied, the synthetic techniques of the present invention adjust the ratio of deoxynucleotides (dNTPs) to the oligonucleotide memory strand being synthesized and the exposure time of dNTPs to the neoplastic memory strands. By controlling the above, the average homopolymer tract length can be effectively controlled. The length of the homopolymer tract can be optimized for the readout technique; the highest data storage density is achieved with the readout resolution of a single nucleotide, while the highest readout speed and accuracy is due to the size of the nucleotide bits. It is achieved by extending a given sequencing technique to a minimal detectable length (eg, 2-10 nucleotides).

Systems and methods of the invention using nanopore sequencing use specialized memory chain constructs such as stoppers (eg, hairpins or polymeric appendages) contained on one or both ends of the chain. obtain. In other nanopore-based methods, memory chains can be circularized and passed through between adjacent nanopores.

Certain aspects of the invention include methods of recording data using nucleic acid memory chains. The steps of this method may include creating an in-silico oligonucleotide sequence that represents a dataset, where each nucleotide of the oligonucleotide sequence corresponds to a unit of said dataset. Nucleic acid memory chains containing multiple homopolymeric tracts can then be synthesized, where each homopolymeric tract corresponds to a nucleotide in the oligonucleotide sequence. Multiple homopolymeric tracts may contain repeating nucleotides between 3 and 10. Each unit of the dataset may be represented by a radix of 2, radix 3, radix 4 or higher, if desired for a particular application.

In certain embodiments, the nucleic acid memory chain can be at least about 200 nucleotides in length to about 5,000 nucleotides in length. The steps of synthesis may include controlling homopolymer length by varying the dNTP concentration. The steps of this method are to modify the first end of the nucleic acid memory chain to prevent the passage of the first end through the nanopores of the nanopore sequencing system; the second end of the nucleic acid memory chain is nanoporeed. It may include the step of passing through the nucleic acid memory chain; and the step of modifying the second end of the nucleic acid memory chain to prevent the passage of the second end through the nanopore.

Other embodiments may use memory chains composed of heteropolymeric tracts of defined stoichiometry or composition to further increase the coding ability of a set number of nucleotide analogs. Further embodiments are nucleotides that use linkers that are similar in structure but can be removed under different conditions, such as ultraviolet or visible light, oxidizing or reducing agents, alkaline or acidic pH, or sequence-specific nucleases. It may be aimed to protect the encoded data in the memory chain by using analogs and thereby hiding the data against them without knowledge of the applicable processes.

The dataset may be selected from the group consisting of text files, image files and audio files. The steps of synthesis can include template-independent synthesis. In certain embodiments, the nucleotidyltransferase enzyme can be used to catalyze the template-independent synthesis. In some embodiments, the polymerase theta can be used to catalyze the template-independent synthesis.

Aspects of the invention may include a method of reading data from a nucleic acid memory strand. The steps of this method are a step of sequencing a nucleic acid memory chain containing a plurality of homopolymer tracts and a step of converting a nucleic acid memory chain sequence into digitized data, each of the plurality of homopolymer tracts. , Which may include a step indicating a nucleotide corresponding to a unit of data, and a step of converting a digitized piece of data into a readable format. The steps of this method may include displaying a readable format. Multiple homopolymeric tracts may include repeats between about 2 nucleotides and about 10 nucleotides. Nucleic acid memory chains can be at least about 200 nucleotides in length and about 5,000 nucleotides in length.

In various embodiments, the sequencing steps can include nanopore sequencing, synthetic sequencing, or mass spectrometry. The sequencing, converting and converting steps can be repeated one or more times with respect to the nucleic acid memory strand.

Other aspects of the invention will be apparent to those skilled in the art in light of the following drawings and detailed description.

FIG. 1 shows the enzymatic synthetic cycle used to form homopolymeric tracts.

FIG. 2 shows a method for synthesizing a nucleic acid memory chain having a homopolymer tract.

FIG. 3 shows a method of reading data from a nucleic acid memory strand having a homopolymer tract.

FIG. 4 shows the relationship between chain / GB and homopolymer length and chain length.

FIG. 5 shows data that can be encoded in a DNA strand composed of 500 polymer tracts as a function of the number of identifiable nucleotide analogs in the memory strand.

FIG. 6 shows the nanopore trapped nucleic acid memory strand of the present invention.

FIG. 7 shows a scheme for changing the encoded data within a chain of homopolymer tracts in response to processing conditions.

FIG. 8 shows a system for synthesizing nucleic acid memory chains with homopolymeric tracts.

FIG. 9 shows a system for parallel synthesis of nucleic acid memory chains with homopolymeric tracts on an array of nanowells.

FIG. 10 gives a more detailed schematic of the components that may appear in the system.

FIG. 11 shows an analysis of enzyme-mediated synthesis of homopolymeric tracts with two different base compositions.

FIG. 12 shows the process for converting character text into a 12-bit nucleic acid sequence.

FIG. 13 shows a 12-cycle enzymatically synthesized polyacrylamide gel electrophoresis (PAGE) analysis.

14A-L show the homopolymer distributions experimentally found for each of the 12-bit homopolymer additions. 14A-L show the homopolymer distributions experimentally found for each of the 12-bit homopolymer additions. 14A-L show the homopolymer distributions experimentally found for each of the 12-bit homopolymer additions. 14A-L show the homopolymer distributions experimentally found for each of the 12-bit homopolymer additions. 14A-L show the homopolymer distributions experimentally found for each of the 12-bit homopolymer additions. 14A-L show the homopolymer distributions experimentally found for each of the 12-bit homopolymer additions. 14A-L show the homopolymer distributions experimentally found for each of the 12-bit homopolymer additions. 14A-L show the homopolymer distributions experimentally found for each of the 12-bit homopolymer additions. 14A-L show the homopolymer distributions experimentally found for each of the 12-bit homopolymer additions. 14A-L show the homopolymer distributions experimentally found for each of the 12-bit homopolymer additions. 14A-L show the homopolymer distributions experimentally found for each of the 12-bit homopolymer additions. 14A-L show the homopolymer distributions experimentally found for each of the 12-bit homopolymer additions.

FIG. 15 illustrates a cycle step for template-independent DNA polymerase-mediated synthesis of homopolymer-encoded information polymers.

FIG. 16 shows a side view of a typical reaction zone in a 2D array of reaction zones showing hydrophobic pattern formation that isolates the liquid reaction droplets.

FIG. 17 shows a side view of a reaction zone with a controlled heater element or an electrochemical element.

FIG. 18 shows a reaction zone showing a light transmissive top cover.

FIG. 19 shows an exemplary device useful for addressable photoactivation of enzymatic DNA synthesis.

FIG. 20 shows the kinetics of UV light decaging of 3'-O- (2-nitro) -benzyl dATP.

FIG. 21 shows optically controlled oligonucleotide elongation. The enzyme reaction mixture containing 3'-orthonitrobenzyl dATP was irradiated with a low power light source over various sections so that the exposure time controlled the amount of decaying. The amount of available natural dATP formed then controlled the average length of the polynucleotide tract.

FIG. 22 shows the structure of a 3'-O-caging modification that allows bicolor light-mediated decaying.

FIG. 23 shows the gel electrophoresis analysis of each of the 12 cycles of homopolymer synthesis using 3'-O- (2-nitro) -benzyl dATP and dCTP, dGTP, dTTP.

FIG. 24 shows an electrophoretic analysis of enzymatic synthetic reactions comparing uptake-regulated dNTP analogs with unmodified dNTPs.

FIG. 25 shows electrophoretic analysis of uptake rate regulated dNTP analogs in two consecutive cycles. The modified analog was used to form the N + 1 homopolymer, followed by the addition of the N + 2 homopolymer. Homopolymer synthesis rate-regulating modifications were removed from the oligonucleotide prior to gel analysis.

FIG. 26 shows an electrophoretogram of the TdT elongation reaction showing homopolymer rate regulated analog uptake followed by a second cycle of homopolymer rate regulated analog uptake. Modified nucleotides are followed by successive modified nucleotides.

FIG. 27 shows the modified dNTP analog used in the information polymer composition.

FIG. 28 shows an electrophoretic analysis of an information polymer composed of modified nucleotides.

FIG. 29 shows the detection of an information polymer (P71) composed of modified nucleotides by translocation via nanopores.

FIG. 30 shows the detection of an information polymer (86B) composed of modified nucleotides by translocation via nanopores.

Detailed Description The present invention presents a system and method for writing data to and reading data from nucleic acids having homopolymeric tracts corresponding to units of digital data. By repeating each nucleotide in the data encoding sequence (eg, 3-10 times), it is only necessary to observe the transition between homopolymeric tracts in the sequencing read, which is in nucleic acid data storage. It enables higher throughput sequencing techniques with lower fidelity that can result in cheaper performance. The advantages of synthesizing nucleic acid homopolymer tract memory strands are: 1) Very long (5-10 kb) strands that allow the use of high throughput long read DNA sequencing techniques for reads. 2) The ability to tolerate errors in sequencing and retrieval techniques, and 3) the ability to fabricate nucleic acid memory chains at a much lower cost than those of conventional chemical synthesis methods. The use of homopolymer nucleic acid memory chains is best realized in long (eg, 5-10 kb) chains that can be efficiently produced using template-independent TdT enzymes or polymerase theta, where homopolymer tract lengths are achieved. Can be controlled by varying the exposure time and the ratio of dNTPs to the polynucleotide memory strand.

The synthesis of homopolymers for encoding data by an enzymatically mediated approach is easily achieved by using non-terminator natural or modified nucleotide triphosphates, the simplest and fastest of DNA synthesis. The method becomes possible. One natural or modified nucleotide triphosphate is delivered to the reaction zone with the nucleotidyltransferase, the reaction occurs and then removed by washing with buffer, one of the data storage illustrated in FIG. Complete the "write" cycle of. Data chain synthesis occurs in a completely aqueous environment, with no toxic or toxic chemicals, thus enabling a practical device suitable for large data storage centers.

FIG. 2 shows a method 101 for synthesizing a nucleic acid memory chain having a homopolymeric tract according to a particular embodiment. Method 101 includes step 103 to create an in-silico oligonucleotide sequence that represents a dataset. The dataset may include digitized data that may represent text, images, video, audio, or any other piece of information that may be digitized. The oligonucleotide sequence can contain any number of natural or modified nucleotides or analogs thereof, depending on the number of unique nucleotides or analogs used in the memory chain, 2, radix 3, radix 4 or more. Data sets can also be encoded using large radix schemes. In a simple embodiment, the encoding scheme may correspond to a binary data scheme traditionally represented by a series of 0's and 1's, where one or more nucleotides or analogs may correspond to 0's and 1 Alternatively, the plurality of other nucleotides may correspond to 1. Nucleic acid memory strands (eg, RNA, single-stranded or double-stranded DNA), each containing a series of homopolymeric tracts corresponding in order to the nucleotides in the in silico oligonucleotide sequence, can then be synthesized 105. In certain embodiments, additional steps include modifying one end of the memory chain 107, passing the memory chain through the nanopores 109, and preventing the ends from passing through the nanopores. Includes step 111 to modify the other end of the chain.

FIG. 3 shows method 203 for reading data from a nucleic acid memory strand having a homopolymer tract. The steps of method 203 are step 203 for sequencing a series of homopolymeric tracts in a nucleic acid memory chain, step 205 for converting the sequence into a dataset, and the dataset in a readable format (eg, image, video, audio clip, or text). Includes a step of converting to (a piece of) and a step 209 of displaying the data in its readable format as needed (eg, on a monitor or using a printer or other input / output device).

Preferably, the systems and methods of the invention are long strands (5-10 kb) of DNA that are either single-stranded or double-stranded and can be naturally occurring or produced by chemical or enzymatic synthesis. ) Is used. In certain embodiments, the nucleic acid memory strand is enzymatically using TdT to create a series of homopolymeric tracts that may be 2-10, 3-10, 4-10 nucleotides or longer. Can be generated in. The homopolymer tract can consist of adenine (A), guanine (G), cytosine (C) or thymine (T), respectively. Alternating sequences of homopolymeric tracts can be used to encode the data stored in the memory chain.

Each nucleotide homopolymer tract can show varying amounts of data, depending on the number of bases used. The number of bits required to make up one byte (256 in decimal) is defined by the following relationship: # bits / byte = 8 / (log2 (n)), in the equation, n = used. Radix. Each tract may correspond to 1 bit when a 2-base encoding is used, or 1/4 of a byte when a 4-base encoding is used. In certain embodiments, the DNA data strand may consist of a homopolymeric tract of 2-10 nucleotides (using a two-group dataset display), which is between 999 and 1000 bases in length. Allows 333 to 100 bits to be shown in the memory chain of. In a preferred embodiment, the nucleobase encoding of the data can be such that a single homopolymer tract of one nucleotide is always adjacent to a homopolymer tract of different nucleotides. For example, the encoding can be such that the homopolymer tract of adenine is not immediately preceded or immediately followed by another adenine homopolymer tract. If two adjacent homopolymeric tracts contain the same nucleotides, a homopolymeric tract that is measurable longer than the average homopolymeric tract showing a single nucleotide in the encoded data sequence can be synthesized. These longer tracts can be created through the manipulation of synthetic reactions described below, for example by increasing the concentration of dNTPs during the reaction or increasing the reaction time. The exact length of two adjacent identical homopolymer tracts only needs to be long enough to be uniquely identified from a single homopolymer tract using a readout device (ie, a nanopore sequencer). Is. In certain embodiments, non-nucleotide homopolymer spacers may be added between A, G, C or T homopolymer tracts to clearly distinguish the same adjacent nucleotide homopolymer tracts from each other. The use of A, G, C and T homopolymeric tracts does not simply use two nucleotides (similar to 0 and 1 in the binary code), but the density of data that can be stored in one contiguous chain. Allows the creation of a 4-bit encoding space. For example, four contiguous homopolymeric tracts can encode 256 numbers (ie, 1 byte) when A, G, C and T are used in a radix 4 scheme. In such an embodiment, when a 3-nucleotide long homopolymer tract or a 10-nucleotide long homopolymer tract is used, respectively, 83 or 25 bytes are shown in a 996 or 1000 nucleotide long nucleic acid memory chain.

In various embodiments, a radix 8 or even radix 12 coding scheme can be used via the incorporation of uniquely modified nucleotide or non-nucleotide analog homopolymeric tracts into the memory chain. These modified nucleotide or non-nucleotide analogs should generate their own digital signals on readout devices such as nanopore sequencers or single molecule ZMW sequencers. The TdTs discussed below can enhance the signals provided by read devices such as nanopores and are therefore extensively modified and can be used to generate nucleic acid memory chains in which data are encoded. The dNTP analog can be imported. Homopolymers of modified nucleotides (eg, A ^* , G ^* , C ^* and T ^* or A ^** , G ^** , C ^** and T ^** ) produce 8-bit or even 12-bit encoding schemes. Can be synthesized using TdT and a modified dNTP analog of each of the four bases (eg, dA ^* TP or dA ^** TP). A higher radix (n) encoding allows for data compression, resulting in a reduction in the number of DNA strands required to encode a given amount of information. The relationships that determine the number of DNA strands per GB of data, the number of groups (n), and the synthesized strand length as a function of the length of the homopolymer tract, as illustrated in FIG. 4, are defined by: : # Chain / GB = (8 / (log2 (n)) ^* 109 ^* Homopolymer length ^* 1 / Chain length.

The number of unique homopolymeric tracts can only be limited by the ability of the readout technique (ie, nanopore or ZMW single molecule sequencing) to determine one homopolymer tract from another homopolymer tract. There are several reports in the literature of the detection of homopolymers composed of unmodified nucleotides by detecting changes in ionic currents between nanopore-mediated translocations (Venta et al, 2013; Feng et al, 2015). Modifications that alter the residence time of DNA in nanopores produce identifiable and characteristic ionic current signals. Singer et al 2010 and Morin et al 2016 use non-covalent bis PNA or γPNA functionalized with 5 kDa or 10 kDa PEG to enhance detection by nanopores. Liu et al 2015 selectively created adamantly 8-oxoG analogs to modify residence time and generate unique signals. Given TdT's tolerance for incorporating bulky modifications in n6 of dATP, N4 of dCTP, N2 or O6 of dGTP, and O4 or N3 of dTTP, acyl or alkyl (alky) modifications at these positions are nanopores. Alternatively, it can be screened and selected to enhance the detection modality of ZMW single molecule sequencing techniques. Detection can be improved via modified nucleotides that enhance differential current blocking in the nanopores or enhance the residence time of modified nucleotides in the active site of DNA polymerase in the ZMW single molecule approach. Other natural and non-natural purine and pyrimidine nucleotide analogs can be used when they generate their own digital signals on a readout device such as a nanopore sequencer or a single molecule ZMW sequencer. Modifications of pyrimidines and purines at C5 or C7, respectively, can be used when they generate their own digital signals on a readout device such as a nanopore sequencer or a single molecule ZMW sequencer. Appropriate modified nucleotide triphosphates are selected to be rapidly incorporated during the enzymatic extension step, providing a substitution-specific residence time with the shortest possible homopolymer during the detection step. Examples of modifications to A, G, C and T bases suitable for expanding the bit encoding space include, but are not limited to, N6-benzoyl dA, N6-benzyl dA, N6-alkyl dA, N6-acyl. dA, N6-substituted alkyl dA, N6-substituted acyl dA, N6-aryl acyl dA, N6-substituted aryl acyl dA, N2-alkyl-dG, N2-acyl dG, N2-aryl acyl dG, N2-substituted alkyl dG, N2-substituted acyl dG, N2-substituted aryl acyl dG, O6 alkyl dG, O4 alkyl dT, N3 alkyl dT, N3 acyl dT, O6 substituted alkyl dG, O4 substituted alkyl dT, C5-propargylamine dT, C5-propargylamine dC , C7-propargylamine dA, C7-propargylamine dG, substituted C5-propargylamine dT, substituted C5-propargylamine dC, substituted C7-propargylamine dA, substituted C7-propargylamine dG. Preferred embodiments of substitution include, but are not limited to, covalent bonds that are perfectly stable to removal, except under the most extreme chemical conditions of pH, temperature and concentration of reactive species. Substitutions that can affect their own current interruptions are, but are not limited to, alkyl, heteroatom-substituted alkyls, aromatic hydrocarbons, alkyl-substituted aromatic hydrocarbons, heteroatom-substituted alkyl-substituted aromatic hydrocarbons, Heteroatom-substituted aromatic hydrocarbons, benzyls, substituted benzyls, or combinations thereof may be included. In some embodiments, the substitution can be polyethylene glycol composed of 2 to 450 monomeric units. In some embodiments, substitutions composed of peptides or peptoids may be appropriate to increase the residence time of the homopolymer in a specific and discernible manner. The efficiency of uptake of modified nucleotides by a template-independent polymerase such as TdT is such as, but not limited to, Co ++, Zn ++, Mg ++, Mn ++, or a mixture of two or more different metal ions. It can be regulated by the use of ionic cofactors. Each modified nucleotide may require different metal ions for optimal performance during enzymatic homopolymer synthesis.

Since the long-term stability of DNA data strands is important, the use of non-purine-based homopolymers has clear advantages because they are subjected to depurination at low pH. .. In some embodiments, the homopolymer bit may consist of only a single nucleotide type (ie, thymine) modified with 2, 3, 4 or more different chemical groups and is unique in current interruption. Each yields a homopolymer tract. Therefore, one nucleotide labeled with four unique modifiers can replace the presence of A, G, C, T. Other embodiments are possible that use only one of the other three nucleotides, having 2, 3, 4 or more different chemical groups.

If the modified nucleotide analog is sufficient to generate a unique residence time for the passage of a single nucleotide through the nanopore, another embodiment uses a single nucleotide bit instead of the homopolymer bit. do. A single modified nucleotide bit is advantageous in that it allows for maximum density information per DNA strand, thus reducing the cost of DNA-based data storage.

The exact length of the homopolymer tract is not important as long as the sequencing technique used for the readout can clearly distinguish the start and stop of one homopolymer tract from another homopolymer tract. It was clear to increase the number of unique nucleotides or bases used (including modified nucleotides or non-nucleotide analogs) and thus reduce the length required to capture a set amount of data. Although there are synthetic and memory density advantages, the lowest cost per DNA data memory synthesis can be achieved by using four naturally occurring nucleotide dNTP monomers during enzymatic synthesis, which are these reagents. Widely used in the fields of molecular biology and sequencing, they are produced in very large batches at the lowest manufacturing costs. The cost of producing dNTP analogs to increase the number of proprietary homopolymeric tracts can be reduced as the use of DNA data storage increases and the scale of analog production increases.

Any method of synthesizing homopolymeric tract segments can be used with the systems and methods of the invention, but a preferred embodiment uses the template-independent enzyme TdT. TdT provides certain benefits as long as it produces homopolymers in a Poisson distribution quickly and inexpensively, where the average size of homopolymers depends on the ratio of [dNTP] to the nascent oligonucleotide memory strand. Can be tightly controlled. In some embodiments, the polymerase theta in the presence of Mn ²⁺ can be used as a template-independent polymerase for synthesizing homopolymeric tract nucleic acid memory chains. In another embodiment, the length of the homopolymer tract segment can be controlled by delivering an excess amount of dNTPs to the reaction zone and then removing the reactants after a carefully controlled time interval.

TdT demonstrates the ability to synthesize homopolymeric tracts of properly defined length by controlling the ratio of dNTP concentration to the concentration at the 3'end of the nucleic acid chain desired to be modified. There is. Inkjet synthesis on array-based formats allows for very low cost phosphoramidite synthesis, but the strands produced are limited to 100-200 base lengths and length to index the sequence. Sequencing of short reads, made on a scale below femtomole concentration, requiring post-synthesis amplification to provide sufficient material for subsequent reads, at the expense of some of the Mainly suitable for readout technology.

Strands of single-stranded DNA synthesized according to the processes of the invention can benefit from the prevention of hairpins or dsDNA, either during synthesis or during readout. Hairpin formation can be prevented by modifying the extracyclic amines of one member of the A: T or G: C base pairs to prevent the hydrogen bonds required for base pairing. In some embodiments, the extracyclic amine can be modified by acylation or alkylation. Any simple and stable modification of the outer amines of A, G or C, which prevents base pairing, can be used to prevent hairpin formation. In certain embodiments, N6 of deoxyadenosine and N2 of deoxyguanosine can be acetylated with acetyl groups that prevent base pairing. In some embodiments, N6 of deoxyadenosine and N4 of deoxycytidine can be modified to prevent base pairing and hairpin formation. In some embodiments, O6 of deoxyguanosine or O6 of deoxythymidine can be modified to prevent base pairing and hairpin formation. In some embodiments, O4 of deoxythymidine or N3 of deoxythymidine can be modified to prevent base pairing and hairpin formation. In some embodiments, modifications to A, G, C or T to generate higher order base encoding schemes also serve the purpose of preventing base pairing and hairpin formation. In some embodiments, the homopolymer bit may consist of only a single nucleotide type (ie, thymine) modified with 2, 3, 4 or more different chemical groups and is unique in current interruption. And prevent the formation of intra-chain or inter-chain double chain regions. In another embodiment, a thermostable version of TdT or another template-independent nucleotidyltransferase can be used to carry out chain synthesis at elevated temperatures, thus intrachain or interchain double chain regions. Prevent the formation of.

Control of homopolymer tract length is optimized for any of the above analogs after determining and calibrating the uptake rate of the dNTP analog to create a reproducible range of homopolymer tract lengths of 2-10 nucleotide lengths. obtain. The use of A ^* , G ^* , C ^* and T ^* and A ^** , G ^** , C ^** and T ^** homopolymeric tracts allows the creation of 8-bit or 12-bit encodings, "0". And increase the density of data that can be stored in one contiguous chain, rather than simply using two nucleotides to encode the "1". Three contiguous homopolymeric tracts can encode 256 numbers (ie, 1 byte) when A, G, C, T, A ^* , G ^* , C ^* and T ^* are used. In such an embodiment, there are 111 or 33 bytes in the 999 or 990 nucleotide length nucleic acid memory strand when a 3 nucleotide length homopolymer tract or a 10 nucleotide length homopolymer tract is used, respectively. Two consecutive homopolymeric tracts are used when A, G, C, T, A ^* , G ^* , C ^* , T ^* , A ^** , G ^** , C ^** and T ^** are used. 256 numbers (ie, 1 byte) can be encoded. In these embodiments, there are 166 or 50 bytes in the 996 or 1000 nucleotide length nucleic acid memory strand when a 3 nucleotide length homopolymer tract or a 10 nucleotide length homopolymer tract is used, respectively.

In certain embodiments, the data can also be encoded in a memory chain heteropolymer tract with random sequences and defined compositions to achieve higher levels of data compression. Heteropolymer stretches can be produced using enzymatic reactions using a mixture of different dNTPs, where dNTP stoichiometry is used to control the composition of the heteropolymer tract. The number and type of heteropolymer tracts is limited only by the combination of the dNTP analog and the ability of the detection modality to identify the composition of different tracts. For m dNTP analogs, there are (m ² -m) / 2 binary combinations for heteropolymer formation. Identifying two different levels of tract composition for each binary combination (eg, tracts in which analogs A and B are present in a ratio of approximately 2: 1 each, and tracts in which they are present in a ratio of 1: 2). The detection modality that can be made allows data to be encoded from a set of m analogs at a rate of radix ^m2 , effectively doubling the coding ability of the memory chain. FIG. 5 illustrates data that can be stored in a memory chain as a function of the number of dNTP analogs available, using either a homopolymer or a binary heteropolymer-based encoding scheme with a two-level tract composition. ..

The data encoding strands of the present invention may not necessarily require precisely defined homopolymer lengths, which make a unique distinction between homopolymer tract segments by high throughput DNA sequencing techniques. It only needs to be long enough (about 2-10 nucleotides) to make it possible. Existing next-generation synthetic sequencing (SBS) systems can easily determine transitions between two adjacent homopolymeric tracts. Again, the exact length of the homopolymer tract is not important for the accurate detection of homopolymer bits. The use of tracts of the same nucleotide provides an advantage in overcoming the most common errors: insertions and deletions in current SBS platforms. Deletions of 1 nucleotide in homopolymeric tracts longer than 2 nt are still interpreted as true homopolymers. Similarly, the insertion of a single nucleotide in a homopolymer tract is not misinterpreted as two adjacent homopolymers, but it allows the insertion of more than one nucleotide between SBSs. This is because it is a low-sexual event. This sequencing error tolerance provides the advantage of reducing the sequencing depth required to ensure correct decoding of the information stored by the DNA data strand. Existing nanopore systems can easily distinguish A, G, C or T homopolymeric tracts from each other based on their differential current interruptions. In certain embodiments, single molecule ZMW sequencing can be used to determine the linear order of homopolymeric tracts on a straight line. The use of either sequencing technique is self-complementary at the 5'end of the single-stranded memory strand synthesized by the DNA initiator to provide primers for single-molecule ZMW sequencing. Complimentary) It may be required to have conforming properties with sequencing techniques such as hairpins. Nanopore sequencing techniques may also require a self-complementary hairpin at the 5'end of the strand to provide a "start" data mark. In various embodiments, the readout technique can be any next-generation sequencing method, such as that provided by Illumina (San Diego, CA). In some embodiments, the readout or sequencing technique can be mass spectrometric based. The technique-specific error rate of a readout technique is as long as the technique can uniquely detect transitions between two different homopolymeric tracts and / or if two identical homopolymeric tracts are adjacent to each other. It is not important as long as the difference between one homopolymer tract length and one 2x length can be uniquely detected.

Certain reading techniques may be preferred over others, based on specific applications of the present invention. Techniques such as nanopore sequencing can be non-destructive, leave nucleic acid memory strands intact, and can be suitable for multiple read cycles. Read-out techniques that rely on synthetic sequencing (SBS), such as the ZMW single molecule, produce a copy of the original template strand and remove the complementary strand, its early stage ready for a subsequent cycle of read-out. Post-reading operations (ie, strand separation by melting) may be required to return the original nucleic acid memory strand to the state. Other read techniques, such as mass spectrometry, are destructive and deplete the pool of nucleic acid memory chains after repeated cycles of sampling and reading.

In various embodiments, the nucleic acid memory strand may include a "stopper". A "stopper" can be a polymeric construct that prevents the passage of single- or double-stranded nucleic acids through nanopores (manrao, et al., 2012, Reading DNA at single, incorporated herein by reference). --nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase, Nature Biotechnology 30, 349-353). Proteins such as Phi 29 DNA polymerase are large enough not to be pulled through the larger pores (about 6.3 nm) on the cis side of the protein nanopores. The pore diameter on the smaller side is estimated to be about 1.2 nm wide. Stoppers can be used on either the 5'end or the 3'end of the nucleic acid memory chains of the invention. For some applications, it may be desirable to have stoppers at both the 3'and 5'ends of the nucleic acid molecule. The stopper can consist of a hairpin (stem-loop) structure with either a protruding 5'-overhang or a 3'-overhang to which the information-encoding nucleic acid is covalently attached. If the stopper consists of a hairpin, the length of the ds stem is sufficient to resist any melting force exerted against it by the electric field used to translocate the memory chain through the nanopores. Can be lengthened. In certain embodiments, the double-stranded stem portion of the hairpin is under the influence of the force exerted against it by an electric field that translocates one base in the double-stranded stem region through the nanopores to the rest of the molecule. It can be crosslinked to its cognate bases forming a base pair so that it is impossible to thaw. To utilize a hairpin stopper for TdT-mediated nucleic acid memory synthesis according to certain embodiments, the stopper is long enough (ie, to allow binding of TdT for template-independent synthesis). It can have a 3'-overhang of> 10 nucleotides).

The stopper can consist of a non-nucleotide macromolecular construct that can be added to either the 3'end or the 5'end of the nucleic acid molecule. The construct is a direct conjugation of the high molecular weight species onto the 3'end of the nucleic acid by a polymerase or a transferase such as TdT (Sorensen, et al., 2013, Enzymatic Ligation of Large Biomolecules, incorporated herein by reference). Incorporation of functionalized nucleotides (incorporated herein by reference) that allows specific modification of nucleic acids by or through their functionality, to DNA, ACS Nano, 7 (9): 8098-8104). It can be synthesized by Winz, et al., 2015, Nucleotidyl transferase assisted DNA labeling with different click chemistries, Nucleic Acids Res. 43 (17): e110). The 5'end stopper is for the oligonucleotide adapter via direct synthesis of the hairpin or through secondary modification of the functional handle introduced as the final step in the synthesis of oligonucleotides from 3'to 5'. It can be easily introduced at the time of chemical synthesis and used as an initiator. Alternatively, the 5'end stopper binds the oligonucleotide initiator to magnetic or non-magnetic beads or particles or nanoparticles via the 5'end to enzymatically synthesize a memory chain containing a homopolymeric tract. It can then be constructed by leaving the memory chain attached to magnetic or non-magnetic beads or particles or nanoparticles.

The stopper is the rest of the molecule to allow the nucleic acid strands to passively diffuse out of the nanopores or to allow the nucleic acid strands to be translocated out of the nanopores via the application of voltage. It can be further modified to allow cutting of the stopper from, thus allowing the chain to be recovered.

In certain embodiments, a template-independent polymerase or transferase modifies a pre-synthesized strand of nucleic acid to allow the use of nanopore devices as "write-once-one-many" memory devices. Can be used for. Some of the inherent problems associated with the use of nanopore devices as DNA sequencers are the high error rates they cause due to inadequate distinction of nanopores. This may be due to the speed of translocation through the pores, or the fact that the approximate depth of the nanopores is 8 nm, allowing multiple bases to be present in the pores at the same time. The homopolymer memory chains of the present invention address this issue through the use of homopolymer iterations, reducing the need for strict sequencing accuracy. In certain embodiments, the drawbacks of nanopore sequencing can be addressed by mounting a hairpin adapter at one end of the double-stranded DNA memory strand, resulting in individual during translocation and base-calling processes. Each sense of the DNA memory strand can be read so that reading the bases and their complementary strands can offset the error rate of reading each base only once. In certain embodiments, the fidelity of nanopore sequencing is at each end of a single-stranded or double-stranded nucleic acid molecule with bulky appendages (eg, proteins or solid states) that do not translocate across the pores. It can be increased by appropriate modifications of 5'and 3'). Molecules can then be trapped within the pores and translocated multiple times forward and backward to allow multiple reads of the same molecule in the same pore, thus reading the sequencing error rate. Can be reduced by the square of the number of (if the sequencing read error is due to a stochastic origin).

Transferases such as TdT can be used to add large, bulky modified nucleotide analogs to the 3'end of the DNA molecule. In certain embodiments, WORM nanopore memory devices can be generated using the following steps: (1) A single piece of DNA that encodes specific information in any high density encoding scheme as discussed above. Steps of covalently modifying the 5'end with a bulky molecular construct that produces a molecule and prevents complete translocation of the DNA molecule via the nanopore; (2) 5'-the modified end is with the nanopore. Steps of contacting and passing DNA molecules through the nanopores to the point where no further translocation is possible; (3) DNA to effectively trap the molecules within the torus of the nanopores using TdT and modified nucleotides. The step of covalently adding one (or more) bulky nucleotide analogs (“stoppers”) to the 3'end of a molecule; (4) 3'-to remove any unmodified DNA molecule. The step of reversing the polarity of the current to the nanopore and thus creating a pure population of "trapped" (5'-and 3'-modified) nucleic acid chains; (5) any untrapped nucleic acid. Is removed from the vicinity of the nanopore via washing or other means; (6) Read the "trapped" DNA strand in either or both directions using the applied voltage. Steps (potentially multiple read steps to reduce the error rate to an acceptable level). In various embodiments, step 6 involves voltage-induced "reading" in one direction and rapid translocation in the opposite direction to "rewind" the data-encoding nucleic acid via the nanopores and subsequent elements. Can consist of another voltage-induced "read" in the direction of. This cycle of "reading"-"rewinding"-"reading" can be repeated as many times as desired.

In some embodiments, the trapped nucleic acid strand can be read during translocation in either direction. In some embodiments, the trapped strand can be translocated to one end of the molecule (either 5'or 3') and read in the opposite direction, so that the reading polarity is higher. Can provide accurate readings.

In certain embodiments, the cyclized nucleic acid memory strand can be generated using the synthetic method described above followed by cyclization. The cyclized chain may comprise a bulky polymer or a particular homopolymer sequence, where the ends of the synthesized chain are joined to specify a start and stop point for data reading. It is a thing. Starting and stopping homopolymer sequences can also be used in linear nucleic acid chains. The cyclic chain 305 has 2 so that the cyclic chain 305 is physically trapped between two nanopores (306 and 309) located on a single membrane 303, as shown in FIG. It may pass between two adjacent nanopores (306 and 309). The circularized memory chain can encode digital information as either a single nucleotide sequence, a homopolymeric tract sequence, a modified nucleotide analog sequence, or a combination of some of them. One nanopore 309 can be used to generate an electrical signal when the information encoded memory strand is translocated through the pore, while the other nanopore 307 is the cis side of the membrane 303 and the first. It can simply function as a portal to allow DNA molecules to return to the nanopores 309. One advantage of this scheme is that the information encoding strand can be recycled for repeated reads and read multiple times, thus reducing any possible read errors.

Other embodiments may encode the data in a chain of memory so that the data can only be accessed under a particular set of conditions. In such cases, the memory chain is at least partially composed of modified nucleotides attached to a cleavable linker. Modifications (eg, chemical protecting groups) and linkers can be selected such that current interruptions differ from the sequences that encode the data when the polymer tract translocates through the nanopores without proper treatment. FIG. 7 outlines a scheme that uses disulfide and amide ligated modifications to dG nucleotides to show how current interruptions and how the data encoded in the memory chain can change in response to processing conditions. Illustrate. G ^* and G ^** are structurally similar in size and flexibility and can result in similar current interruptions on the nanopore platform, but are eliminated under different conditions. Although G ^* and G ^*** are structurally different, these modifications share the same removal conditions. Other embodiments may use other modifications or linkers that can be cleaved using different treatments such as light of a particular wavelength, acidic or alkaline pH, oxidative or reducing conditions, or sequence-specific nucleases. Some embodiments may use the presence or absence of a memory chain modification for encryption or as a chemical marker of previous access or modification of data. Most linker cleavage reactions are effectively irreversible, so this approach can be sufficient for a single molecule to encode data without redundancy write-one read many. Best suited for the system.

Many possible information encoding schemes useful in the readout schemes of the present invention are possible and may be apparent to those skilled in the art based on the present disclosure.

Synthesis can be accomplished using acoustic delivery of drops into the wells of the plate (eg, 1.5 μL each of the 1536 well plates). In various embodiments, the nucleic acid memory chain can be synthesized on the beads or magnetic beads or the surface and, depending on the application, can be left on the beads or the magnetic beads or the surface after the full-length synthesis is complete, or synthesized. It can be removed from the support.

In certain embodiments, the system for the synthesis of long (5-10 kb) data chains may use inkjet delivery into an array of wells (eg, wells of multiple nanoliter volumes). In other embodiments, a plurality of pneumatically controlled actuators may be positioned above each well to simultaneously deliver reagents to each location in the array. Each actuator is used to identify a bit of a DNA data strand, a selector valve that selects between each of two or more nucleotides or modified nucleotides formulated with a template-independent polymerase. Served by. One or more additional selector valve ports are dedicated to one or more cleaning reagents as needed. An array of nanoliter volume wells can be open at both ends as long as the diameter of the wells is such that the delivered liquid is trapped by capillary action within the length of the open end wells. After each round of nucleotide-enzyme formulation has been delivered to the open end wells, each well is rinsed with the reaction mixture, thus preparing the array for the next cycle of enzymatic synthesis. A rinsing reagent or enzyme reaction termination reagent can be flushed across and through the lower opening of the array of wells. In other embodiments, a vacuum source is used to rapidly remove one reagent from the capillary nanowell prior to delivery of the next reagent.

Certain embodiments may use highly parallel nanofluid chambers with valve controlled reagent delivery. An exemplary microfluidic nucleic acid memory chain synthesis device is shown in FIG. 8 for exemplary purposes, but not at a constant scaling ratio. The microfluidic channel 255 containing the regulator 257 couples the reservoir 253 into the reaction chamber 251 and the outlet channel 259 containing the regulator 257 removes waste from the reaction chamber 251. Microfluidic devices for nucleic acid memory chain synthesis may include, for example, channel 255, reservoir 253 and / or regulator 257. Nucleic acid memory chain synthesis may include beads or other substrates anchored or attached to the inner surface of the reaction chamber, capable of releasing the polynucleotide initiator, if desired. It can occur in a microfluidic reaction chamber 251 that may include an anchored synthesized nucleotide initiator. The reaction chamber 251 may include at least one uptake channel and one outlet channel 259 so that reagents can be added to and removed from the reaction chamber 254. The reaction chamber 251 must be temperature controlled to maintain optimal and reproducible enzymatic synthetic conditions. The microfluidic device may include a reservoir 253 for each respective dNTP or analog used in the memory chain coding scheme. Each of these reservoirs 253 may also contain an appropriate amount of TdT or any other enzyme that extends DNA or RNA strands in a template-independent manner. The additional reservoir 253 may include reagents for washing or other tasks.

The reservoir 253 may be coupled to the reaction chamber 254 via a separate channel 255, and the flow of reagents through each channel 255 into the reaction chamber 254 uses the use of gates, valves, pressure regulators or other means. Can be individually adjusted through. The flow from the reaction chamber 254 through the outlet channel 259 can be regulated as well. The reservoir 253 may be a dNTP suspended in a fluid at a known concentration, a modified dNTP or the above, so that the concentration of the reagent can be tightly controlled based on the volume of the reagent flowing into the reaction chamber 254. It can hold any of those analogs. Therefore, the length of each homopolymer tract can be controlled through control of reagent concentration.

In certain examples, reagents, especially dNTPs and enzyme reagents, can be recycled. Reagents are introduced from the reaction chamber 254 into their respective reservoirs 253 via the same channel 255 they entered by inducing regurgitation using gates, valves, vacuum pumps, pressure regulators or other regulators 257. Can be pulled back. Alternatively, the reagents can be returned from the reaction chamber 254 to their respective reservoirs 253 via independent return channels. The microfluidic device may include a controller capable of operating the gates, valves, pressures or other regulators 257 described above.

An exemplary microfluidic nucleic acid memory chain synthesis reaction involves the desired dNTP (in the nucleic acid memory chain of the invention) prior to removing the NTP reagent from the reaction chamber 254 via the exit channel 259 or the return channel (not shown). Reagents (used everywhere to refer to any component molecule used to encode data) at a given concentration (calculated to produce the desired homopolymer length) for a given amount of time. The step of flowing the cleaning reagent into the reaction chamber 254; the step of removing the cleaning reagent from the reaction chamber 254 via the outlet channel 259; calculated to achieve the desired homopolymer tract ratio. It may include the step of running the next NTP reagent through the desired memory chain sequence under the above conditions; and the step of repeating until the desired nucleic acid memory chain is synthesized. After the desired nucleic acid memory chain has been synthesized, it can be released from the reaction chamber anchor or substrate and collected via exit channel 259 or other means.

Due to the significant number of homopolymer-encoded DNA strands required to encode the amount of data available, highly parallel methods of DNA synthesis are required. In some embodiments, as shown in FIG. 9, the flow cell (9-1) containing the array of wells forms a plurality of hydrophilic wells (?) Bounded by hydrophobic regions. It is formed on a suitable substrate by patterning horizontal and vertical stripes (9-2) of the hydrophobic material. Typical dimensions of hydrophilic wells can be 300 x 300 nm to 1000 x 1000 nm. This hydrophilic array forms the floor of the flow cell with a properly sized gap between the floor and the optically permeable cover. A solution of cold (ie, below optimal enzyme-specific temperature) nucleotidyltransferase, one of the natural or modified nucleotide triphosphates, and any required cofactor, upon cessation of fluid flow, The flow cell via the inlet (9-3) so that the enzyme-nucleotide triphosphate solution beaded into spatially defined droplets (9-4) located above each hydrophilic region. Shed inside. The IR source (9-5) projects a beam (9-7) onto a DLP (digital light projection) device (9-8) via a molded lens (9-6), which is specific. Used to simultaneously direct the IR beam (9-9) to each of the hydrophobically bound droplets (9-4) selected to which the nucleotides are added, homopolymers of the desired length. Rapid heating of the polymerase extension reaction formulation to a temperature for maximum enzymatic activity over a defined period of time for synthesis occurs. After some well-defined reaction time, the IR source is switched off and cold rinse buffer is rapidly injected into the flow cell and drained through the outlet (9-10), so that The reaction is quenched to end one "write" cycle. This sequence of steps is performed multiple times for each "write" cycle so that each nascent data strand is randomly accessed according to its spatial position and the selected nucleotides are added until the full length homopolymer data strand is complete. Repeated. In some embodiments, a DLP device with a 1920x1080 directional mirror can be used to simultaneously randomly access approximately 2M synthetic positions in a synthetic flow cell. In some embodiments, the template-independent polymerase used is thermophilic at an optimum reaction temperature well above room temperature, so that the enzymatic activity is the rapid heating of the droplets by the IR source. Previously, it is minimized in hydrophobically bound droplets. In some embodiments, the bottom surface of the flow cell, which has a hydrophobically defined hydrophilic well, is a hydrophobic well at a reduced temperature to prevent enzymatic activity until the temperature is raised by the IR source. It borders on a cooling device that keeps the droplets inside.

In another embodiment, an array of wells formed on a suitable substrate by patterning horizontal and vertical stripes of hydrophobic material forming multiple hydrophilic spots bounded by hydrophobic regions. Flow cell composed of. Typical dimensions of hydrophilic spots can be 300 x 300 nm to 1000 x 1000 nm. Each hydrophilic spot is located on an individually addressable CMOS heater. This hydrophilic-CMOS heater array forms the floor of the flow cell with a properly dimensional gap between the floor and the optically permeable cover. A solution of cold (ie, below optimal enzyme-specific temperature) nucleotidyltransferase, and one of the natural or modified nucleotide triphosphates is associated with the polymerase extension reaction solution during fluid flow arrest. It is flushed into the flow cell in a beaded manner into spatially defined droplets located above each hydrophilic region having a CMOS heater. Each of the hydrophobically bound droplets selected to which a particular nucleotide is added is up to the temperature for maximum enzymatic activity over a defined period of time to synthesize homopolymers of the desired length. , Heated quickly. After some well-defined reaction time, the heater is switched off and cold rinse buffer is rapidly injected into the flow cell, thus quenching the reaction and one "write" cycle. To end. This sequence of steps is performed multiple times for each "write" cycle so that each nascent data strand is randomly accessed according to its spatial position and the selected nucleotides are added until the full length homopolymer data strand is complete. Repeated. In some embodiments, the enzyme used is thermophilic at an optimum temperature well above room temperature, resulting in hydrophobic binding prior to rapid heating of the droplets by a CMOS heater. The likelihood of unwanted nucleotide additions in the droplet is low.

Those skilled in the art recognize that the systems and methods of the invention are necessary or best suited, but the systems and methods of the invention are one or more processors 309 that communicate with each other via a bus ( A computing as shown in FIG. 10, which may include, for example, a central processing unit (CPU), a graphics processing unit (GPU), etc.), a computer-readable storage device 307 (eg, main memory, static memory, etc.), or a combination thereof. It may include a wing device. Computing devices may include mobile devices 101 (eg, mobile phones), personal computers 901 and server computers 511. In various embodiments, the computing devices may be configured to communicate with each other via network 517.

Computing devices synthesize memory chains, read sequenced memory chains, and sequence data between human or machine-readable formats, digitized data, and nucleic acid sequences between other steps described herein. Can be used to control the compilation or conversion of. Computing devices can be used to display data in readable format.

The processor 309 may be any suitable processor known in the art, such as a processor sold under the trademark Xeon E7 by Intel (Santa Clara, CA), or the trademark OPTERON 6200 by AMD (Sunnyvale, CA). Processors sold below may be included.

The memory 307 preferably includes at least one tangible non-transient medium capable of storing: one or more executables for causing the system to perform the functions described herein. A set of instructions (eg, software that embodies any methodology or function found herein); data (eg, data encoded in a memory chain); or both. A computer-readable storage device can be a single medium in an exemplary embodiment, but the term "computer-readable storage device" is a single medium or multiple media (eg, centralized) for storing instructions or data. Or it should be interpreted as including a distributed database and / or associated caches and servers). The term "computer-readable storage device" means, without limitation, solid state memory (eg, subscriber identification module (SIM) card, secure digital card (SD card), micro SD card or solid state drive (SSD)), optical and It shall be construed as appropriate to include magnetic media, hard drives, disk drives, and any other tangible storage medium.

Any suitable service, such as Amazon Web Services, memory 307 of server 511, cloud storage, another server, or other computer-readable storage may be used for storage 527. Cloud storage can refer to a data storage scheme, where data can be stored in a logical pool and physical storage can span multiple servers and locations. Storage 527 may be owned and managed by the hosting company. Preferably, storage 527 is used to store records 399 to perform and support the operations described herein, as needed.

Input / output devices 305 according to the present invention include video display units (eg, liquid crystal display (LCD) or cathode line tube (CRT) monitors), alphanumeric input devices (eg, keyboards), cursor control devices (eg, mice or tracks). A network that can be a pad), a disk drive unit, a signal generation device (eg, a speaker), a touch screen, a button, an accelerometer, a microphone, a cellular radio frequency antenna, such as a network interface card (NIC), a Wi-Fi card or a cellular modem. It may include one or more of the interface devices, or any combination thereof.

Those skilled in the art will recognize that any suitable development environment or programming language can be used to enable the operability described herein for the various systems and methods of the invention. For example, the systems and methods herein are Perl, Python, C ++, C #, Java®, JavaScript®, Visual Basic, Ruby on Rails, Groovy and Grails, or any other suitable. Can be implemented using tools. For the computing device 101, it may be preferable to use native xCode or Android Java®.

FIG. 11 shows polyacrylamide gel electrophoresis analysis of two different single homopolymeric tracts made via enzymatic synthesis. Lane A is a sample of 20 mer starting oligonucleotides used in all of the following lanes. Lane B is a sample from a TdT reaction containing a 20-mer oligonucleotide and a lossy terminator ddATP showing 21-mer formation. Lane C is a sample from a TdT reaction containing 20 mars and the natural nucleotide dATP after 1 minute at 37 ° C. Lane D is a sample of the same reaction mixture in Lane D after 5 minutes at 37 ° C. Lane E is a sample of the same reaction mixture in Lane C after 15 minutes at 37 ° C. These three lanes show homopolymer length control due to consumption of input dATP at about 5 minutes during the TdT elongation reaction, and no homopolymer growth observed between 5 and 15 minutes. Lane F is a sample from the TdT reaction containing the 20-mer oligonucleotide and the nucleotide analog N6-benzoyl-dATP after 1 minute at 37 ° C. Lane G is a sample of the reaction mixture in Lane F after 5 minutes at 37 ° C. Lane H is a sample of the same reaction mixture in Lane F after 15 minutes at 37 ° C. There are qualitative differences between the lengths of the dA ^Bz homopolymers formed in lanes F to H, but the same length control has also been demonstrated using N6-modified dATP analogs. There is.

Writing digital data into a molecular storage format based on a molecular approach offers advantages over currently used storage media such as tapes or discs. DNA-based memory has sparked interest due to its high achievable information density, extremely long lifespan, and low energy consumption during rest periods. To date, most attempts to use synthetic DNA as a storage medium involve chemical synthesis using common phosphoramidite methods.

DNA-based data storage can require a much larger number of strands than are currently synthesized for existing research markets. Any synthetic technique (chemical or enzymatic) that relies on nucleotide blockade or removal of the terminator imposes additional steps and complexity, in order for the reagent to orient the correct nucleotide in the correct position on the array. This is because they need to be delivered to an array of synthetic features (ie, wells or spots) in an addressable manner. Array-based methods of synthesis can be carried out in one of several methods: 1) bulk delivery of the activated reactants followed by selective removal of blocking groups, or 2) activity. Addressable delivery of the transformed reactants and subsequent bulk blocking group removal, or 3) bulk delivery of the Inactive reactants and subsequent addressable activation. Addressable delivery of reagents to large ( ^{104-10 6 )} 2-D arrays is generally achieved using inkjet deposition at each desired location. If the data encoding scheme uses 4 nucleotides, 4 separate writeheads are used and must be indexed in a complex XY mechanical manner. In addition, the use of inkjet delivery limits the dimensions between each spot (well or spot) to as low as tens of microns. The challenge in this process is to reduce the step time for each synthetic cycle, no matter how it is achieved.

In certain embodiments, the systems and methods of the invention deliver the Inactive Reaction Mixture to all features on the 2-D array, followed by features requiring the addition of A or G or C or T. It may include the step of selectively activating only. Addressable methods of mechanical operation-independent delivery, activation or blockade removal are preferred. A preferred embodiment uses bulk delivery of the reactants and selective activation of specific synthetic features by removal of blocking groups from nucleotide analogs, which is then rapidly mediated by DNA polymerase as shown in FIG. It allows for sex uptake and the formation of homopolymer bits, thus allowing highly parallel and rapid synthesis of homopolymer-encoded nucleic acid memory chains. Homopolymer-encoded nucleic acid information Polymers have several other advantages: 1) Homopolymer-encoded bits overcome error profiles associated with next-generation sequencing 2) Obtained polymers Is a non-natural nucleic acid and therefore cannot be diverted to bioterrorist activity.

Delivery and selective activation of template-independent polymerase DNA synthesis sequentially (delivering A-> activating and initiating homopolymer synthesis addressably-> washing; delivering C-> homopolymer synthesis. Addressably activate and initiate-> wash; deliver G-> addressably activate and initiate homopolymer synthesis-> wash; deliver T-> address homopolymer synthesis Activate and initiate-> wash) or in parallel (deliver all four nucleotides to all features at the same time-> activate A, C, G, T in an addressable manner simultaneously or sequentially- > Wash), can be carried out. In this method, the synthetic cycle becomes very efficient and involves only three steps: reaction delivery, uptake reaction activation, and then washing before the start of the next cycle. The reaction is stopped by rapid removal of the reactants with a gas or liquid, or by rapid delivery of the quenching reagent. In a preferred embodiment, the quenching reagent is a metal chelator.

A preferred design for equipment that can be used to synthesize homopolymer bit-encoded memory chains is two of the hydrophobically patterned wells that are appropriately modified to support template-independent enzymatic synthesis. -Consists of a flow cell composed of a D array. After delivering the reactants to the 2-D array of hydrophobic wells, the liquid beals and defines spatially separate reaction zones as shown in FIG.

A preferred embodiment is a covalently bound oligonucleotide initiator with a 5'-> 3'orientation in which the bottom surface of the well, whose four sides are surrounded by hydrophobic patterning, is bound to the bottom surface of the well with a 5'end. It is an embodiment modified by. In another embodiment, the wells are physically formed by etching depressions on the bottom surface of the flow cell, where the covalent oligonucleotide initiator is covalently bonded to the surface (bottom and sides) of the wells. Will be done. In some cases, the wells are open at the bottom to allow the flow of liquid through the wells. In other cases, the wells are closed at the bottom.

Nucleotide analogs protected in 3'-OH are generally inactive with commercially available or WT TdT enzymes (US Pat. No. 10,059,929). Thus, a mixture of 3'-blocked dNTP analogs, TdT proteins and suitable cofactors can be mixed together in the presence of initiator oligonucleotides at 37 ° C. with little to no homopolymerization. .. When 3'-OH is "decagged" (ie, unblocked) by removal of protecting or blocking groups, the resulting nucleotides are of free running incorporation and homopolymerization. Is available for. Decaging that can be achieved by the "no delivery" method is preferred. Analogs constructed to allow addressable 3'-OH decay by the administration of or exposure to activation energies such as light, heat, pH changes, or reducing agents are preferred. Each of these decaging or non-blocking reactions includes mechanically directional light through a light transmissive cover (eg, FIG. 17), individually addressable heaters (eg, FIG. 18), electrochemically. It can be achieved in a well-constructed flow cell using either the induced pH change or the generation of reducing conditions.

Each of the illustrated flow cell designs is compatible with the method of addressable decaying of nucleotides contained in the droplets bound by hydrophobic patterning around them. FIG. 19 illustrates an apparatus that can be used with a flow cell designed for the use of photodecayed nucleotide analogs. Other embodiments and configurations known to those of skill in the art are possible for this purpose.

Each decugging mechanism requires a nucleotide analog specifically designed for the physicochemical process selected for use in the system:

In some embodiments, a suitable nucleotide analog for photomediated decay is 3'-O- (2-nitro-benzyl) -dNTP. In some embodiments, a suitable nucleotide analog for heat-mediated decaying is 3'-O- (tetrahydrofuranyl) -dNTP. In some embodiments, a suitable nucleotide analog for reduction-mediated decaying is 3'-O-methyl-dithiomethyl-dNTP. As long as the resulting 3'-OH modified nucleotide analog is not a substrate for a template-independent polymerase and is easily removed by a "delivery-free" method, eg, a photo, thermal or electrochemically produced reactant. , Many other 3'-OH protecting groups are suitable. As long as the 3'-O modifications described herein are explicitly stated to be substrates for template-independent polymerases and are referred to as reversible terminators, the composition of these dNTP analogs is described in WO 2016/034807. Different from what is done. The subject of the present invention is not an explicit polymerase substrate, but a 3'-O-blocking group that functions to cage dNTPs until they are removed and allow the polymerization to proceed. Mathews AS et al (2016) described the use of 3'-O- (2-nitrobenzyl) -dNTP analogs as reversible terminators for the controlled enzymatic synthesis of natural non-homopolymer oligonucleotides. ing. They explicitly teach the use of such nucleotides as substrates for DNA synthesis by using extremely long (about 1 hour) enzymatic reaction times, as well as enzymatic polymerization of multiple nucleotides. In contrast to the subject matter of this patent, which teaches the use of these analogs as caging groups to initiate, they explicitly teach their use as reversible terminators. Initiation of free-continuation homopolymer synthesis can be achieved by methods other than caged dNTP analogs. Some embodiments may use a fluid pulse of the polymerase to start and stop ssDNA synthesis, as described in Church 9,928,869 (2018). In Reza et al WO2017 / 196783, template-dependent enzymatic synthesis is initiated by the activation of polymerase by electrochemically generated pH changes. Although modified nucleotide analogs containing 3'-O-reversible terminators have been described, neither patent teaches the activation of homopolymer synthesis using the decaging of caged dNTP analogs. Church 9,928,869 (2018) describes free-continuation homopolymer synthesis using natural dNTPs, in which the length of homopolymers formed is controlled by reaction duration. Lee HR et al (2018) demonstrated the length of homopolymerization by depositing natural nucleotides in multiple reaction zones on a 2D array and aggressively disrupting unreacted unmodified dNTPs with apillases. Describes the use of mechanical delivery to control. In some embodiments of the subject matter of this patent, homopolymer rate-regulated modified dNTP analogs with unmodified 3'-OH are fluid pulsed or mechanically XY delivered or pH activated template-independent. Used in combination with any of the DNA polymerases.

20 and 21 show the dynamics of photomediated decaying. The 3'-caged dNTPs in the template-independent polymerase reaction mixture are polymerized into nascent homopolymer chains when converted to the polymerase substrate. Since the available uncaged dNTPs are easily incorporated by the polymerase, complete conversion of the caged dNTPs to uncaged (substrate) dNTPs is not required. Once decaged, the length of the desired homopolymer is controlled by controlling the concentration of decaged dNTPs produced and / or the time of the enzymatically mediated uptake reaction.

FIG. 22 shows an example of two nucleotide analogs caged with a 3'-O modification that can be decaged with visible wavelength light and exhibits adjustable photophysical properties (Peterson J.A., et al J). Amer. Chem. Soc. 2018 140: 7343-6), which allows the simultaneous introduction, decugging and synthesis of two different homopolymer chain sequences. In some embodiments, nucleotide analogs modified with two different 3'-caging species that can be decaged by two different wavelengths of light are delivered simultaneously to the 2D array at the synthetic position. In another embodiment, four dNTP analogs modified with four different photoremovable 3'-caging species are simultaneously delivered to the 2D array at the synthetic position and decaged with four different wavelengths of light. The decagging reaction can be carried out sequentially for one set of positions on the 2D array, followed by delivery of the two remaining dNTPs and subsequent sequential decugging, thus the length of all information polymers. Complete one round of increasing the number by one nucleotide. In an alternative embodiment, four dNTP analogs modified with four different 3'-caging species can be delivered simultaneously by simultaneous exposure of each synthetic feature to one of four different wavelengths of light. Can be decayed. Hardware configurations, such as those shown in FIG. 19, direct two or more wavelength-specific light sources, and one of two or more light sources on a 2D light orientation system. Can be used for multicolor decay by incorporating a suitable shutter mechanism for.

FIG. 23 is a 12-bit consisting of a continuous cycle of homopolymer synthesis using dCTP, dGTP, dTTP incorporating UV photodecagged 3'-oNBn-dATP in cycles # 1, 6, 8, 10, 12 The synthesis of the information chain of is shown. The uptake reaction can be terminated by several methods, including but not limited to the rapid removal of enzymatic reaction components by the introduction of either a gaseous or liquid bolus. In some embodiments, the liquid simply rinses away the reaction components, but in other embodiments, the rinse liquid contains an active quenching agent such as EDTA or other enzyme inhibitor.

In another embodiment, the method of caged dNTPs can be mediated by a steric mechanism involving modification to nucleotide bases instead of 3'-OH. In a preferred embodiment, the nucleotide with unmodified 3'-OH is a removable steric blocking group (uptake blocking factor) that cages dNTPs at N6, N4, N2, O4 of A, C, G or T, respectively. Qualified by. Homopolymer synthesis is carried out by either photo-mediated, heat-mediated or reduction-mediated cleavage of the stereoblocking group, and thus by "uncaging" of natural nucleotides suitable for free-continuation homopolymer synthesis. Can be started.

In some embodiments, the modified nucleotide is composed of a linker containing one moiety that keeps the dNTP caged until removed, thus making it incapable of uptake, and the other portion is composed of multiple homopolymers. It remains covalent throughout tract synthesis. In some embodiments, the purine or pyrimidine base is modified at two positions, one modification cages the nucleotide and prevents enzymatic uptake, while the other modification is a covalent bond throughout polymer synthesis. Remains; each modification can be removed by a different mechanism that allows for each selective removal. In some embodiments, the purine or pyrimidine base is modified at two positions, and each modification can be removed by a different mechanism that allows for selective removal of each.

Depending on the sequence detection modality used to read the homopolymer bit data strand, a suitable dNTP analog modification that yields nucleotides without or with scratches can be designed. For synthetic sequencing (SBS) -dependent readout methods, all modifications to purine or pyrimidine must be removed after homopolymer synthesis but prior to SBS readout. Modifications to purines and pyrimidines that are readily distinguishable from each other are desired for current modulation-dependent readout methods between polymer translocations via one or more nanopores. In some embodiments, modifications are made to purine and pyrimidine bases that regulate polymerase kinetics during homopolymer synthesis. These "velocity control" modifications can also act as current modulators for nanopore detection or can be removed for SBS detection.

Regardless of the mechanism of activation, it is important to control the length of free-continuing homopolymer synthesis. Ideally, homopolymers of 2-4 nucleotides are desirable. In the presence of native nucleotides, TdT tends to form homopolymers based on the nucleobases corresponding to Km (A> T> G> C) of the individual nucleotides. As pointed out by Lee HR et al (2018), it is difficult to limit homopolymer growth without increasing the frequency of deletion. The above authors report a deletion error of approximately 66% in an attempt to limit homopolymer growth to 2-3 nucleotides. TdT has been reported to operate in a dispersive manner, resulting in a Poisson distribution of homopolymer length in free-continuation synthesis, but in practice, in the absence of long enzymatic elongation reaction times, during homopolymer synthesis. It is difficult to drive the complete conversion of initiator nucleic acids. Free-continuing homopolymer synthesis reaction times that are too short result in bit deletion errors, as reported above. Too long homopolymer synthesis reaction times result in overly long homopolymer formation and inefficient data density. One solution is to use modified nucleotide analogs that regulate the rate of multiple base uptake but do not require removal at every step, thus resulting in complete conversion and formation of the starting nucleic acid. Allows control of the length of homopolymers and maintains the simple two-step cycle that is the subject of this patent. Ideally, the homopolymer tract in the information polymer is greater than 2 nucleotides but has a nucleotide length of 4 or less.

If desired, homopolymer length limiting modifications can be removed from all nucleotides at the end of synthesis, thus producing a natural DNA molecule suitable for SBS detection. Modifications that are chemically compatible with the decaging conditions described above are most desirable; they must be removable by chemical conditions that are orthogonal to those used for decaging. In some embodiments, 3'-OH is caged by 3'-O- (2-nitrobenzyl), but N6 of dA, N4 of dC, N2 of dG and O4 or N3 of dT are free. It is modified with a non-terminating moiety that regulates multiple additions during continuous enzymatic homopolymer synthesis. FIG. 24 shows a comparison of free-continuation homopolymer synthesis in the presence and absence of rate-regulating modifications. In some embodiments, the homopolymer synthesis rate-regulating modifications are the same for all four nucleotide analogs, but in other embodiments, the modifications are different for each of the dATP, dCTP, dGTP and dTTP analogs. In some embodiments, the rate limiting modification remains in that position during total synthesis.

Figures 25 and 26 show the results of two consecutive addition cycles of two differently modified nucleotide analogs. In another embodiment, the nascent information polymer is exposed to periodic or alternating cycles with chemical conditions that result in partial or complete removal of homopolymer rate limiting modifications. In some embodiments of the use of class 2 dNTP analogs, previously incorporated rate-regulating modifications are removed at the same time as polymerase extension by including a mild reducing agent in the alternating cycle of the enzymatic extension reaction mixture. Will be done.

If a non-SBS method of readout (ie, nanopore) is used, the homopolymer rate-regulating modification can be covalently bonded using a non-cleavable link and can be left in that position during the readout. In some embodiments, homopolymer rate-regulating modifications can also be used to encode information during the readout process. In a preferred embodiment, the homopolymer synthesis rate-regulating analog is also designed to regulate the current between nanopore translocations, and also provides two or more detectable and identifiable current interruption levels. Is selected to do. In another embodiment, only a single type of nucleotide (ie, A or C or G or T) is used in information chain synthesis, and homopolymer bits are two or more differential currents. Encoded by blockade generative modification.

Figures 27-30 show examples of detection by solid-state nanopores of enzymatically synthesized homopolymers of peptide-modified dNTP analogs. Using a dTTP analog (N-Ac-CYPEE) modified by a cleavable disulfide ligation to a 5-amino acid peptide, a fully modified molecule is produced via free-continuation homopolymer synthesis longer than 100 nt in length. did. Translocation via a 30 nm thick nanopore of 2D silicon nitride at 500 mV resulted in a large signal runout compared to unmodified dU homopolymers (courtesy of Goeppert LLC, Philadelphia, PA).

The table below lists six different classes of modified dNTP analogs that are useful for three different "no delivery" homopolymer synthesis activation approaches and two different homopolymer-encoded nucleic acid memory chain readout techniques. Describe.

Examples of the four photomediated decagging nucleotide analogs that make up Class I are shown below:

This class of analogs represents a 3'-O-caged dNTP that becomes a natural unmodified dNTP during photomediated decaying. Once decaged, the rate of homopolymer formation does not differ from that of native nucleotides, and the length of homopolymers formed needs to be regulated by additives to the template-independent polymerase reaction formulation. .. In some embodiments, tetrahydropyranyl, 4-methoxy-tetrahydropyranyl, tetrahydrofuranyl, acetyl, methoxyacetyl or phenoxyacetyl modifications are due to the heat-induced decaying of the 3'-O blocked dNTP analog. It is useful. In some embodiments, 3'-O-analogs such as -O-CH ₂ -SSR are useful for electrochemically mediated decaging by reducing conditions. Class I dNTP analogs are characterized by nucleotides that give rise to unmodified homopolymers suitable for retrieval by either classical synthetic sequencing or ratchet-style nanopores. In both cases, accuracy in homopolymer length is not required, but accurate detection of transitions between homopolymers is required.

Examples of four Class II photomediated decaging nucleotide analogs with orthogonally removable peptide rate-regulating modifications are shown below:

Peptide modifications for use with Class II dNTP analogs are SBS-dependent, sensitive enough to detect the transition from one homopolymer type (A, C, G, T) to the next homopolymer type. It is intended to be removed at the end of information polymer synthesis due to subsequent sequencing methods or subsequent queries by translocation via nanopores. Peptides suitable for use in this application slow down the rate of uptake of modified nucleotide analogs to prevent long homopolymer formation prior to complete conversion of unmodified or modified strands of different composition. In some embodiments, the uptake rate regulatory modification can consist of 1, 2, 3 or 4 different peptides for 4 nucleotide analogs. In some embodiments, the peptide is linked to a nucleotide via a disulfide linker that has a self-sacrificing wound that can be cleaved under mild chemical conditions. In some embodiments, the peptides are, but are not limited to, Ac-EECGY, Ac-EEGCGW, Ac-EEGCGGW, Ac-EC-pNA, Ac-CWEE, Ac-CYPEE, Ac-EEGCPPW, Ac-CPYEE, It can consist of Ac-CPWEE and Ac-CWPEE. Many other peptide sequences and compositions are available to those of skill in the art. Peptides with an overall anionic composition appear to be most suitable; peptides with a cationic composition accelerate the rate of nucleotide uptake, as previously pointed out by Finn PS et al 2003. Peptides with covalent linkages to nucleotides other than those via disulfides to cysteine amino acids, as long as the ability to remove them from the nucleotides of the completed homopolymer DNA strands without leaving residual scars is maintained. Is possible. Disulfide cleavage-mediated self-sacrificing linkers, which are eliminated under mild conditions by the formation of thiolactone, are particularly useful.

The following are examples of four Class II nucleotides with photomediated decaying and orthogonally removable non-peptide homopolymer synthesis rate regulatory modifications:

Non-peptide modifications to adenine N6, cytidine N4, guanine N2 and thymine N3 can act as uptake rate modulators. In some embodiments, acetyl, diacetyl, isobutyryl, proprionyl, pivaloyl, benzoyl, cyclohexyl and other organic modifiers are useful. This type of modifier is removed after the synthesis of the information polymer by ammonolysis, but not limited to. Class II dNTP analogs are characterized by nucleotides that give rise to unmodified homopolymers suitable for retrieval by either classical synthetic sequencing or ratchet-style nanopores. In both cases, accuracy in homopolymer length is not required, but accurate detection of transitions between homopolymers is required.

The following are examples of two Class III nucleotide analogs with unmodified 3'-OH and removable uptake caging modifications on purines or pyrimidines:

In some embodiments, useful caging modifications to 2-nitrobenzyl functional groups are bulky modifications such as, but not limited to, peptides, cyclic peptides, PEGs, branched chain PEGs, star PEGs, dendrimers, nanoparticles and the like. Is. Class III dNTP analogs are also characterized by nucleotides that give rise to unmodified homopolymers suitable for either classical synthetic sequencing or ratchet-style nanopore readout. In both cases, accuracy in homopolymer length is not required, but accurate detection of transitions between homopolymers is required.

It has a removable uptake blocking factor at positions other than unmodified 3'-OH and 3'-OH, and an orthogonally removable homopolymer synthesis rate modulator modification, which is also not located at 3'-OH1 Examples of two class IV nucleotide analogs are:

Class IV nucleotide analogs are designed to have one or more uptake blocking modifications that cage the nucleotides from enzymatic uptake until they are removed by light, heat, or electrochemical means. Large bulky modifications that render the nucleotide analog inactive against polymerase uptake are preferred. In some embodiments, one or more modifications are derivatives of 2-nitrobenzyl that can be removed by photolysis. Removal of the caging modification in all cycles leaves an uptake rate-regulating modification that is removed by a chemical method orthogonal to the method used to decatenate the nucleotide analog upon completion of information polymer synthesis. In a preferred embodiment, the uptake blocking modification is covalently linked to an uptake rate regulating modification. In some embodiments, a caging modification to a 2-nitrobenzyl functional group, which is a bulky modification and acts as a steric blocker, is useful. Examples of modifications that can act as steric blockers are, but are not limited to, peptides, proteins, peptoids, PEGs, branched chain PEGs, star PEGs, dendrimers or nanoparticles.

Examples of class V pyrimidine nucleotides with 3'-O-caging modifications and irremovable base modifications are:

If nanopore detection by direct translocation is desired as a readout method, the nucleotide is a break current modulator between a suitable removable 3'-O-caging group and direct translocation via non-indexed nanopores. It is doubly modified with a covalent, irremovable modification that acts as. Suitable modifications can be peptide or non-peptide. The ideal embodiment of a breaking current regulator is the smallest possible modification that produces the most unique signal with the shortest possible homopolymer stretch. An important innovation in this class of dNTP analogs is that only one type of modified nucleotide is required, which is that the homopolymer "sequence" is not the nucleotides themselves, but two or more. This is because it is encoded by an array of current-regulated modifications.

The following are unmodified 3'-OH, removable uptake blocking (caging) modifications, and two or more different types of irremovable current regulation useful for detection by direct nanopore sequencing. An example of a class VI nucleotide analog with modifications. An important innovation in class VI dNTP analogs is that only one type of modified nucleotide is required, which is that the homopolymer "sequence" is not the nucleotide itself, but two or more currents. This is because it is encoded by an array of regulatory modifications. The encoding is not limited to radix 2 or radix 4 but is limited only by the number of current regulation modifications that can be made to a single purine or pyrimidine nucleotide.

In some embodiments, the uptake blocking (caging) modification can be removed by either light, heat or electrochemical means, but two or more nanopore detection elements cannot be removed. Survive repeated exposure to the decugging conditions used in all cycles during homopolymer chain synthesis. Caging modifications to 2-nitrobenzyl that act as steric blockers are bulky modifications such as, but not limited to, peptides, proteins, peptoids, PEGs, branched chain PEGs, star PEGs, dendrimers or nanoparticles.

(Example 1)
N ⁶ -benzoyl-deoxyadenosine triphosphate was prepared by adding N ⁶ -benzoyl-2'-deoxyadenosine (0.055 g, 0.16 mmol) to the vial under dry N ₂ and trimethyl phosphate (0). .435 mL) was added. To the resulting solution was added tributylamine (0.077 mL, 0.32 mmol) and the reaction mixture was flushed with dry N ₂ for 30 minutes while maintaining at −5 ° C. Anhydrous phosphorus oxychloride (0.018 mL, 0.19 mmol) was added to the vial via a syringe and the reaction mixture was stirred at −5 ° C. for 3 minutes. A second aliquot of anhydrous phosphorus oxychloride (0.009 mL, 0.10 mmol) was added via syringe and the reaction mixture was stirred at −5 ° C. for 8 minutes. To the second vial, tributylamine pyrophosphate (0.075 g, 0.14 mmol) was added, dried _N2 was flushed, anhydrous acetonitrile (0.609 mL) was added, followed by tributylamine (0.231 mL, 0). .97 mmol) was added. The prepared tributylamine pyrophosphate mixture was cooled to −20 ° C., added to the reaction mixture and reacted for 10 minutes. The reaction was quenched by the instillation of _H2O (4.35 mL). The contents of the flask were combined with 0.87 mL of H2O and extracted with dichloromethane (3 x 150 mL). The aqueous phase was adjusted to pH 6.5 with concentrated NH ₄ OH and stirred at 4 ° C. for 12 hours. The mixture was transferred to a 250 mL round bottom flask with 50 mL of water and concentrated under reduced pressure. The residue was dissolved in 40 mL water and purified via ion exchange chromatography (AKTA FPLC, Fractogel DEAE 48 mL column volume, gradual gradient 0-> 70% TEAB in water, pH 7.5). Fractions containing the desired product were pooled, concentrated at A260 concentration under reduced pressure, and residual triethylammonium bicarbonate was removed via repeated concentration (5 x 50 mL) from water until drying to remove N. ⁶ -Benzoyl-2'-deoxyadenosine triphosphate was obtained.

Controlled synthesis of homopolymeric tracts composed of modified nucleotides with nucleotidyltransferase TdT was performed in the following manner. Stock solutions of 1 mM deoxyadenosine triphosphate (TriLink Biosciences) and N6 ^- benzoyl-deoxyadenosine triphosphate, respectively, were prepared in _H2O .

Each of 0.5 μL (500 pmol) of different triphosphates, separately 1.5 μL (30 U) of commercially available TdT (Thermo Scientific), 0.5 μL (50 pmol) of 5'-TAATAATAATAATTTTTT-3'( IDT), 2 μL of commercially available TdT Rxn buffer (Thermo Scientific-1M potassium cacodylate, 0.125 M Tris, 0.05% (v / v) Triton X100, 5 mM Co Cl2 (pH 7.2 at 25 _° C.)) And 4.5 μL of H2O. The reaction was incubated at 37 ° C. 30 μL aliquots were removed after 1 minute, 5 minutes and 15 minutes and quenched with 20 μL 5 mM EDTA, respectively. Each sample was dried under vacuum and reconstituted in 100 μL H2O. Each 10 μL time point was mixed with 10 μL of denaturing load buffer (100% formamide and 0.1% Orange G) and applied to wells of a 1 mm × 20 cm × 14 cm 20% polyacrylamide gel. After electrophoresis at 400 V for 3.5 hours, the band was visualized by Thermo Scientific and photographed under UV irradiation (UV blocking Wratten 2A filter 405 nm cutoff, UVP, LLC).

For the synthesis of multiple homopolymeric tracts, the initiator oligonucleotide can be attached to the beads to allow multiple rounds of enzymatic synthesis incorporating the removal of previous reactants and wash liquor. 5'-biotin-TAATAATAATAATTTT-3'(IDT) can be incubated with streptavidin-coated magnetic sepharose microbeads (GE Healthcare Life Sciences). Beads to which oligonucleotides have been added can be prepared by removing the aliquot of the bead slurry and transferring it to a filter cup. The beads can then be washed 5 times with 1 x PBS (using a 2 x volume of bead slurry) vortex and each rinse solution can be spun down. A 1/2 bead slurry volume of 1 × PBS can then be added and the biotinylated oligonucleotide can be spiked with 1/10 of the published bead binding capacity. The mixture can be incubated at 37C for 2 hours, vortexing every 30 minutes. After 2 hours, a small amount of supernatant can be removed and A260 can be measured for any unbound oligonucleotide. The beads can be washed 5 times with MQ water where A260 shows less than 10% of the remaining oligonucleotide. The washed beads can be made to the desired concentration in MQ water.

Homopolymer synthesis can be performed as described above using 2-10 x molar equivalents of the desired dNTPs on the bead-bound oligonucleotide. The reaction mixture containing beads, dNTP, TdT and buffer can be incubated at 37 ° C. for 15 minutes. The reaction can be stopped with 10 μl EDTA and rinsed 3 times with water. A new cycle of homopolymer synthesis can be initiated by adding new TdT enzyme, dNTP, buffer and incubating at 37 ° C. for 15 minutes. After terminating the reaction with EDTA and rinsing with water three times, the cycle of homopolymer synthesis can be repeated as many times as desired. After a final EDTA quench and 3 rinses with water, the support-bound alternating homopolymers can be cleaved from the solid support using 100 μl concentrated ammonium and the supernatant is prepared by Gen-vac. It can be dried and then stored at −20 ° C. until ready for analysis using a polyacrylamide gel as described above.

In another experiment, the two-letter message "MA" was converted to binary and then to a nucleotide code of radix 2, as shown in FIG. Each letter symbol was converted to a corresponding number from 1 to 26 (A → 1, ... Z → 26). Each number was then converted to a 6-bit binary number by adding two zeros to the usual binary representation from 1 to 26 (eg, "M" = 00110; "A" = 000001). Each bit in binary notation was converted to a nucleotide representation of radix 2 according to the table below:

Thus, "MA" was converted to 01101 000001, then to the single nucleotide string ACTGAGACACAG, which was converted to _An C _n T _n G _n A _n G _n A _n C _{n A n A.} Synthesized as _n G _n , where each nucleotide is synthesized as a variable length homopolymer.

Synthesis of 12-bit homopolymer-encoded nucleic acids with 5'-biotinylated 39 nt-length oligonucleotide initiators bound to 34 um streptavidin-Sepharose beads (GE Healthcare) at approximately 20 pmol / ul beads: 5 It was carried out using'Biotin-CAGGTCCTAUC GATATC TGTGAGCTTAATGTCCTTATGT-3'.

This oligonucleotide contains two features for releasing the final product from the solid support used during synthesis: 1) a single deoxy that allows cleavage by the USER enzyme system (New England Biolabs). Uridine residues, and 2) Eco RV endonuclease restriction site.

Starting with a bead-bound initiator of about 2 nmol, each variable length homopolymer was enzymatically synthesized using TdT and one of four modified nucleotide triphosphates. Each reaction was incubated at 37 ° C. for 2.5-20 minutes with 40-100 uM modified dNTP (40 uMA; 100 uM-C; 50 uM-T; 100 uM-G), 20 UTdT (Thermo-Fisher Scientific). ), With a total volume of 750 ul containing 1 × TdT buffer (Thermo-Fisher Scientific). After each enzymatic extension step, the reaction was quenched by the addition of 500 ul of 250 mM EDTA in 10 mM Tris buffer (pH 6.8). The beads were collected by centrifugation at 10000 xg and removal of the supernatant. FIG. 13 shows a PAGE analysis of each cycle of enzymatic synthesis of a 12-bit homopolymer data chain. Each lane begins with an "N" indicating an unreacted 39nt initiator and is marked by the number of cycles. Black arrows indicate size markers for 60 nt oligonucleotides.

After removal of the full-length data strand from the solid support, NGS library preparation was performed using ACCEL-NGS® 1S PLUS DNA LIBRARY KIT (Swift Bioscience) according to the manufacturer's instructions, followed by. , Illumina MiSeq System was used for sequencing. 14A-L show a histogram of the observed base composition of each of the 12 nucleotide additions (as labeled) generated during enzymatic synthesis.

ニトロベンジル－シトシン

ニトロベンジル－グアノシン

ニトロベンジル－チミジン

(Example 2)
Procedures for synthesizing class I-purines and pyrimidine dNTP analogs Schemes for synthesizing class I dNTP analogs Nitrobenzyl-adenosine

Nitrobenzyl-cytosine

Nitrobenzyl-guanosine

Nitrobenzyl-thymidine

９－［β－Ｄ－５’－ヒドロキシ－２’－デオキシリボフラノシル］－６－クロロプリン（１．００ｇ、３．６９ｍｍｏｌ）およびイミダゾール（５５４ｍｇ、８．１２ｍｍｏｌ）を、無水ギ酸ジメチル（１８ｍＬ）中に溶解させ、その後、ｔｅｒｔ－ブチルジメチルシリルクロリド（６１１ｍｇ、３．９３ｍｍｏｌ）を添加した。反応混合物を、アルゴン下で室温で２０時間撹拌した。乾燥させた残渣を、シリカ上に含浸させ、フラッシュカラムクロマトグラフィー（ヘキサン／酢酸エチル、２：１）によって精製して、９－［β－Ｄ－５’－Ｏ－（ｔｅｒｔ－ブチルジメチルシリル）－２’－デオキシリボフラノシル］－６－クロロプリンを得た。

(Example 3)
Detailed Procedure for 3'-O-Nitrobenzyl Deoxyadenosine:

9- [β-D-5'-Hydroxy-2'-deoxyribofuranosyl] -6-chloropurine (1.00 g, 3.69 mmol) and imidazole (554 mg, 8.12 mmol) in dimethyl anhydride (18 mL). It was dissolved in and then tert-butyldimethylsilyl chloride (611 mg, 3.93 mmol) was added. The reaction mixture was stirred under argon at room temperature for 20 hours. The dried residue was impregnated on silica and purified by flash column chromatography (hexane / ethyl acetate 2: 1) to 9- [β-D-5'-O- (tert-butyldimethylsilyl). -2'-Deoxyribofuranosyl] -6-chloropurine was obtained.

9- [β-D-5'-O- (tert-butyldimethylsilyl) -2'-deoxyribofuranosyl] -6-chloropurine (1.73 g, 4.48 mmol) in anhydrous dichloromethane (135 mL). Dissolved. Tetrabutylammonium bromide (722 mg, 2.24 mmol), 2-nitrobenzyl bromide (2.41 g, 11.2 mmol) and 40% aqueous sodium hydroxide (65 mL) were added to the prepared solution. The reaction mixture was stirred at room temperature for 1 hour and diluted with ethyl acetate (300 mL). The layers were separated. The aqueous layer was extracted with ethyl acetate (125 mL x 2). The combined organic layers were dried over anhydrous sodium sulfate. The organic layer was impregnated on silica gel and then purified by flash column chromatography (hexane / ethyl acetate 2: 1) to 9- [β-D-5'-O- (tert-butyldimethylsilyl). -3'-O- (2-nitrobenzyl) -2'-deoxyribofuranosyl] -6-chloropurine was obtained.

9- [β-D-5'-O- (tert-butyldimethylsilyl) -3'-O- (2-nitrobenzyl) -2'-deoxyribofuranosyl] -6-chloropurine (2.22 g, 3 .83 mmol) was dissolved in anhydrous tetrahydrofuran (40 mL) and cooled to 0 ° C. Then, 1.0 M tetrabutylammonium fluoride solution (4.20 mL, 4.20 mmol) in tetrahydrofuran was added dropwise. The reaction mixture was stirred at room temperature for 1 hour. After the reaction mixture was dried, the residue was dissolved in dioxane and 7N ammonia (40 mL) in ethanol. The reaction mixture was stirred at 90 ° C. for 18 hours in a sealed round bottom. The reaction mixture was impregnated on silica and purified by flash column chromatography (dichloromethane / methanol, 20: 1) to give 3'-O- (2-nitrobenzyl) -2'-deoxyadenosine.

3'-O- (2-nitrobenzyl) -2'-deoxyadenosine (15 mg, 1 eq, 38 μmol) was co-evaporated with pyridine (1 mL x 3) and dried in high vacuum overnight. It was then dissolved in 1.5 mL of trimethyl phosphate and 0.60 mL of dry pyridine and cooled in an ice bath under argon. A first aliquot of 6 uL of phosphoryl trichloride (18 mg, 11 μL, 3 eq, 0.11 mmol) was added. After 5 minutes, a 5 uL second aliquot was added. The mixture was stirred for an additional 30 minutes. A solution of tetrabutylammonium hydrogen diphosphate (0.14 g, 4 equivalents, 0.15 mmol) in 1.5 mL dry DMF was prepared under Ar and cooled in an ice bath. This was added dropwise to the rxn mixture over 30 seconds. Immediately, pre-weighed N1, N1, N8, N8-tetramethylnaphthalene-1,8-diamine (33 mg, 4 eq, 0.15 mmol) was added at once as a solid. The mixture was stirred for 30 minutes after this addition and quenched with 8 mL cold 0.1 M TEAB buffer. The mixture was stirred in an ice bath for 10 minutes and then transferred to a separatory funnel. The solution was extracted once with 10 mL of EtOAc. The aqueous layer was transferred to a canal for FPLC separation and this separation was performed immediately after Extraction of EtOAc. Final purification was by reverse phase HPLC.

３’，５’－ジ－Ｏ－（ｔｅｒｔ－ブチルジメチルシリル）－２’－デオキシウリジン（１．００ｇ、２．１２ｍｍｏｌ）を、無水アセトニトリル（９０ｍＬ）中に溶解させ、アルゴン下で０℃に冷却した。三塩化ホスホリル（１．４９ｍＬ、２．１２ｍｍｏｌ）を、２分間かけて滴加した。１０分後、トリエチルアミン（１１．１ｍＬ、７９．７ｍｍｏｌ）を、３分間かけて滴加した。１５分後、反応混合物を、室温で２時間撹拌した。反応混合物を、０℃に冷却し、トリアゾール（４．４０ｇ、６３．７ｍｍｏｌ）を、固体として一度に添加した。沈殿物が観察され、懸濁物を３０分間撹拌した。室温で２時間攪拌した後、反応混合物を、乾燥するまで濃縮した。残渣を、ジクロロメタン（３０ｍＬ）中に溶解させ、重炭酸ナトリウムの飽和溶液（２５ｍＬ×２）、ブライン（２５ｍＬ）で洗浄した。有機層を、無水硫酸ナトリウムで乾燥させ、乾燥するまで濃縮した。残渣を、ジクロロメタン中に溶解させ、その後、アリルアルコール（２．００ｍＬ、２９．４ｍｍｏｌ）およびトリエチルアミン（２．６７ｍＬ、１８．９ｍｍｏｌ）を添加した。反応混合物を、０℃で１５分間撹拌した。ＤＢＵ（０．３３ｍＬ、２．１７ｍｍｏｌ）を添加し、室温で６時間撹拌した。反応混合物を、ジクロロメタン（１７ｍＬ）で希釈し、ブライン（１５ｍＬ）で洗浄した。有機層を、無水硫酸ナトリウムで乾燥させた。有機層を、シリカ上に含浸させ、フラッシュカラムクロマトグラフィー（ヘキサン／酢酸エチル、４：１）によって精製して、４－Ｏ－アリル－３’，５’－ジ－Ｏ－（ｔｅｒｔ－ブチルジメチルシリル）－２’－デオキシウリジンを得た。

(Example 4)
Detailed Procedure for 3'-O-Nitrobenzyl Deoxycytidine:

3', 5'-di-O- (tert-butyldimethylsilyl) -2'-deoxyuridine (1.00 g, 2.12 mmol) was dissolved in anhydrous acetonitrile (90 mL) and brought to 0 ° C. under argon. Cooled. Phosphoryl trichloride (1.49 mL, 2.12 mmol) was added dropwise over 2 minutes. After 10 minutes, triethylamine (11.1 mL, 79.7 mmol) was added dropwise over 3 minutes. After 15 minutes, the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was cooled to 0 ° C. and triazole (4.40 g, 63.7 mmol) was added at once as a solid. A precipitate was observed and the suspension was stirred for 30 minutes. After stirring at room temperature for 2 hours, the reaction mixture was concentrated to dryness. The residue was dissolved in dichloromethane (30 mL) and washed with a saturated solution of sodium bicarbonate (25 mL x 2) and brine (25 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated to dryness. The residue was dissolved in dichloromethane and then allyl alcohol (2.00 mL, 29.4 mmol) and triethylamine (2.67 mL, 18.9 mmol) were added. The reaction mixture was stirred at 0 ° C. for 15 minutes. DBU (0.33 mL, 2.17 mmol) was added and the mixture was stirred at room temperature for 6 hours. The reaction mixture was diluted with dichloromethane (17 mL) and washed with brine (15 mL). The organic layer was dried over anhydrous sodium sulfate. The organic layer is impregnated on silica and purified by flash column chromatography (hexane / ethyl acetate, 4: 1) to 4-O-allyl-3', 5'-di-O- (tert-butyldimethyl). Cyril) -2'-deoxyuridine was obtained.

4-O-allyl-3', 5'-di-O- (tert-butyldimethylsilyl) -2'-deoxyuridine (3.37 g, 5.27 mmol) was dissolved in dry tetrahydrofuran (50 mL). .. Triethylamine (1.98 mL, 14.2 mmol) was added, followed by triethylammonium fluoride dihydrofluoride (2.32 mL, 14.2 mmol) under argon. The reaction mixture was stirred at room temperature for 29 hours and then concentrated. The residue was dissolved in dichloromethane (100 mL) and washed with 1.5 M ammonium carbonate (75 mL x 1) and brine (75 mL). The organic layer was dried over anhydrous sodium sulfate, impregnated on silica and purified by flash column chromatography (dichloromethane / methanol, 9: 1) to give 4-O-allyl-2'-deoxyuridine.

4-O-allyl-2'-deoxyuridine (1.18 g, 4.40 mmol) is dissolved in anhydrous pyridine (37 mL), followed by tert-butyldimethylsilyl chloride (815 mg, 5.41 mmol) under argon. Was added. The reaction mixture was stirred at room temperature for 20 hours. After concentration, the residue was dissolved in dichloromethane and impregnated on silica. The crude product was purified by flash column chromatography (dichloromethane / methanol, 9: 1) to give 4-O-allyl-5'-O- (tert-butyldimethylsilyl) -2'-deoxyuridine.

4-O-allyl-5'-O- (tert-butyldimethylsilyl) -2'-deoxyuridine (1.28 g, 3.35 mmol) in dichloromethane / water (20 mL, 1: 1), tetrabutylammonium hydroxy To a mixture of de (1.5 mL, 55-60% in water) and sodium iodide (50.0 mg, 0.335 mmol) was added a 1.0 M sodium hydroxide solution (10 mL) under argon. The reaction mixture was stirred at room temperature for 10 minutes, after which 2-nitrobenzyl bromide (1.45 g, 6.70 mmol) in 10 mL dichloromethane was added over 5 minutes. After stirring at room temperature for 7 hours, the reaction mixture was diluted with dichloromethane (150 mL). The organic layer was washed with brine (20 mL) and dried over anhydrous sodium sulfate. The organic layer is impregnated on silica and purified by flash column chromatography (hexane / ethyl acetate, 1: 1) to 4-O-allyl-5'-O- (tert-butyldimethylsilyl) -3'. -O- (2-nitrobenzyl) -2'-deoxyuridine was obtained.

4-O-allyl-5'-O- (tert-butyldimethylsilyl) -3'-O- (2-nitrobenzyl) -2'-deoxyuridine (1.55, 3.00 mmol) in ethanol at 7 N Was dissolved in ammonia (55 mL) and stirred at 55 ° C. for 20 hours in a sealed round bottom. The reaction mixture is impregnated on silica and purified by flash column chromatography (dichloromethane / methanol, 20: 1) to 5'-O- (tert-butyldimethylsilyl) -3'-O- (2-nitro). Benzyl) -2'-deoxycytidine was obtained.

5'-O- (tert-butyldimethylsilyl) -3'-O- (2-nitrobenzyl) -2'-deoxycytidine (2.22 g, 3.83 mmol) was dissolved in anhydrous tetrahydrofuran (40 mL). , Cooled to 0 ° C. Then, 1.0 M tetrabutylammonium fluoride solution (4.20 mL, 4.20 mmol) in tetrahydrofuran was added dropwise. The reaction mixture was stirred at room temperature for 1 hour. After the reaction, the mixture was impregnated on silica and purified by flash column chromatography (dichloromethane / methanol, 8: 2) to give 3'-O- (2-nitrobenzyl) -2'-deoxycytidine. ..

3'-O- (2-nitrobenzyl) -2'-deoxycytidine (14 mg, 1 eq, 38 μmol) was co-evaporated with pyridine (1 mL x 3) and dried in high vacuum overnight. It was then dissolved in 1.5 mL of trimethyl phosphate and 0.60 mL of dry pyridine and cooled in an ice bath under argon. A first aliquot of 6 uL of phosphoryl trichloride (18 mg, 11 μL, 3 eq, 0.11 mmol) was added. After 5 minutes, a 5 uL second aliquot was added. The mixture was stirred for an additional 30 minutes. A solution of tetrabutylammonium diphosphate (0.14 g, 4 eq, 0.15 mmol) in 1.5 mL dry DMF was prepared under Ar and cooled in an ice bath. This was added dropwise to the rxn mixture over 30 seconds. Immediately, pre-weighed N1, N1, N8, N8-tetramethylnaphthalene-1,8-diamine (33 mg, 4 eq, 0.15 mmol) was added at once as a solid. The mixture was stirred for 30 minutes after this addition and quenched with 8 mL cold 0.1 M TEAB buffer. The mixture was stirred in an ice bath for 10 minutes and then transferred to a separatory funnel. The solution was extracted once with 10 mL of EtOAc. The aqueous layer was transferred to a canal for FPLC separation and this separation was performed immediately after Extraction of EtOAc. Final purification was by reverse phase HPLC.

２－アミノ－６－クロロ－９－［β－Ｄ－２’－デオキシリボフラノシル］プリン（１．００ｇ、３．５０ｍｍｏｌ）およびイミダゾール（７１５ｍｇ、１０．５０ｍｍｏｌ）を、無水ギ酸ジメチル（１８ｍＬ）中に溶解させ、その後、ｔｅｒｔ－ブチルジメチルシリルクロリド（６８６ｍｇ、４．６０ｍｍｏｌ）を添加した。反応混合物を、アルゴン下で室温で１２時間撹拌した。乾燥させた残渣を、シリカ上に含浸させ、フラッシュカラムクロマトグラフィー（ジクロロメタン／メタノール、９：１）によって精製して、２－アミノ－６－クロロ－９－［β－Ｄ－５’－Ｏ－（ｔｅｒｔ－ブチルジメチルシリル）－２’－デオキシリボフラノシル］プリンを得た。

(Example 5)
Detailed Procedure for 3'-O-Nitrobenzyl Deoxyguanosine:

2-Amino-6-chloro-9- [β-D-2'-deoxyribofuranosyl] purine (1.00 g, 3.50 mmol) and imidazole (715 mg, 10.50 mmol) in dimethyl anhydride (18 mL). After that, tert-butyldimethylsilyl chloride (686 mg, 4.60 mmol) was added. The reaction mixture was stirred under argon at room temperature for 12 hours. The dried residue was impregnated on silica and purified by flash column chromatography (dichloromethane / methanol, 9: 1) to 2-amino-6-chloro-9- [β-D-5'-O-. (Tert-Butyldimethylsilyl) -2'-deoxyribofuranosyl] purine was obtained.

2-Amino-6-chloro-9- [β-D-5'-O- (tert-butyldimethylsilyl) -2'-deoxyribofuranosyl] purine (1.19 g, 3.00 mmol) was added in anhydrous tetrahydrofuran (1.9 g, 3.00 mmol). It was dissolved in 8.0 mL), after which N, N-dimethylformamide dimethyl acetal (3.10 mL, 18.0 mmol) was added at room temperature. The reaction mixture was stirred at 40 ° C. for 3 hours. The reaction mixture was impregnated on silica and purified by flash column chromatography (dichloromethane / methanol, 9: 1) to 6-chloro-N ² -[(dimethylaminomethylene) amino] -9- [β-D. A -5'-O- (tert-butyldimethylsilyl) -2'-deoxyribofuranosyl] purine was obtained.

6-Chloro-N ² -[(dimethylaminomethylene) amino] -9- [β-D-5'-O- (tert-butyldimethylsilyl) -2'-deoxyribofuranosyl] purine (1.09 g, 2 .40 mmol) was dissolved in anhydrous acetonitrile (3.5 mL), after which sodium hydride powder (60%) (122 mg, 4.80 mmol) in mineral oil was added at 0 ° C. After stirring at room temperature for 1 hour, a solution of 2-nitrobenzyl bromide (1.04 g, 4.80 mmol) in anhydrous acetonitrile (1.5 mL) was added. After stirring at room temperature for 2 hours, the reaction mixture was filtered. The filtrate was dried and the resulting residue was dissolved in ethyl acetate (100 mL). The organic layer was washed with saturated sodium bicarbonate solution (50 mL), brine (50 mL) and dried over anhydrous sodium sulfate. The organic layer was impregnated on silica and purified by flash column chromatography (hexane / ethyl acetate 4: 6) to 6-chloro-N ² -[(dimethylaminomethylene) amino] -9- [β-D. A -5'-O- (tert-butyldimethylsilyl) -3'-O- (2-nitrobenzyl) -2'-deoxyribofuranosyl] purine was obtained.

6-Chloro-N ² -[(dimethylaminomethylene) amino] -9- [β-D-5'-O- (tert-butyldimethylsilyl) -3'-O- (2-nitrobenzyl) -2' -Deoxyribofuranosyl] purine (1.02 g, 1.73 mmol) was dissolved in dimethyl anhydride (15 mL) and then under argon, cesium acetate (996 mg, 5.19 mmol), 1,4-diazabicyclo [ 2.2.2] Octane (194 mg, 1.73 mmol) and triethylamine (0.72 mL, 5.19 mmol) were added. The reaction mixture was stirred at room temperature for 18 hours. Acetic anhydride (5 mL) was added and the mixture was stirred for 0.5 hours. The reaction mixture was quenched with water (100 mL) and extracted with ethyl acetate (100 mL). The organic layer was dried over anhydrous sodium sulfate, impregnated on silica and purified by flash column chromatography (dichloromethane / methanol, 20: 1) to 5'-O- (tert-butyldimethylsilyl) -N ² -[(Dimethylamino) methylene] -3'-O- (2-nitrobenzyl) -2'-deoxyguanosine was obtained.

5'-O- (tert-butyldimethylsilyl) -N ² -[(dimethylamino) methylene] -3'-O- (2-nitrobenzyl) -2'-deoxyguanosine (732 mg, 1.28 mmol), It was dissolved in anhydrous tetrahydrofuran (8 mL) under argon and cooled to 0 ° C. Then, 1.0 M tetrabutylammonium fluoride solution (2.56 mL, 2.56 mmol) in tetrahydrofuran was added dropwise. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was poured into cold water (50 mL) and extracted with ethyl acetate (50 mL x 2). The organic layers were combined and dried over anhydrous sodium sulfate. The organic layer is impregnated on silica and purified by flash column chromatography (dichloromethane / methanol, 10: 1) to N ² -[(dimethylamino) methylene] -3'-O- (2-nitrobenzyl). -2'-Deoxyguanosine was obtained.

N ² -[(dimethylamino) methylene] -3'-O- (2-nitrobenzyl) -2'-deoxyguanosine (17 mg, 1 eq, 38 μmol) was co-evaporated with pyridine (1 mL x 3) to make it high. It was dried overnight in vacuum. It was then dissolved in 1.5 mL of trimethyl phosphate and 0.60 mL of dry pyridine and cooled in an ice bath under argon. A first aliquot of 6 uL of phosphoryl trichloride (18 mg, 11 μL, 3 eq, 0.11 mmol) was added. After 5 minutes, a 5 uL second aliquot was added. The mixture was stirred for an additional 30 minutes. A solution of tetrabutylammonium diphosphate (0.14 g, 4 eq, 0.15 mmol) in 1.5 mL dry DMF was prepared under Ar and cooled in an ice bath. This was added dropwise to the rxn mixture over 30 seconds. Immediately, pre-weighed N1, N1, N8, N8-tetramethylnaphthalene-1,8-diamine (33 mg, 4 eq, 0.15 mmol) was added at once as a solid. The mixture was stirred for 30 minutes after this addition and quenched with 8 mL cold 0.1 M TEAB buffer. The mixture was stirred in an ice bath for 10 minutes and then transferred to a separatory funnel. The solution was extracted once with 10 mL of EtOAc. The aqueous layer was transferred to a canal for FPLC separation and this separation was performed immediately after Extraction of EtOAc. Final purification was by reverse phase HPLC.

チミジン（２．５０ｇ、１０．３ｍｍｏｌ）を、アルゴン下で室温でジメチルホルムアミド（６０ｍＬ）中に懸濁した。この懸濁物に、イミダゾール（４．２２ｇ、６１．９ｍｍｏｌ）およびｔｅｒｔ－ブチルクロロジメチルシラン（４．６６ｇ、３０．１ｍｍｏｌ）を添加した。２時間攪拌した後、反応混合物を、メタノール（８ｍＬ）でクエンチし、酢酸エチル（２００ｍＬ）で希釈した。有機層を、水（１００ｍＬ×２）、重炭酸ナトリウムの飽和溶液（１００ｍＬ）およびブライン（１００ｍＬ）で洗浄した。有機層を、無水硫酸ナトリウムで乾燥させ、シリカ上に含浸させ、フラッシュカラムクロマトグラフィー（ヘキサン／酢酸エチル、８：２）によって精製して、３’，５’－ジ－Ｏ－（ｔｅｒｔ－ブチルジメチルシリル）チミジンを得た。

(Example 6)
Detailed procedure for 3'-O-nitrobenzyldeoxythymidine:

Thymidine (2.50 g, 10.3 mmol) was suspended in dimethylformamide (60 mL) under argon at room temperature. To this suspension was added imidazole (4.22 g, 61.9 mmol) and tert-butylchlorodimethylsilane (4.66 g, 30.1 mmol). After stirring for 2 hours, the reaction mixture was quenched with methanol (8 mL) and diluted with ethyl acetate (200 mL). The organic layer was washed with water (100 mL x 2), saturated solution of sodium bicarbonate (100 mL) and brine (100 mL). The organic layer was dried over anhydrous sodium sulfate, impregnated on silica and purified by flash column chromatography (hexane / ethyl acetate, 8: 2) to 3', 5'-di-O- (tert-butyl). Dimethylsilyl) thymidine was obtained.

3', 5'-di-O- (tert-butyldimethylsilyl) thymidine (4.34 g, 9.22 mmol) and dimethyl-4-aminopyridine (1.12 g, 9.22 mmol) in anhydrous dichloromethane (140 mL). Dissolved in. Triethylamine (5.14 mL, 36.9 mmol) was added and the reaction mixture was cooled to 0 ° C. Benzyl chloride (3.21 mL, 27.7 mmol) was added dropwise and warmed to room temperature (warp up). After stirring for 14 hours, a saturated solution of sodium bicarbonate (80 mL) was added and the layers were separated. The aqueous layer was extracted with dichloromethane (200 mL x 2). The combined organic layers were washed with water (300 mL). The organic layer was dried over anhydrous sodium sulfate, impregnated on silica and purified by flash column chromatography (hexane / ethyl acetate, 8: 2) to 3-N-benzoyl-3'5'-di-O. -(Tert-Butyldimethylsilyl) thymidine was obtained.

3-N-benzoyl-3'5'-di-O- (tert-butyldimethylsilyl) thymidine (3.03 g, 5.27 mmol) was dissolved in dry tetrahydrofuran (50 mL). Triethylamine (1.98 mL, 14.2 mmol) was added, followed by triethylammonium fluoride dihydrofluoride (2.32 mL, 14.2 mmol) under argon. The reaction mixture was stirred at room temperature for 29 hours and then concentrated. The residue was dissolved in dichloromethane (100 mL) and washed with 1.5 M ammonium carbonate (75 mL) and brine (75 mL). The organic layer was dried over anhydrous sodium sulfate, impregnated on silica and purified by flash column chromatography (dichloromethane / methanol, 9: 1) to give 3-N-benzoylthymidine.

3-N-benzoylthymidine (XXg, 4.40 mmol) was dissolved in anhydrous pyridine (37 mL), after which tert-butyldimethylsilyl chloride (815 mg, 5.41 mmol) was added under argon. The reaction mixture was stirred at room temperature for 20 hours. After concentration, the residue was dissolved in dichloromethane and impregnated on silica. The crude product was purified by flash column chromatography (dichloromethane / methanol, 9: 1) to give 3-N-benzoyl-5'-O- (tert-butyldimethylsilyl) thymidine.

To 3-N-benzoyl-5'-O- (tert-butyldimethylsilyl) thymidine (1.18 g, 2.56 mmol) was added an aqueous solution of tetrabutylammonium hydroxide (10 mL, 60%), followed by sodium iodide. Sodium iodide (76.7 mg, 0.51 mmol), dichloromethane (10 mL), water (10 mL), and an aqueous solution of 1 M sodium hydroxide (10 mL) were added. The mixture was added dropwise to a solution of 2-nitrobenzyl bromide (718 mg, 3.32 mmol) in dichloromethane (10 mL). The reaction mixture was stirred at room temperature for 6 hours and then water (10 mL) was added. The aqueous layer was extracted with dichloromethane (50 mL x 3). The organic layers were combined, washed with brine (100 mL) and dried over anhydrous sodium sulfate. After filtration and concentration, the residue is dissolved in ethyl acetate, impregnated on silica and purified by flash column chromatography (hexane / ethyl acetate, 6: 4) to 3-N-benzoyl-5'-O. -(Tert-Butyldimethylsilyl) -3'-O- (2-nitrobenzyl) thymidine was obtained.

3-N-benzoyl-5'-O- (tert-butyldimethylsilyl) -3'-O- (2-nitrobenzyl) thymidine (1.24 g, 2.08 mmol) is dissolved in ethanol (15 mL). After that, a 30% ammonium hydroxide solution (1.5 mL, 12.3 mmol) was added. The reaction mixture is stirred at room temperature for 1 hour, impregnated on silica and purified by flash column chromatography (hexane / ethyl acetate, 6: 4) to 5'-O- (tert-butyldimethylsilyl) -3. '-O- (2-nitrobenzyl) thymidine was obtained.

5'-O- (tert-butyldimethylsilyl) -3'-O- (2-nitrobenzyl) thymidine (681 mg, 1.39 mmol) was dissolved in anhydrous tetrahydrofuran (12 mL) under argon to 0 ° C. Cooled. Then, 1.0 M tetrabutylammonium fluoride solution (2.78 mL, 2.78 mmol) in tetrahydrofuran was added dropwise. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was poured into cold water (50 mL) and extracted with ethyl acetate (50 mL x 3). The organic layers were combined and dried over anhydrous sodium sulfate. The organic layer was impregnated on silica and purified by flash column chromatography (dichloromethane / methanol, 10: 1) to give 3'-O- (2-nitrobenzyl) thymidine.

3'-O- (2-nitrobenzyl) thymidine (15 mg, 1 eq, 38 μmol) was co-evaporated with pyridine (1 mL x 3) and dried in high vacuum overnight. It was then dissolved in 1.5 mL of trimethyl phosphate and 0.60 mL of dry pyridine and cooled in an ice bath under argon. A first aliquot of 6 uL of phosphoryl trichloride (18 mg, 11 μL, 3 eq, 0.11 mmol) was added. After 5 minutes, a 5 uL second aliquot was added. The mixture was stirred for an additional 30 minutes. A solution of tetrabutylammonium diphosphate (0.14 g, 4 eq, 0.15 mmol) in 1.5 mL dry DMF was prepared under Ar and cooled in an ice bath. This was added dropwise to the rxn mixture over 30 seconds. Immediately, pre-weighed N1, N1, N8, N8-tetramethylnaphthalene-1,8-diamine (33 mg, 4 eq, 0.15 mmol) was added at once as a solid. The mixture was stirred for 30 minutes after this addition and quenched with 8 mL cold 0.1 M TEAB buffer. The mixture was stirred in an ice bath for 10 minutes and then transferred to a separatory funnel. The solution was extracted once with 10 mL of EtOAc. The aqueous layer was transferred to a canal for FPLC separation and this separation was performed immediately after Extraction of EtOAc. Final purification was by reverse phase HPLC.

非ペプチド前駆体 － シトシン

クラスＩＩペプチド－ｄＮＴＰアナログの合成のためのスキーム
ジスルフィドペプチド前駆体 － アデノシン

ジスルフィドペプチド前駆体 － シトシン

(Example 6)
Procedure for synthesizing class II-purines and pyrimidine dNTP analogs.
Scheme for the synthesis of class II non-peptide dNTP analogs Non-peptide precursor-adenosine

Non-peptide precursor-cytosine

Scheme for the synthesis of class II peptides-dNTP analogs Disulfide peptide precursors-adenosine

Disulfide Peptide Precursor-Cytosine

９－［β－Ｄ－５’－Ｏ－（ｔｅｒｔ－ブチルジメチルシリル）－３’－Ｏ－（２－ニトロベンジル）－２’－デオキシアデノシン（０．２４ｍｍｏｌ）を、アルゴン下で室温で無水テトラヒドロフラン（４ｍＬ）中に溶解させた。水素化ナトリウム（４８ｍｇ、１．２１ｍｍｏｌ）を、一度に添加した。１時間攪拌した後、反応混合物を０℃に冷却し、塩化アシル（３当量、０．７２３ｍｍｏｌ）を滴加した。反応混合物を、室温で１８時間撹拌した。反応混合物を、飽和重炭酸ナトリウムおよびジクロロメタンの冷溶液中に注いだ。層を分離した。有機層を、無水硫酸ナトリウムで乾燥させ、シリカ上に含浸させ、フラッシュカラムクロマトグラフィー（ヘキサン／酢酸エチル、２：８）によって精製して、所望の産物を得た。

(Example 7)
Detailed Procedures for the Synthesis of Class II Non-Peptide dATP Constructs:

9- [β-D-5'-O- (tert-butyldimethylsilyl) -3'-O- (2-nitrobenzyl) -2'-deoxyadenosine (0.24 mmol) is anhydrous under argon at room temperature. It was dissolved in tetrahydrofuran (4 mL). Sodium hydride (48 mg, 1.21 mmol) was added all at once. After stirring for 1 hour, the reaction mixture was cooled to 0 ° C. and acyl chloride (3 eq, 0.723 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was poured into a cold solution of saturated sodium bicarbonate and dichloromethane. The layers were separated. The organic layer was dried over anhydrous sodium sulfate, impregnated on silica and purified by flash column chromatography (hexane / ethyl acetate 2: 8) to give the desired product.

The N-substituted nucleoside (5.27 mmol) was dissolved in dry tetrahydrofuran (50 mL). Triethylamine (1.98 mL, 14.2 mmol) was added, followed by triethylammonium fluoride dihydrofluoride (2.32 mL, 14.2 mmol) under argon. The reaction mixture was stirred at room temperature for 29 hours and then concentrated. The residue was dissolved in dichloromethane (100 mL) and washed with 1.5 M ammonium carbonate (75 mL x 1), brine (75 mL). The organic layer was dried over anhydrous sodium sulfate, impregnated on silica and purified by flash column chromatography (dichloromethane / methanol, 9: 1) to N-substituted 3'-O- (2-nitrobenzyl)-. 2'-deoxyadenosine was obtained.

N-substituted 3'-O- (2-nitrobenzyl) -2'-deoxyadenosine (1 eq, 38 μmol) was co-evaporated with pyridine (1 mL x 3) and dried in high vacuum overnight. It was then dissolved in 1.5 mL of trimethyl phosphate and 0.60 mL of dry pyridine and cooled in an ice bath under argon. A first aliquot of 6 uL of phosphoryl trichloride (18 mg, 11 μL, 3 eq, 0.11 mmol) was added. After 5 minutes, a 5 uL second aliquot was added. The mixture was stirred for an additional 30 minutes. A solution of tetrabutylammonium diphosphate (0.14 g, 4 eq, 0.15 mmol) in 1.5 mL dry DMF was prepared under Ar and cooled in an ice bath. This was added dropwise to the rxn mixture over 30 seconds. Immediately, pre-weighed N1, N1, N8, N8-tetramethylnaphthalene-1,8-diamine (33 mg, 4 eq, 0.15 mmol) was added at once as a solid. The mixture was stirred for 30 minutes after this addition and quenched with 8 mL cold 0.1 M TEAB buffer. The mixture was stirred in an ice bath for 10 minutes and then transferred to a separatory funnel. The solution was extracted once with 10 mL of EtOAc. The aqueous layer was transferred to a canal for FPLC separation and this separation was performed immediately after Extraction of EtOAc. Final purification was by reverse phase HPLC.

５’－Ｏ－（ｔｅｒｔ－ブチルジメチルシリル）－３’－Ｏ－（２－ニトロベンジル）－２’－デオキシシチジン（５２４．３ｍｇ、１．１０ｍｍｏｌ）およびカルボン酸（１．３２ｍｍｏｌ）を、アルゴン下で無水ギ酸ジメチル中に溶解させた。Ｎ，Ｎ－ジイソプロピルエチルアミン（０．４８ｍＬ、２．７４ｍｍｏｌ）および２－（３Ｈ－［１，２，３］トリアゾロ［４，５－ｂ］ピリジン－３－イル）－１，１，３，３－テトラメチルイソウロニウムヘキサフルオロホスフェート（Ｖ）（４１７ｍｇ、１．１０ｍｍｏｌ）を添加した。反応混合物を、室温で１８時間撹拌し、酢酸エチル（３０ｍＬ）で希釈した。有機層を、飽和重炭酸ナトリウム溶液（３０ｍＬ）で洗浄し、無水硫酸ナトリウムで乾燥させ、シリカ上に含浸させ、フラッシュカラムクロマトグラフィー（ヘキサン／酢酸エチル、４：６）によって精製して、Ｎ－置換５’－Ｏ－（ｔｅｒｔ－ブチルジメチルシリル）－３’－Ｏ－（２－ニトロベンジル）－２’－デオキシシチジンを得た。

(Example 8)
Detailed Procedures for Synthesis of Class II Non-Peptide dCTP Constructs:

5'-O- (tert-butyldimethylsilyl) -3'-O- (2-nitrobenzyl) -2'-deoxycytidine (524.3 mg, 1.10 mmol) and carboxylic acid (1.32 mmol), argon Underneath, it was dissolved in dimethyl anhydride. N, N-diisopropylethylamine (0.48 mL, 2.74 mmol) and 2- (3H- [1,2,3] triazolo [4,5-b] pyridin-3-yl) -1,1,3,3 -Tetramethylisouronium hexafluorophosphate (V) (417 mg, 1.10 mmol) was added. The reaction mixture was stirred at room temperature for 18 hours and diluted with ethyl acetate (30 mL). The organic layer was washed with saturated sodium bicarbonate solution (30 mL), dried over anhydrous sodium sulfate, impregnated on silica and purified by flash column chromatography (hexane / ethyl acetate 4: 6) to N-. Substitution 5'-O- (tert-butyldimethylsilyl) -3'-O- (2-nitrobenzyl) -2'-deoxycitidine was obtained.

N-substituted 5'-O- (tert-butyldimethylsilyl) -3'-O- (2-nitrobenzyl) -2'-deoxycytidine (3.37 g, 5.27 mmol) in dry tetrahydrofuran (50 mL). Was dissolved in. Triethylamine (1.98 mL, 14.2 mmol) was added, followed by triethylammonium fluoride dihydrofluoride (2.32 mL, 14.2 mmol) under argon. The reaction mixture was stirred at room temperature for 29 hours and then concentrated. The residue was dissolved in dichloromethane (100 mL) and washed with 1.5 M ammonium carbonate (75 mL x 1), brine (75 mL). The organic layer was dried over anhydrous sodium sulfate, impregnated on silica and purified by flash column chromatography (dichloromethane / methanol, 9: 1) to N-substituted 3'-O- (2-nitrobenzyl)-. 2'-Deoxycytidine was obtained.

N-substituted 3'-O- (2-nitrobenzyl) -2'-deoxycytidine (1 eq, 38 μmol) was co-evaporated with pyridine (1 mL x 3) and dried in high vacuum overnight. It was then dissolved in 1.5 mL of trimethyl phosphate and 0.60 mL of dry pyridine and cooled in an ice bath under argon. A first aliquot of 6 uL of phosphoryl trichloride (18 mg, 11 μL, 3 eq, 0.11 mmol) was added. After 5 minutes, a 5 uL second aliquot was added. The mixture was stirred for an additional 30 minutes. A solution of tetrabutylammonium diphosphate (0.14 g, 4 eq, 0.15 mmol) in 1.5 mL dry DMF was prepared under Ar and cooled in an ice bath. This was added dropwise to the rxn mixture over 30 seconds. Immediately, pre-weighed N1, N1, N8, N8-tetramethylnaphthalene-1,8-diamine (33 mg, 4 eq, 0.15 mmol) was added at once as a solid. The mixture was stirred for 30 minutes after this addition and quenched with 8 mL cold 0.1 M TEAB buffer. The mixture was stirred in an ice bath for 10 minutes and then transferred to a separatory funnel. The solution was extracted once with 10 mL of EtOAc. The aqueous layer was transferred to a canal for FPLC separation and this separation was performed immediately after Extraction of EtOAc. Final purification was by reverse phase HPLC.

９－［β－Ｄ－５’－Ｏ－（ｔｅｒｔ－ブチルジメチルシリル）－３’－Ｏ－（２－ニトロベンジル）－２’－デオキシアデノシン（５２４．３ｍｇ、１．１０ｍｍｏｌ）および４－（ピリジン－２－イルジスルファニル（sulfaneyl））ブタン酸（３０２．７ｍｇ、１．３２ｍｍｏｌ）を、アルゴン下で無水ギ酸ジメチル中に溶解させた。Ｎ，Ｎ－ジイソプロピルエチルアミン（０．４８ｍＬ、２．７４ｍｍｏｌ）および２－（３Ｈ－［１，２，３］トリアゾロ［４，５－ｂ］ピリジン－３－イル）－１，１，３，３－テトラメチルイソウロニウムヘキサフルオロホスフェート（Ｖ）（４１７ｍｇ、１．１０ｍｍｏｌ）を添加した。反応混合物を、室温で１８時間撹拌し、酢酸エチル（３０ｍＬ）で希釈した。有機層を、飽和重炭酸ナトリウム溶液（３０ｍＬ）で洗浄し、無水硫酸ナトリウムで乾燥させ、シリカ上に含浸させ、フラッシュカラムクロマトグラフィー（ヘキサン／酢酸エチル、４：６）によって精製して、Ｎ－（４－（ピリジン－２－イルジスルファニル）ブタニリル（butanyryl））－５’－Ｏ－（ｔｅｒｔ－ブチルジメチルシリル）－３’－Ｏ－（２－ニトロベンジル）－２’－デオキシアデノシンを得た。

(Example 9)
Detailed Procedures for the Synthesis of Peptide-dATP Conjugates:

9- [β-D-5'-O- (tert-butyldimethylsilyl) -3'-O- (2-nitrobenzyl) -2'-deoxyadenosine (524.3 mg, 1.10 mmol) and 4- ( Pyridine-2-yldisulfaneyl butanoic acid (302.7 mg, 1.32 mmol) was dissolved in dimethyl anhydride under argon. N, N-diisopropylethylamine (0.48 mL, 2.74 mmol) and 2- (3H- [1,2,3] triazolo [4,5-b] pyridin-3-yl) -1,1,3,3 -Tetramethylisouronium hexafluorophosphate (V) (417 mg, 1.10 mmol) was added. The reaction mixture was stirred at room temperature for 18 hours and diluted with ethyl acetate (30 mL). The organic layer was washed with saturated sodium bicarbonate solution (30 mL), dried over anhydrous sodium sulfate, impregnated on silica and purified by flash column chromatography (hexane / ethyl acetate 4: 6) to N-. (4- (Pyridin-2-yldisulfanyl) butanyryl) -5'-O- (tert-butyldimethylsilyl) -3'-O- (2-nitrobenzyl) -2'-deoxyadenosin was obtained. ..

N- (4- (Pyridine-2-yldisulfanyl) butanilyl) -5'-O- (tert-butyldimethylsilyl) -3'-O- (2-nitrobenzyl) -2'-deoxyadenosine (3.75 g) 5.27 mmol) was dissolved in dry tetrahydrofuran (50 mL). Triethylamine (1.98 mL, 14.2 mmol) was added, followed by triethylammonium fluoride dihydrofluoride (2.32 mL, 14.2 mmol) under argon. The reaction mixture was stirred at room temperature for 29 hours and then concentrated. The residue was dissolved in dichloromethane (100 mL) and washed with 1.5 M ammonium carbonate (75 mL x 1), brine (75 mL). The organic layer was dried over anhydrous sodium sulfate, impregnated on silica and purified by flash column chromatography (dichloromethane / methanol, 9: 1) to N- (4- (pyridin-2-yldisulfanyl) butaniryl). -3'-O- (2-nitrobenzyl) -2'-deoxyadenosine was obtained.

N- (4- (Pyridine-2-yldisulfanyl) butanilyl) -3'-O- (2-nitrobenzyl) -2'-deoxyadenosine (23 mg, 1 equivalent, 38 μmol) with pyridine (1 mL x 3) It was co-evaporated and dried in high vacuum overnight. It was then dissolved in 1.5 mL of trimethyl phosphate and 0.60 mL of dry pyridine and cooled in an ice bath under argon. A first aliquot of 6 uL of phosphoryl trichloride (18 mg, 11 μL, 3 eq, 0.11 mmol) was added. After 5 minutes, a 5 uL second aliquot was added. The mixture was stirred for an additional 30 minutes. A solution of tetrabutylammonium diphosphate (0.14 g, 4 eq, 0.15 mmol) in 1.5 mL dry DMF was prepared under Ar and cooled in an ice bath. This was added dropwise to the rxn mixture over 30 seconds. Immediately, pre-weighed N1, N1, N8, N8-tetramethylnaphthalene-1,8-diamine (33 mg, 4 eq, 0.15 mmol) was added at once as a solid. The mixture was stirred for 10 minutes after this addition and quenched with 8 mL cold 0.1 M TEAB buffer. The mixture was stirred in an ice bath for 10 minutes and then transferred to a separatory funnel. The solution was extracted once with 10 mL of EtOAc. The aqueous layer was transferred to a canal for FPLC separation and this separation was performed immediately after Extraction of EtOAc. Final purification was by reverse phase HPLC.

５’－Ｏ－（ｔｅｒｔ－ブチルジメチルシリル）－３’－Ｏ－（２－ニトロベンジル）－２’－デオキシシチジン（５２４．３ｍｇ、１．１０ｍｍｏｌ）および４－（ピリジン－２－イルジスルファニル）ブタン酸（１．３２ｍｍｏｌ）を、アルゴン下で無水ギ酸ジメチル中に溶解させた。Ｎ，Ｎ－ジイソプロピルエチルアミン（０．４８ｍＬ、２．７４ｍｍｏｌ）および２－（３Ｈ－［１，２，３］トリアゾロ［４，５－ｂ］ピリジン－３－イル）－１，１，３，３－テトラメチルイソウロニウムヘキサフルオロホスフェート（Ｖ）（４１７ｍｇ、１．１０ｍｍｏｌ）を添加した。反応混合物を、室温で１８時間撹拌し、酢酸エチル（３０ｍＬ）で希釈した。有機層を、飽和重炭酸ナトリウム溶液（３０ｍＬ）で洗浄し、無水硫酸ナトリウムで乾燥させ、シリカ上に含浸させ、フラッシュカラムクロマトグラフィー（ヘキサン／酢酸エチル、４：６）によって精製して、Ｎ－（４－（ピリジン－２－イルジスルファニル）ブタニリル）－５’－Ｏ－（ｔｅｒｔ－ブチルジメチルシリル）－３’－Ｏ－（２－ニトロベンジル）－２’－デオキシシチジンを得た。

(Example 10)
Detailed Procedures for the Synthesis of Peptide-dCTP Conjugates:

5'-O- (tert-butyldimethylsilyl) -3'-O- (2-nitrobenzyl) -2'-deoxycytidine (524.3 mg, 1.10 mmol) and 4- (pyridine-2-yldisulfanyl) Butanoic acid (1.32 mmol) was dissolved in dimethyl anhydride under argon. N, N-diisopropylethylamine (0.48 mL, 2.74 mmol) and 2- (3H- [1,2,3] triazolo [4,5-b] pyridin-3-yl) -1,1,3,3 -Tetramethylisouronium hexafluorophosphate (V) (417 mg, 1.10 mmol) was added. The reaction mixture was stirred at room temperature for 18 hours and diluted with ethyl acetate (30 mL). The organic layer was washed with saturated sodium bicarbonate solution (30 mL), dried over anhydrous sodium sulfate, impregnated on silica and purified by flash column chromatography (hexane / ethyl acetate 4: 6) to N-. (4- (Pyridin-2-yldisulfanyl) butanilyl) -5'-O- (tert-butyldimethylsilyl) -3'-O- (2-nitrobenzyl) -2'-deoxycitidine was obtained.

N- (4- (Pyridine-2-yldisulfanyl) butanilyl) -5'-O- (tert-butyldimethylsilyl) -3'-O- (2-nitrobenzyl) -2'-deoxycytidine (3.62 g) 5.27 mmol) was dissolved in dry tetrahydrofuran (50 mL). Triethylamine (1.98 mL, 14.2 mmol) was added, followed by triethylammonium fluoride dihydrofluoride (2.32 mL, 14.2 mmol) under argon. The reaction mixture was stirred at room temperature for 29 hours and then concentrated. The residue was dissolved in dichloromethane (100 mL) and washed with 1.5 M ammonium carbonate (75 mL x 1), brine (75 mL). The organic layer was dried over anhydrous sodium sulfate, impregnated on silica and purified by flash column chromatography (dichloromethane / methanol, 9: 1) to N- (4- (pyridin-2-yldisulfanyl) butaniryl). -3'-O- (2-nitrobenzyl) -2'-deoxycytidine was obtained.

N- (4- (Pyridine-2-yldisulfanyl) butanilyl) -3'-O- (2-nitrobenzyl) -2'-deoxycytidine (22 mg, 1 equivalent, 38 μmol) with pyridine (1 mL x 3) It was co-evaporated and dried in high vacuum overnight. It was then dissolved in 1.5 mL of trimethyl phosphate and 0.60 mL of dry pyridine and cooled in an ice bath under argon. A first aliquot of 6 uL of phosphoryl trichloride (18 mg, 11 μL, 3 eq, 0.11 mmol) was added. After 5 minutes, a 5 uL second aliquot was added. The mixture was stirred for an additional 30 minutes. A solution of tetrabutylammonium diphosphate (0.14 g, 4 eq, 0.15 mmol) in 1.5 mL dry DMF was prepared under Ar and cooled in an ice bath. This was added dropwise to the rxn mixture over 30 seconds. Immediately, pre-weighed N1, N1, N8, N8-tetramethylnaphthalene-1,8-diamine (33 mg, 4 eq, 0.15 mmol) was added at once as a solid. The mixture was stirred for 10 minutes after this addition and quenched with 8 mL cold 0.1 M TEAB buffer. The mixture was stirred in an ice bath for 10 minutes and then transferred to a separatory funnel. The solution was extracted once with 10 mL of EtOAc. The aqueous layer was transferred to a canal for FPLC separation and this separation was performed immediately after Extraction of EtOAc. Final purification was by reverse phase HPLC.

(Example 11)
Class III-Procedures for synthesizing purine and pyrimidine dNTP analogs.
Scheme for synthesis of class III dNTP analogs

乾燥トルエン（１００ｍＬ）中のホスゲン（６．４６ｇ、２当量、６５．３ｍｍｏｌ）溶液に、２３℃で、２０ｍＬ乾燥ＴＨＦ中の（２－ニトロフェニル）メタノール（５．００ｇ、１当量、３２．６ｍｍｏｌ）を添加した。反応物を、２３℃で２４時間撹拌した。次いで、反応物を、ＮａＯＨ水溶液でトラップしながら、真空下で乾燥するまで濃縮した。残存した琥珀色の油状物を、さらなる精製なしに反応において直接使用した。

(Example 12)
Detailed Procedures for Class III-Prince dNTP Analogs:

In a solution of phosgene (6.46 g, 2 eq, 65.3 mmol) in dry toluene (100 mL) at 23 ° C., (2-nitrophenyl) methanol (5.00 g, 1 eq, 32.6 mmol) in 20 mL dry THF. ) Was added. The reaction was stirred at 23 ° C. for 24 hours. The reaction was then concentrated under vacuum until dry, trapped in aqueous NaOH solution. The remaining amber oil was used directly in the reaction without further purification.

9-((2R, 4S, 5R) -4-((tert-butyldimethylsilyl) oxy) -5-(((tert-butyldimethylsilyl) oxy) methyl) tetrahydrofuran-2- in dry DMF (100 mL)) N, N-diisopropylethylamine (3.60 g, 4.9 mL, 1.2) in a solution of -9H-purine-6-amine (12.2 g, 1.1 eq, 25.5 mmol) at 0 ° C. Equivalent (27.8 mmol) was added. The reaction is stirred for 30 minutes, then 2-nitrobenzyl carbonate (5.00 g, 1 eq, 23.2 mmol) is slowly added dropwise over 30 minutes, keeping the temperature below 5C. And added. The reaction was then warmed to room temperature and stirred overnight. The reaction was poured into a cold solution of 5% Na2CO3 and EtOAc. The EtOAc layer was dried over sodium sulphate and then concentrated to dryness. The crude product was chromatographed on silica gel using a hexane / EtOAc mixture to give a purified product, which was used in the next reaction.

(9-((2R, 4S, 5R) -4-((tert-butyldimethylsilyl) oxy) -5-(((tert-butyldimethylsilyl) oxy) methyl) tetrahydrofuran-2-yl) -9H-purine 2-Ninitrobenzyl carbamate (5.00 g, 1 eq, 7.59 mmol) was dissolved in THF at room temperature and then cooled to OC under a blanket of dry argon. Tetrabutylammonium fluoride (4.96 g, 2.5 eq, 19.0 mmol) was then added to the mixture. The mixture was stirred at 0 C for 2 hours and then warmed to 23 C for 1 hour. The solution was poured into a cold solution of 10% NaHCO3 and extracted with DCM. The DCM layer was concentrated and the crude product was purified on silica gel eluting with 5-50% DCM / MeOH to give a product suitable for triphosphorylation.

(9-((2R, 4S, 5R) -4-hydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl) -9H-purine-6-yl) 2-nitrobenzyl carbamic acid (28 mg, 1 equivalent, 65 μmol) ) Was dissolved in trimethyl phosphate (1.5 mL) and 0.60 mL of dry pyridine and cooled under argon in an ice bath. A first aliquot of phosphoryl trichloride (30 mg, 18 μL, 3 eq, 0.20 mmol) was added. After 5 minutes, a 10 uL second aliquot was added. The mixture was stirred for an additional 30 minutes. A solution of tetrabutylammonium hydrogen diphosphate (0.23 g, 4 eq, 0.26 mmol) in dry DMF was prepared under Ar and cooled in an ice bath. This was added dropwise to the reaction mixture over 30 seconds at rxn t = 35 minutes. Immediately, pre-weighed N1, N1, N8, N8-tetramethyl-naphthalene-1,8-diamine (56 mg, 4 eq, 0.26 mmol) were added at once as a solid. The mixture was stirred for 30 minutes after this addition and quenched with 8 mL cold 0.1 M TEAB buffer. The mixture was stirred for 30 minutes and then transferred to a separatory funnel. The solution was extracted once with 10 mL of EtOAc. The aqueous layer was transferred to a canal for FPLC separation.

乾燥ＤＭＦ（１００ｍＬ）中の４－アミノ－１－（（２Ｒ，４Ｓ，５Ｒ）－４－（（ｔｅｒｔ－ブチルジメチルシリル）オキシ）－５－（（（ｔｅｒｔ－ブチルジメチルシリル）オキシ）－メチル）テトラヒドロフラン－２－イル）ピリミジン－２（１Ｈ）－オン（９．３０ｇ、１．１当量、２０．４ｍｍｏｌ）の溶液に、０℃で、Ｎ，Ｎ－ジイソプロピルエチルアミン（２．８８ｇ、３．９ｍＬ、１．２当量、２２．３ｍｍｏｌ）を添加した。反応物を、３０分間撹拌し、次いで、カルボノクロリド酸２－ニトロベンジル（４．００ｇ、１当量、１８．６ｍｍｏｌ）を、温度を５Ｃより下に維持しながら、３０分間かけてゆっくりと滴下して加えた。次いで、反応物を室温に温め、一晩撹拌した。反応物を、５％Ｎａ２ＣＯ３およびＥｔＯＡｃの冷却溶液中に注いだ。ＥｔＯＡｃ層を、硫酸ナトリウムで乾燥させ、次いで、乾燥するまで濃縮した。粗製産物を、ヘキサン／ＥｔＯＡｃ混合物を使用するシリカゲル上でのクロマトグラフィーにかけて、精製された産物を得、これを次の反応で使用した。

(Example 13)
Detailed Procedures for Class III-Pyrimidine dNTP Analogs:

4-Amino-1-((2R, 4S, 5R) -4-((tert-butyldimethylsilyl) oxy) -5-(((tert-butyldimethylsilyl) oxy) -methyl) in dry DMF (100 mL) ) Tetrahydrofuran-2-yl) In a solution of pyrimidine-2 (1H) -one (9.30 g, 1.1 eq, 20.4 mmol) at 0 ° C., N, N-diisopropylethylamine (2.88 g, 3. 9 mL, 1.2 eq, 22.3 mmol) was added. The reaction is stirred for 30 minutes, then 2-nitrobenzyl carbonochloride (4.00 g, 1 eq, 18.6 mmol) is slowly added dropwise over 30 minutes, keeping the temperature below 5C. And added. The reaction was then warmed to room temperature and stirred overnight. The reaction was poured into a cold solution of 5% Na2CO3 and EtOAc. The EtOAc layer was dried over sodium sulphate and then concentrated to dryness. The crude product was chromatographed on silica gel using a hexane / EtOAc mixture to give a purified product, which was used in the next reaction.

(1-((2R, 4S, 5R) -4-((tert-butyldimethylsilyl) oxy) -5-(((tert-butyldimethylsilyl) oxy) methyl) -tetrahydrofuran-2-yl) -2- 2-Nitrobenzyl carbamic acid (5.00 g, 1 eq, 7.88 mmol) oxo-1,2-dihydropyrimidine-4-yl) was dissolved in 25 mL THF at room temperature and then under a blanket of dry argon. It was cooled to OC. Tetrabutylammonium fluoride (5.15 g, 2.5 eq, 19.7 mmol) was then added to the mixture. The mixture was stirred at 0 C for 2 hours and then warmed to 23 C for 1 hour. The solution was poured into a cold solution of 10% NaHCO3 and extracted with DCM. The DCM layer was concentrated and the crude product was purified on silica gel eluting with 5-50% DCM / MeOH to give a product suitable for triphosphorylation.

(1-((2R, 4S, 5R) -4-hydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl) -2-oxo-1,2-dihydropyrimidine-4-yl) 2-nitrobenzyl carbamate (35.0 mg, 1 eq, 86.1 μmol) was dissolved in trimethyl phosphate (1.5 mL) and 0.60 mL of dry pyridine and cooled under argon in an ice bath. A first aliquot of phosphoryl trichloride (39.6 mg, 3 eq, 258 μmol) was added. After 5 minutes, a 10 uL second aliquot was added. The mixture was stirred for an additional 30 minutes. A solution of tetrabutylammonium hydrogen diphosphate (311 mg, 4 eq, 345 μmol) in dry DMF was prepared under Ar and cooled in an ice bath. This was added dropwise to the reaction mixture over 30 seconds at rxn t = 35 minutes. Immediately, pre-weighed N1, N1, N8, N8-tetramethylnaphthalene-1,8-diamine (73.8 mg, 4 equivalents, 345 μmol) were added at once as a solid. The mixture was stirred for 30 minutes after this addition and quenched with 8 mL cold 0.1 M TEAB buffer. The mixture was stirred for 30 minutes and then transferred to a separatory funnel. The solution was extracted once with 10 mL of EtOAc. The aqueous layer was transferred to a canal for FPLC separation.

クラスＩＶ ｄＣＴＰアナログのための詳細な手順：

(Example 14)
Procedure for synthesizing class IV-dCTP analogs.
Scheme for synthesis of class IV dCTP analogs

Detailed Procedures for Class IV dCTP Analogs:

Methyl 3,4,5-trihydroxybenzoate (10 g, 1 eq, 54 mmol) was dissolved in 50 mL of acetone. Sodium iodide (0.81 g, 0.1 eq, 5.4 mmol) and potassium carbonate (38 g, 5 eq, 270 mmol) were added as solids at ambient temperature. 1- (Chloromethyl) -2-nitrobenzene (34 g, 3.6 eq, 0.20 mol) was added dropwise over 10 minutes as a solution in 40 mL acetone. The mixture was stirred for 1 hour and then heated to 50 ° C. for 6 hours. The mixture was cooled to ambient temperature and the bulk solvent was removed on a rotary evaporator. The residue was suspended in 200 mL of EtOAc and washed sequentially with 200 mL of water and saturated aqueous NaCl solution. The EtOAc layer was dried over sodium sulphate and evaporated. The crude product was chromatographed on silica using a hexane / EtOAc mixture to give a purified product that could be used in the next reaction.

Methyl 3,4,5-tris ((2-nitrobenzyl) oxy) benzoate (25 g, 1 eq, 42 mmol) was dissolved in 300 mL of THF. Aqueous 2M NaOH (105 mL, 210 mmol) was added and the mixture was stirred at ambient temperature for 18 hours. Bulk THF was removed on a rotary evaporator and the residue was slowly acidified with 6M HCl until the pH was 1 or less. The resulting solid was filtered, washed thoroughly with water, dried on a filter funnel for 5 hours and then dried under high vacuum for 18 hours. The product proceeded to the next reaction without further purification.

4-Amino-1-((2R, 4S, 5R) -4-((tert-butyldimethylsilyl) oxy) -5-(((tert-butyldimethylsilyl) oxy) methyl) tetrahydrofuran-2-yl) pyrimidin -2 (1H) -one (12 g, 1 eq, 26 mmol) and 3,4,5-tris ((2-nitrobenzyl) oxy) benzoic acid (15 g, 1 eq, 26 mmol) at ambient temperature in an argon atmosphere. Was dissolved in 50 mL of dry DMF. N-Ethyl-N-isopropylpropane-2-amine (5.1 g, 6.8 mL, 1.5 eq, 39 mmol) was added, followed by 1-((dimethylamino) (dimethyli)) in 10 mL of dry DMF. A solution of minio) methyl) -1H- [1,2,3] triazolo [4,5-b] pyridine3-oxide hexafluorophosphate (V) (12 g, 1.2 eq, 31 mmol) at ambient temperature for 5 minutes. Dropped over. The mixture was stirred at ambient temperature for 18 hours. The mixture was dissolved in 300 mL of EtOAc and washed sequentially with 200 mL of water (2x) and saturated aqueous NaCl solution. EtOAc was dried over sodium sulphate, first evaporated on a rotary evaporator and then evaporated under high vacuum for 18 hours. The residue was chromatographed on silica using a mixture of dichloromethane and methanol to give the desired product as a colorless foam.

N-(1-((2R, 4S, 5R) -4-((tert-butyldimethylsilyl) oxy) -5-(((tert-butyldimethylsilyl) oxy) methyl) tetrahydrofuran-2-yl) -2 -Oxo-1,2-dihydropyrimidine-4-yl) -3,4,5-tris ((2-nitrobenzyl) oxy) benzamide (5 g, 1 equivalent, 5 mmol) under argon at ambient temperature 25. It was dissolved in 0 mL of dry THF. Triethylamine (4 g, 6 mL, 8 eq, 4e + 1 mmol) was added rapidly, followed by triethylammonium fluoride dihydrofluoride (5 g, 5 mL, 6 eq, 3e + 1 mmol) also rapidly added at ambient temperature. The mixture was stirred at ambient temperature for 24 hours. Silica gel (20 g) is added and the mixture is evaporated on a rotary evaporator to a fine powder, then loaded on a 100 g silica column and eluted with a mixture of dichloromethane and methanol to give a slightly yellow foam. Obtained as dichloromethane.

N-(1-((2R, 4S, 5R) -4-hydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl) -2-oxo-1,2-dihydropyrimidine-4-yl) -3,4 , 5-Tris ((2-nitrobenzyl) oxy) benzamide (30 mg, 1 eq, 38 μmol) was co-evaporated with pyridine (1 mL × 3) and dried in high vacuum overnight. It was then dissolved in 1.5 mL of trimethyl phosphate and 0.60 mL of dry pyridine and cooled in an ice bath under argon. A first aliquot of 6 uL of phosphoryl trichloride (18 mg, 11 μL, 3 eq, 0.11 mmol) was added. After 5 minutes, a 5 uL second aliquot was added. The mixture was stirred for an additional 30 minutes. A solution of tetrabutylammonium diphosphate (0.14 g, 4 eq, 0.15 mmol) in 1.5 mL dry DMF was prepared under Ar and cooled in an ice bath. This was added dropwise to the rxn mixture over 30 seconds. Immediately, pre-weighed N1, N1, N8, N8-tetramethylnaphthalene-1,8-diamine (33 mg, 4 eq, 0.15 mmol) was added at once as a solid. The mixture was stirred for 30 minutes after this addition and quenched with 8 mL cold 0.1 M TEAB buffer. The mixture was stirred in an ice bath for 10 minutes and then transferred to a separatory funnel. The solution was extracted once with 10 mL of EtOAc. The aqueous layer was transferred to a canal for FPLC separation and this separation was performed immediately after Extraction of EtOAc. Final purification was by reverse phase HPLC.

非ペプチド－チミジンｄＮＴＰコンジュゲートの合成のためのスキーム

(Example 15)
Procedure for synthesizing class V peptides and non-peptide analogs.
Scheme for the synthesis of peptide-thymidine dNTP conjugate

Scheme for synthesis of non-peptide-thymidine dNTP conjugate

４－（ブロモメチル）ベンゼンチオール（５．００ｇ、１当量、２４．６ｍｍｏｌ）を、メタノール（５０ｍＬ）中に溶解させ、０℃に冷却した。次いで、混合物に、１，２－ジ（ピリジン－２－イル）ジスルファン（５．４２ｇ、１当量、２４．６ｍｍｏｌ）を加え、０Ｃで１８時間撹拌した。次いで、反応物を直接濃縮し、ヘキサン／酢酸エチル（０～１００％ＥｔＯＡｃ）で溶出させるシリカゲル（silica get）上で精製して、産物を白色固体として得、これを次の反応において直接使用した。

(Example 16)
Detailed procedure for peptide-dTTP analogs:

4- (Bromomethyl) benzenethiol (5.00 g, 1 eq, 24.6 mmol) was dissolved in methanol (50 mL) and cooled to 0 ° C. Then, 1,2-di (pyridin-2-yl) disulfan (5.42 g, 1 equivalent, 24.6 mmol) was added to the mixture, and the mixture was stirred at 0 C for 18 hours. The reaction was then concentrated directly and purified on silica gel eluting with hexane / ethyl acetate (0-100% EtOAc) to give the product as a white solid, which was used directly in the next reaction. ..

1-((2R, 4S, 5R) -4-((tert-butyldimethylsilyl) oxy) -5-(((tert-butyldimethylsilyl) oxy) methyl) tetrahydrofuran-2-yl) -5-methylpyrimidine -2,4 (1H, 3H) -dione (5.00 g, 1 eq, 10.6 mmol) was dissolved in 50 mL DMF and then cooled to 0C. The reaction was stirred at 0C for 30 minutes, then sodium hydride (306 mg, 1.2 eq, 12.7 mmol) was added. The reaction was then stirred at 0C for an additional 30 minutes and then warmed to 23C. 2-((4- (Bromomethyl) phenyl) -disulfanyl) -pyridine (3.32 g, 1 eq, 10.6 mmol) was then added to the mixture and stirring was continued at 23 C for an additional 2 hours. The reaction was then poured into a cold solution of 10% NaHCO3 and DCM. The DCM layer was separated, dried over sodium sulphate and concentrated to dryness. The mixture was then purified on silica gel eluting with 0-20% DCM / methanol to give the desired product.

1-((2R, 4S, 5R) -4-((tert-butyldimethylsilyl) oxy) -5-(((tert-butyldimethylsilyl) oxy) -methyl) tetrahydrofuran-2-yl) -5-methyl -3- (4- (Pyridine-2-yldisulfanyl) benzyl) pyrimidine-2,4 (1H, 3H) -dione (2.00 g, 1 equivalent, 2.85 mmol) was dissolved in THF to 0C. Cooled. Tetrabutylammonium fluoride (2.23 mg, 3 eq, 8.55 mmol) was then added to the mixture at 0 C. The reaction was continuously stirred at 0C for 2 hours and then warmed to 23C for an additional hour. The reaction was then cooled to 0C again and added at 0C into a pre-cooled solution of 10% NaHCO3 and DCM. The DCM layer was then separated, dried over sodium sulphate, concentrated and purified on silica gel eluting with 5-50% DCM / methanol to give a pure product.

1-((2R, 4S, 5R) -4-hydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl) -5-methyl-3- (4- (pyridin-2-yldisulfanyl) benzyl) pyrimidine-2 , 4 (1H, 3H) -dione (5.00 g, 1 eq, 10.6 mmol) was dissolved in 20 mL THF at 23 C. Triethylamine (1.07 g, 1.5 mL, 1 eq, 10.6 mmol) was then added to the mixture and cooled to 0C. TBS-Cl (1.59 g, 1 eq, 10.6 mmol) was then added to the mixture and stirring was continued at 0 C for an additional 2 hours. The mixture was then added to a pre-cooled mixture of 10% aqueous NaCl and DCM. The DCM layer was dried over sodium sulphate, concentrated and dried to give an amber oil. The crude product was then purified on silica gel eluting with 5-50% DCM / methanol to give a pure product.

1-((2R, 4S, 5R) -5-(((tert-butyldimethylsilyl) oxy) methyl) -4-hydroxytetrahydro-2-yl) -5-methyl-3- (4- (pyridine-2) -Ildisulfanyl) benzyl) pyrimidine-2,4 (1H, 3H) -dione (2.00 g, 1 equivalent, 3.40 mmol) was dissolved in 20 mL DMF and then cooled to 0C. Sodium hydride (98.0 mg, 1.2 eq, 4.08 mmol) was then added to the mixture and stirring was continued at 0 C for an additional 30 minutes. 1- (Bromomethyl) -2-nitrobenzene (735 mg, 1 eq, 3.40 mmol) was then added to the reaction and stirring was continued at 0 C for an additional hour. The reaction was then added to a pre-cooled mixture of 10% NaCl and EtOAc. The EtOAc layer was separated, dried over sodium sulphate and concentrated to dryness. The crude product was purified on silica gel eluting with 0-50 hexane / EtOAc to give the desired product.

1-((2R, 4S, 5R) -5-(((tert-butyldimethylsilyl) oxy) methyl) -4-((2-nitrobenzyl) oxy) tetrahydrofuran-2-yl) -5-methyl-3 -(4- (Pyridine-2-yldisulfanyl) benzyl) pyrimidine-2,4 (1H, 3H) -dione (1.00 g, 1 equivalent, 1.38 mmol) was dissolved in THF and cooled to 0C. .. The reaction was then added TBAF (362 mg, 1 eq, 1.38 mmol) at 0 C, stirred at 0 C for 1 hour, then warmed to room temperature over 2 hours. The mixture was then poured into a pre-cooled solution of 10% NaHCO3 and DCM. The DCM layer was separated and dried over sodium sulfate. The crude product was purified on silica gel eluting with 5-25% DCM / methanol to give the desired product.

(1-((2R, 4S, 5R) -5- (hydroxymethyl) -4-((2-nitrobenzyl) oxy) tetrahydrofuran-2-yl) -2-oxo-1,2-dihydropyrimidine-4- Il) Carbamic acid 4- (pyridin-2-yl) benzyl (35.0 mg, 1 equivalent, 61.0 μmol) is dissolved in trimethyl phosphate (1.5 mL) and 0.60 mL of dry pyridine and bathed in an ice bath. Cooled under medium argon. A first aliquot of phosphoryl trichloride (39.6 mg, 3 eq, 258 μmol) was added. After 5 minutes, a 10 uL second aliquot was added. The mixture was stirred for an additional 30 minutes. A solution of tetrabutylammonium hydrogen diphosphate (311 mg, 4 eq, 345 μmol) in dry DMF was prepared under Ar and cooled in an ice bath. This was added dropwise to the reaction mixture over 30 seconds at rxn t = 35 minutes. Immediately, pre-weighed N1, N1, N8, N8-tetramethylnaphthalene-1,8-diamine (73.8 mg, 4 equivalents, 345 μmol) were added at once as a solid. The mixture was stirred for 30 minutes after this addition and quenched with 8 mL cold 0.1 M TEAB buffer. The mixture was stirred for 30 minutes and then transferred to a separatory funnel. The solution was extracted once with 10 mL of EtOAc. The aqueous layer was transferred to a canal for FPLC separation.

１－（（２Ｒ，４Ｓ，５Ｒ）－４－（（ｔｅｒｔ－ブチルジメチルシリル）オキシ）－５－（（（ｔｅｒｔ－ブチルジメチルシリル）－オキシ）メチル）テトラヒドロフラン－２－イル）－５－メチルピリミジン－２，４（１Ｈ，３Ｈ）－ジオン（５．００ｇ、１当量、１０．６ｍｍｏｌ）を、５０ｍＬ ＤＭＦ中に溶解させ、次いで、０Ｃに冷却した。反応物を、０Ｃで３０分間撹拌し、次いで、水素化ナトリウム（３０６ｍｇ、１．２当量、１２．７ｍｍｏｌ）を加えた。反応物を、０Ｃでさらに３０分間撹拌し、次いで、２３Ｃに温めた。次いで、混合物に、（ブロモメチル）ベンゼン（１．８２ｇ、１当量、１０．６ｍｍｏｌ）を加え、撹拌を、２３Ｃでさらに２時間継続した。次いで、反応物を、１０％ＮａＨＣＯ３およびＤＣＭの冷溶液中に注いだ。ＤＣＭ層を分離し、硫酸ナトリウムで乾燥させ、乾燥するまで濃縮した。次いで、混合物を、０～２０％ＤＣＭ／メタノールで溶出させるシリカゲル上で精製して、所望の産物を得た。

(Example 17)
Detailed Procedures for Non-Peptide-dTTP Analogs:

1-((2R, 4S, 5R) -4-((tert-butyldimethylsilyl) oxy) -5-(((tert-butyldimethylsilyl) -oxy) methyl) tetrahydrofuran-2-yl) -5-methyl Pyrimidine-2,4 (1H, 3H) -dione (5.00 g, 1 eq, 10.6 mmol) was dissolved in 50 mL DMF and then cooled to 0C. The reaction was stirred at 0C for 30 minutes, then sodium hydride (306 mg, 1.2 eq, 12.7 mmol) was added. The reaction was stirred at 0C for an additional 30 minutes and then warmed to 23C. (Bromomethyl) benzene (1.82 g, 1 eq, 10.6 mmol) was then added to the mixture and stirring was continued at 23 C for an additional 2 hours. The reaction was then poured into a cold solution of 10% NaHCO3 and DCM. The DCM layer was separated, dried over sodium sulphate and concentrated to dryness. The mixture was then purified on silica gel eluting with 0-20% DCM / methanol to give the desired product.

3-Benzyl-1-((2R, 4S, 5R) -4-((tert-butyldimethylsilyl) oxy) -5-(((tert-butyldimethylsilyl) oxy) -methyl) tetrahydrofuran-2-yl) -5-Methylpyrimidine-2,4 (1H, 3H) -dione (4.00 g, 1 eq, 7.13 mmol) was dissolved in THF and cooled to 0C. Tetrabutylammonium fluoride (3.72 g, 2 eq, 14.26 mmol) was then added to the mixture at 0 C. The reaction was continuously stirred at 0C for 2 hours and then warmed to 23C for an additional hour. The reaction was then cooled to 0C again and added at 0C into a pre-cooled solution of 10% NaHCO3 and DCM. The DCM layer was then separated, dried over sodium sulphate, concentrated and purified on silica gel eluting with 5-50% DCM / methanol to give a pure product.

3-Benzyl-1-((2R, 4S, 5R) -4-hydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl) -5-methylpyrimidine-2,4 (1H, 3H) -dione (1. 00 g, 1 eq, 3.01 mmol) was dissolved in 20 mL THF at 23 C. Triethylamine (304 mg, 0.42 mL, 1 eq, 3.01 mmol) was then added to the mixture and cooled to 0C. TBS-Cl (453 mg, 1 eq, 3.01 mmol) was then added to the mixture and stirring was continued at 0 C for an additional 2 hours. The mixture was then added to a pre-cooled mixture of 10% aqueous NaCl and DCM. The DCM layer was dried over sodium sulphate, concentrated and dried to give an amber oil. The crude product was then purified on silica gel eluting with 5-50% DCM / methanol to give a pure product.

3-Benzyl-1-((2R, 4S, 5R) -5-(((tert-butyldimethylsilyl) oxy) methyl) -4-hydroxytetrahydro-2-yl) -5-methylpyrimidine-2,4 ( 1H, 3H) -dione (6.00 g, 1 eq, 13.4 mmol) was dissolved in 20 mL DMF and then cooled to 0C. Sodium hydride (387 mg, 1.2 eq, 16.1 mmol) was then added to the mixture and stirring was continued at 0 C for an additional 30 minutes. 1- (Bromomethyl) -2-nitrobenzene (2.90 g, 1 eq, 13.4 mmol) was then added to the reaction and stirring was continued at 0C for an additional hour. The reaction was then added to a pre-cooled mixture of 10% NaCl and EtOAc. The EtOAc layer was separated, dried over sodium sulphate and concentrated to dryness. The crude product was purified on silica gel eluting with 0-50 hexane / EtOAc to give the desired product.

3-Benzyl-1-((2R, 4S, 5R) -5-(((tert-butyldimethylsilyl) oxy) methyl) -4-((2-nitrobenzyl) oxy) -tetrahydrofuran-2-yl)- 5-Methylpyrimidine-2,4 (1H, 3H) -dione (1.50 g, 1 equivalent, 2.58 mmol) was dissolved in THF and cooled to 0C. The reaction was then added TBAF (674 mg, 1 eq, 2.58 mmol) at 0 C, stirred at 0 C for 1 hour, then warmed to room temperature over 2 hours. The mixture was then poured into a pre-cooled solution of 10% NaHCO3 and DCM. The DCM layer was separated and dried over sodium sulfate. The crude product was purified on silica gel eluting with 5-25% DCM / methanol to give the desired product.

(1-((2R, 4S, 5R) -5- (hydroxymethyl) -4-((2-nitrobenzyl) oxy) tetrahydrofuran-2-yl) -2-oxo-1,2-dihydropyrimidine-4- Il) benzyl carbamate (35.0 mg, 1 eq, 70.5 μmol) was dissolved in trimethyl phosphate (1.5 mL) and 0.60 mL of dry pyridine and cooled under argon in an ice bath. A first aliquot of phosphoryl trichloride (39.6 mg, 3 eq, 258 μmol) was added. After 5 minutes, a 10 uL second aliquot was added. The mixture was stirred for an additional 30 minutes. A solution of tetrabutylammonium hydrogen diphosphate (311 mg, 4 eq, 345 μmol) in dry DMF was prepared under Ar and cooled in an ice bath. This was added dropwise to the reaction mixture over 30 seconds at rxn t = 35 minutes. Immediately, pre-weighed N1, N1, N8, N8-tetramethylnaphthalene-1,8-diamine (73.8 mg, 4 equivalents, 345 μmol) were added at once as a solid. The mixture was stirred for 30 minutes after this addition and quenched with 8 mL cold 0.1 M TEAB buffer. The mixture was stirred for 30 minutes and then transferred to a separatory funnel. The solution was extracted once with 10 mL of EtOAc. The aqueous layer was transferred to a canal for FPLC separation.

非ペプチドｄＧＴＰアナログのための詳細な手順：

(Example 18)
Procedure for synthesizing class VI peptide and non-peptide dGTP analogs.
Scheme for synthesis of class VI-dGTP constructs

Detailed Procedures for Non-Peptide dGTP Analogs:

2-Amino-9-((2R, 4S, 5R) -4-((tert-butyldimethylsilyl) oxy) -5-(((tert-butyldimethylsilyl) oxy) methyl) tetrahydrofuran-2-yl) -1 , 9-Dihydro-6H-purine-6-one (0.50 g, 1 eq, 1.0 mmol) was dissolved in 5.0 mL of dry dimethylacetamide under argon. Oxylane (0.13 g, 3 eq, 3.0 mmol) was added at ambient temperature, followed by sodium hydroxide (40 mg, 1 eq, 1.0 mmol) as a solid. The mixture was stirred at ambient temperature for 4 hours. The mixture was diluted with 50 mL EtOAc and washed sequentially with 100 mL water and 100 mL brine. The EtOAc layer was dried over sodium sulphate and evaporated, leaving a yellow oil. This was chromatographed on 40 g of silica using a dichloromethane / methanol mixture as the eluent to give white foam.

2-Amino-9-((2R, 4S, 5R) -4-((tert-butyldimethylsilyl) oxy) -5-(((tert-butyldimethylsilyl) oxy) methyl) tetrahydrofuran-2-yl)- 1- (2-Hydroxyethyl) -1,9-dihydro-6H-purine-6-one (200 mg, 1 eq, 370 μmol) was suspended in 5 mL of dry pyridine under argon at ambient temperature. Chloroformate (1 eq) was added as a solid. The mixture was heated to 95 C for 8 hours and cooled to ambient temperature. The solvent was removed in vacuo and the residue was diluted with 50 mL EtOAc and washed sequentially with 50 mL water and 50 mL brine. The EtOAc layer was dried over sodium sulphate and evaporated, leaving a yellow oil. This was chromatographed on 40 g of silica using a dichloromethane / methanol mixture as the eluent to give white foam.

The alcohol starting material was dissolved in dry THF under argon at ambient temperature. Two equivalents of triethylamine were added. The acyl chloride solution in THF was added dropwise at ambient temperature and the mixture was stirred for 18 hours. The solvent was removed in vacuo and the residue was diluted with 50 mL EtOAc and washed sequentially with 50 mL water and 50 mL brine. The EtOAc layer was dried over Na2SO4 and evaporated, leaving a light brown solid. This was chromatographed on a silica column using a dichloromethane / methanol mixture as the eluent to give the corresponding ester.

Bis-silyl ether (1 eq) was dissolved in dry THF under argon at ambient temperature. Triethylamine (8 eq) was added rapidly, after which triethylammonium fluoride dihydrofluoride (6 eq) was also added rapidly at ambient temperature. The mixture was stirred at ambient temperature for 24 hours. Silica gel is added and the mixture is evaporated on a rotary evaporator to a fine powder, then loaded onto a silica column and eluted with a mixture of dichloromethane and methanol to give nucleoside as a slightly yellow foam. rice field.

The nucleoside was co-evaporated with pyridine (1 mL x 3) and dried in high vacuum overnight. It was then dissolved in 1.5 mL of trimethyl phosphate and 0.60 mL of dry pyridine and cooled in an ice bath under argon. A first aliquot (1.5 eq) of phosphoryl trichloride was added. After 5 minutes, 1.5 equivalents of a second aliquot was added. The mixture was stirred for an additional 30 minutes. A solution of tetrabutylammonium hydrogen diphosphate (4 eq) in 1.5 mL dry DMF was prepared under Ar and cooled in an ice bath. This was added dropwise to the rxn mixture over 30 seconds. Immediately, pre-weighed N1, N1, N8, N8-tetramethylnaphthalene-1,8-diamine (4 equivalents) were added at once as a solid. The mixture was stirred for 30 minutes after this addition and quenched with 8 mL cold 0.1 M TEAB buffer. The mixture was stirred in an ice bath for 10 minutes and then transferred to a separatory funnel. The solution was extracted once with 10 mL of EtOAc. The aqueous layer was transferred to a canal for FPLC separation and this separation was performed immediately after Extraction of EtOAc. Final purification was by reverse phase HPLC.

Detailed Procedures for Peptide-dGTP Analogs:

2-Amino-9-((2R, 4S, 5R) -4-((tert-butyldimethylsilyl) oxy) -5-(((tert-butyldimethylsilyl) oxy) methyl) tetrahydrofuran-2-yl)- 1,9-Dihydro-6H-purine-6-one (1.00 g, 1 eq, 2.02 mmol) was dissolved in 30 mL of dry N, N-dimethylacetamide under argon. 4-Bromobutane acid (337 mg, 1 eq, 2.02 mmol) was added at ambient temperature, followed by sodium hydroxide (161 mg, 2 eq, 4.03 mmol) as a solid. The mixture was heated to 80 C and stirred for 12 hours. The mixture was cooled to ambient temperature, diluted with 100 mL EtOAc and washed sequentially with 50 mL water and 50 mL brine. The EtOAc layer was dried over Na2SO4 and evaporated, leaving a light brown solid. This was chromatographed on a silica column using a dichloromethane / methanol mixture as the eluent to 4- (2-amino-9-((2R, 4S, 5R) -4-((tert-butyldimethylsilyl)). Oxy) -5-(((tert-butyldimethylsilyl) oxy) methyl) tetrahydrofuran-2-yl) -6-oxo-6,9-dihydro-1H-purine-1-yl) butanoic acid is obtained as a white solid. rice field.

4- (2-Amino-9-((2R, 4S, 5R) -4-((tert-butyldimethylsilyl) oxy) -5-(((tert-butyldimethylsilyl) oxy) methyl) tetrahydrofuran-2- Il) -6-oxo-6,9-dihydro-1H-purine-1-yl) butanoic acid (1 eq) was suspended in 5 mL of dry pyridine under argon at ambient temperature. Chloroformate (1 eq) was added as a solid. The mixture was heated to 95 C for 8 hours and cooled to ambient temperature. The solvent was removed in vacuo and the residue was diluted with 50 mL EtOAc and washed sequentially with 50 mL water and 50 mL brine. The EtOAc layer was dried over sodium sulphate and evaporated, leaving a yellow oil. This was chromatographed on 40 g of silica using a dichloromethane / methanol mixture as the eluent to give white foam.

The carboxylic acid was dissolved or suspended in dry THF at ambient temperature. To this solution was added 1.3 equivalents of triethylamine, followed by 1.1 equivalents of diphenylphosphoryl azide. The mixture was heated to reflux for 20 hours and cooled to ambient temperature. Silica gel was added to the mixture and the solvent was evaporated to give a fine powder. It was loaded onto a column of silica gel and eluted with a mixture of EtOAc and dichloromethane to give the desired isocyanate as a colorless oil.

Bis-silyl ether (1 eq) was dissolved in dry THF under argon at ambient temperature. Triethylamine (8 eq) was added rapidly, after which triethylammonium fluoride dihydrofluoride (6 eq) was also added rapidly at ambient temperature. The mixture was stirred at ambient temperature for 24 hours. Silica gel is added and the mixture is evaporated on a rotary evaporator to a fine powder, then loaded onto a silica column and eluted with a mixture of dichloromethane and methanol to give nucleoside as a slightly yellow foam. rice field.

The nucleoside was co-evaporated with pyridine (1 mL x 3) and dried in high vacuum overnight. It was then dissolved in 1.5 mL of trimethyl phosphate and 0.60 mL of dry pyridine and cooled in an ice bath under argon. A first aliquot (1.5 eq) of phosphoryl trichloride was added. After 5 minutes, 1.5 equivalents of a second aliquot was added. The mixture was stirred for an additional 30 minutes. A solution of tetrabutylammonium hydrogen diphosphate (4 eq) in 1.5 mL dry DMF was prepared under Ar and cooled in an ice bath. This was added dropwise to the rxn mixture over 30 seconds. Immediately, pre-weighed N1, N1, N8, N8-tetramethylnaphthalene-1,8-diamine (4 equivalents) were added at once as a solid. The mixture was stirred for 30 minutes after this addition and quenched with 8 mL cold 0.1 M TEAB buffer. The mixture was stirred in an ice bath for 10 minutes and then transferred to a separatory funnel. The solution was extracted once with 10 mL of EtOAc. The aqueous layer was transferred to a canal for FPLC separation and this separation was performed immediately after Extraction of EtOAc. Final purification was by reverse phase HPLC.

Decaging of 3'-O- (2-nitro-benzyl) -dATP and homopolymer synthesis are shown in FIGS. 20 and 21. 25 uM 3'-O- (2-nitro-benzyl) -dATP (TriLink Technologies, San Diego, CA), 1 x TdT reaction buffer (Thermo-Fisher), 2 U / uL terminal deoxynucleotidyl transferase (Thermo) -Fisher) and 0.002 U / uL inorganic pyrophosphatase (Thermo-Fisher) were mixed with a 1 uM oligonucleotide initiator (5'-biotin-TTTTTTGGCCCTTTUTATAATAATAATAATAATTTTTT, IDT). The reaction volume was exposed to light at 365 nm at 20-22 mW / cm2 over various sections and then placed at 37 oC for 30 minutes. After quenching with the addition of 0.1 M EDTA, each time point was mixed with an equal volume of 2 × Novex TBE-urea gel loading buffer (Thermo-Fisher) and analyzed by polyacrylamide gel electrophoresis (15%). It was stained with Cybr Gold (Thermo-Fisher) and photographed with an ultraviolet transilluminator.

Incorporation by Reference References and citations to other documents such as patents, patent applications, patent gazettes, journals, books, treatises and web content have been made through this disclosure. All such documents are incorporated herein by reference in their entirety for all purposes.

Equivalents In addition to those shown and described herein, various modifications of the invention, and many further embodiments thereof, include references to the scientific and patent documents cited herein. The entire contents of the document will be apparent to those skilled in the art. The subject matter of this specification includes important information, illustrations and guidance that may be applied for the practice of the present invention in its various embodiments and their equivalents.

Claims (26)

A method of synthesizing multiple nucleic acid memory chains,
A step of providing an array of two or more substrate-linked nucleic acids with addressable delivery of activation energy to each of the substrate-linked nucleic acids.
One of the substrate-linked nucleic acids by delivering addressable activation energy to one or more of the substrate-linked nucleic acids in the presence of multiple blocked nucleotide analogs and template-independent polymerases. Or a step of extending a plurality with a homopolymeric tract of two or more repeating nucleotides, wherein the template-independent polymerase incorporates an unblocked nucleotide analog, but the blocked nucleotide analog. Is not incorporated and the addressable activation energy converts the blocked nucleotide analog into an unblocked nucleotide analog, comprising a step. The method of claim 1, wherein the blocked nucleotide analog is converted to an unblocked nucleotide analog by removal of the blocking group. The method of claim 1, wherein the addressable activation energy comprises light, reducing conditions, pH changes or heat. The method of claim 2, wherein the removable blocking group is in the blocked nucleotide analog 3'-OH. The method of claim 2, wherein the removable blocking group is on the purine or pyrimidine base of the nucleotide analog. Claim that the blocked nucleotide analog comprises a removable blocking group on deoxyribose or ribose 3'-OH of nucleotide triphosphate and an irremovable modification on the purine or pyrimidine base of the nucleotide analog. The method according to 1. With the multiple blocked nucleotide analogs, a removable 3'-O-blocking group and two or more non-removable molecular modifications that allow the distinction between modified nucleotide analogs of the same nucleobase. The method of claim 1, which is a modified nucleotide of the same nucleobase, comprising. The step of stopping the extension and
Two or more of the homopolymeric tracts by delivering addressable activation energy to the homopolymeric tracts in the presence of a plurality of blocked nucleotide analogs and said template-independent polymerases. The method of claim 1, further comprising the step of extending many repeating nucleotides with additional homopolymeric tracts. 8. The method of claim 8, wherein the extension is stopped after a predetermined length of time to obtain the desired length for the homopolymer tract. The method of claim 1, wherein the rate of elongation is regulated by modification to the blocked nucleotide analog. 10. The method of claim 10, wherein the rate-regulating modification is removed from the homopolymeric tract after elongation. The method of claim 1, wherein the rate-regulating modification is removed during elongation. The method of claim 1, wherein the repeating nucleotide of the homopolymer tract is between 2 and about 10. The method of claim 8, further comprising synthesizing a nucleic acid memory chain by repeating the stopping step and the extending step. The method of claim 14, wherein the nucleic acid memory chain is from about 200 nucleotides in length to about 5,000 nucleotides in length. The method of claim 1, wherein a predetermined concentration of the blocked nucleotide analog is provided in the stretching step to obtain the desired length for the homopolymer tract. The method of claim 8, wherein the homopolymer tract and the additional homopolymer tract contain different nucleobases. 14. The method of claim 14, wherein the nucleic acid memory strand encodes a dataset selected from the group consisting of text files, image files and audio files. 18. The method of claim 18, further comprising displaying the readable format of the dataset. The method of claim 14, wherein the unit of data is radix 2. The method of claim 14, wherein the unit of data is radix 3. The method of claim 14, wherein the unit of data is radix 4. The method of claim 14, wherein the unit of data is indicated by a radix greater than radix 4. 14. The method of claim 14, wherein the data is indicated by the degree of decaying or the tract length obtained at each step of memory chain synthesis. The method of claim 14, wherein the data encoded in the nucleic acid memory strand is read by DNA sequencing. 14. The method of claim 14, wherein the data encoded in the nucleic acid memory chain is read by passing the nucleic acid memory chain through a nanopore.

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