Patent Application
20230313255
A Sequence Listing is provided herewith in a text file “DNAS-025_SEQ_LIST_June_2023_ST25” created on Jun. 20, 2023, and having a size of 552.047 bytes. The contents of the text file are incorporated herein by reference in their entirety.
Inkjet printing is a low-cost versatile technology for non-contact delivery of defined quantities of liquids to precise locations with minimal wastage, e.g. Le. Recent Progress in Ink Jet Technologies II, chapter 1 (1999); Derby. Annu. Rev. Mater. Res., 40: 395-414 (2010); Singh et al, Advanced Materials, 22: 673-685 (2010); Tekin et al, Soft Matter, 4: 703-713 (2008); Calvert, Chem Mater., 13: 3299-3305 (2001). The technology has been applied to synthesis of oligonucleotide microarrays using phosphoramidite chemistry (e.g. Brennan, U.S. Pat. No. 5,474,796; Blanchard et al. Biosensors & Bioelectronics, 11(6/7): 687-690 (1996); Hughes et al. Nature Biotechnology, 19:342-347 (2001)) and has been employed to directly print enzymes onto substrates in the production of enzyme-based biosensors, e.g. Derby, J. Mater. Chem. 18:5717-5721 (2008) Setti et al, Biosensors & Bioelectronics, 20: 2019-2026 (2005). In regard to the latter applications of inkjet printing, it has been observed that not only is enzyme activity affected by shear forces and the rheological requirements for droplet formation, but also by the changing enzyme concentration and buffer conditions from evaporative loss when, for example, enzyme-containing fluids are printed to microarrays, e.g. Derby (cited above, 2008); Di Risio et al, Macromolecular Rapid Comm., 28(18-19): (2007); Nishioka et al, J. Amer. Chem. Soc., 126(50): 16320-16321 (2004).
Recently there has been an interest in applying enzyme-based polynucleotide synthesis to problems which are ill-suited for conventional chemically based DNA synthesis, largely because of the mild aqueous reaction conditions of the enzymatic process. e.g. Church et al. Science, 10.1126/science.1226355 (2012); Lee et al. bioRxiv, http://dx.doi.org/10.1101/348987 (2018); Godron et al. International patent publication WO2020/120442. However, in addition to the above-mentioned difficulties of inkjet-delivery of enzymes, the use of enzymes presents a host of additional problems for any automated multi-step synthesis process including, enzyme adhesion to surfaces, the need for stringent temperature and pH control to maintain enzyme activity, aggregation of enzymes resulting in loss of activity and/or clogging of tubing, reaction sites or nozzles, variations in enzyme activity in or near synthesis supports, batch to batch differences in enzyme specific activity, the formation of foams or bubbles that inhibit reagent transfer and separation, loss of efficiency from reaction of certain protection groups with environmental contaminants, such as formaldehyde, and the like.
If the above challenges could be overcome, the ability to carry out inkjet reagent delivery for enzyme-based synthesis of dense arrays of polynucleotides would provide not only a convenient desk top synthesis method using aqueous reagents without the need of extensive environmental controls, but also significant advances in several diverse fields, including DNA data storage and cell and tissue analysis, such as, by direct labeling of viable biological cells, direct synthesis of spatial barcodes on tissues, and the like, e.g. Horgan et al, International patent publication WO2020/020608; Godron et al. International patent publication WO2020/120442.
The present invention is directed to methods and compositions for inkjet-based template-free enzymatic synthesis of polynucleotides.
In some embodiments, the invention is directed to a method of enzymatically synthesizing a plurality of polynucleotides each having a predetermined sequence at distinct reaction sites on a planar substrate comprising the steps of: (a) providing a planar substrate having initiators at a plurality of distinct reaction sites, wherein each initiator has a free 3′-hydroxyl and wherein each polynucleotide of the plurality is assigned to a different reaction site for synthesis; (b) dispensing through one or more inkjet pumps at least one droplet of at least one synthesis reagent to each reaction site of the plurality to perform a reaction cycle comprising the steps of (i) reacting under elongation conditions the initiator or elongated fragments having free 3′-O-hydroxy is with a 3′-O-protected nucleoside triphosphate and a template-free polymerase so that the initiator or elongated fragments are elongated by incorporation of a 3′-O-protected nucleoside triphosphate to form 3′-O-protected elongated fragments, and (ii) deprotecting the elongated fragments to form elongated fragments having free 3′-hydroxyls, wherein the synthesis reagent comprises a template-free polymerase, a 3′-O-protected nucleoside triphosphate, a mixture of a template-free polymerase and a 3′-O-protected nucleoside triphosphate, or a deprotection solution; and (c) repeating step (b) until the plurality of polynucleotides is synthesized. In some embodiments, the polynucleotides comprise poly-2′-deoxyribonucleotides and the template-free polymerase is a template-free DNA polymerase, such as, terminal deoxynucleotidyl transferase (TdT).
In some embodiments, the invention is directed to printable template-free polymerase inks comprising: an aqueous template-free polymerase solution having a concentration of template-free polymerase in a range of from 2.0 μM to 20 μM, or from 2.0 μM to 100 μM; wherein whenever the ink is printed to a substrate, printed droplets each have a volume in the range of from 0.1 μL to 5 nL of the aqueous template-free polymerase solution and is characterized by a viscosity of about 1 centipoise to about 20 centipoise when viscosity is measured at room temperature; and a surface tension of about 15 dynes/cm and about 50 dynes/cm when measured at room temperature. In particular, such template-free polymerase inks comprise at least one terminal deoxynucleotidyltransferase (TdT) variant, a polyA polymerase (PAP) variant, or a polyU polymerase (PUP) variant. More particularly, such template-free polymerase inks comprise at least one TdT variant. In some such embodiments, the TdT inks further include a viscosity modifier. In still further embodiments, such TdT inks include a viscosity modifier and a surface tension modifier. In some embodiments, the TdT inks comprise a viscosity modifier that is also a humectant. In further embodiments, the template-free polymerase inks of the invention comprise one or more dyes. Such dyes may be fluorescent or non-fluorescent, or a mixture thereof. In some embodiments, the one or more dyes are fluorescent dyes. In some embodiments, at least one of such dyes is a separate component of an ink (which may be either a template-free polymerase ink or a non-template-free polymerase ink) for delivering a synthesis reagent, such as, a template-free polymerase, such that the dye permits optical detection to provide information as to the size, spacing and possible overlap of reaction sites, quantity of synthesis reagent delivered, and the like.
The present invention further includes the use of dye-labeled 3′-O-reversibly protected nucleoside triphosphates as monomers, wherein a coupling reaction cycle comprises the steps of (i) reacting under elongation conditions an initiator or elongated fragments having free 3′-O-hydroxyls with a base-labeled-3′-O-protected nucleoside triphosphate and a template-free DNA polymerase so that the initiator or elongated fragments are elongated by incorporation of a base-labeled 3′-O-protected nucleoside triphosphate to form 3′-O-protected elongated fragments each having a labeled base. (ii) measuring a quantity of labeled bases at each reaction site, (iii) removing labels of the labeled bases, and (iv) deprotecting the elongated fragments to form elongated fragments having free 3′-hydroxyls.
In embodiments employing 3′-O-amino-nucleoside triphosphate monomers, template, template-free polymerase inks of the invention further comprise an effective amount of an aldehyde scavenger to minimize or eliminate the generation of spuriously capped elongated fragments when adventitious aldehydes, ketones, and like compounds, react with 3′-O-amines to form oximes.
While the invention is amenable to various modifications and alternative forms, specifics thereof have been shown by way of example in the drawings and will be described in detail. It should be understood, however, that the intention is not to limit the invention to the particular embodiments described. The intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention. Guidance for selecting materials and components to carry out particular functions may be found in available treatises and references on scientific instrumentation including, but not limited to, Moore et al, Building Scientific Apparatus, Third Edition (Perseus Books, Cambridge. MA); Hiermanson, Bioconjugate Techniques, 3rd Edition (Academic Press, 2013); and like references.
In some embodiments, the invention is directed to methods and compositions for inkjet assisted synthesis of a plurality of polynucleotides each at a distinct reaction site on a substrate using template-free polymerases, such as, terminal deoxynucleotidyl transferases (TdTs). Typically, such synthesis takes place on substrates comprising a planar surface, such as, glass, silica, silicon oxide, plastic, or like surfaces, but it may also take place on other surfaces, such as, for example, biological tissues, or surface-immobilized cDNAs extracted from tissues. As used herein, “inkjet assisted synthesis” means that one or more synthesis reagents are delivered to reaction sites in droplets generated by one or more inkjet pumps. “Synthesis reagents” include any reagent used in a synthesis cycle to couple a monomer, particularly a 3′-O-protected-nucleoside triphosphate, to an initiator or elongated fragment, such as, buffers comprising a template-free polymerase, buffers comprising 3′-O-protected-nucleotide monomers, buffers comprising a mixture of a template-free polymerase and one or more 3′-O-protected nucleoside triphosphates, deprotection (or deblocking) buffers, and the like. The terms “deprotection” agent, buffer, solution, or the like, are used synonymously herein to the terms “deblocking” agent, buffer, solution, or the like, respectively. Likewise, the term “protected” in reference to compounds, such as, dNTPs, is used synonymously with the term “blocked” in reference to compounds. As used herein, the term “deprotection solution” (or its equivalent terms) means a reagent that brings about or promotes the removal of a protection group, for example, a 3′-O-protecting group of a nucleotide. As described more fully below, the composition of a deprotection solution (and deprotection reaction conditions) depends on the nature of the protecting group (or blocking group) which is to be removed. In various embodiments, a deprotection solution may contain specific reagents that chemically react with a protection group and/or protected moiety (such as, a reducing agent like TCEP (tris(2-carboxyethyl)phosphine)), enzymes for enzymatic cleavage, scavengers, co-factors, or the like. In some embodiments, a deprotection solution may not contain specific reagents that react with a protection group, but may contain components. e.g. pH buffers, that are compatible with or promote physical cleavage of a protecting group, such as in the case of a photocleavable protecting group. Typically, in a reaction cycle for elongating a polynucleotide fragment, in a deprotecting step a deprotection solution is incubated with 3′-O-protected elongated fragments for a predetermined incubation time. Typical incubation times (i.e. durations of incubating steps) are in the range of from 1 minute to 30 minutes; or in the range of from 3 minutes to 30 minutes; or in the range of from 3 minutes to 15 minutes. Typical elongation reaction temperatures are in the range of from room temperature (RT) to 80° C.; or from 200′C to 80° C.; or from 20° C. to 60° C. “Synthesis reagents” also include reagents for preparing a substrate for polynucleotide synthesis, such as, reagents for defining reaction sites, initiators, capping reagents, and the like. Usually, a “distinct reaction site” on a substrate is a discrete site in that it is separated from other reaction sites; that is, a discrete site does not have a border with, or overlap with, another reaction site, such as exemplified in
In some embodiments, the plurality of polynucleotides may be in the range of from 2 to 500,000; or in the range of from 100 to 400,000; or in the range of from 100 to 200.000; or in the range of from 100 to 100.000. The plurality of polynucleotides may be the same or different than the plurality of reaction sites. In some embodiments, the plurality of reaction sites may be greater than the plurality of polynucleotides. In some embodiments, the above pluralities of reaction sites each have a density equivalent to that if uniformly deposited on an area equivalent to that of a standard 25 mm×75 mm microscope slide. In some embodiments, an array of reaction sites formed by uniform deposition may be a rectilinear array; and in other embodiments, an array of reaction sites formed by uniform deposition may be a hexagonal array.
The basic steps of template-free enzymatic synthesis of polynucleotides is illustrated in
In accordance with the invention, inkjet assisted enzymatic synthesis of polynucleotides may be implemented in a variety of embodiments in which different reagents are delivered by inkjet pumps. Some of these embodiments are described in
An embodiment of
An embodiment of
An embodiment of
In some embodiments of
In some embodiments, the droplet evaporation problem may be addressed by using a droplet-in-oil array as described by Sun et al (LabChip, 11: 2429-2436(2015)), in which aqueous reagents are delivered onto silicone oil droplets: (1) Mineral oil is first printed in a pattern on a silanized silicon dioxide surface with excellent hydrophobic and oleophobic properties using a 50 μm nozzle. (2) A first round printing of aqueous reagent A is performed on top of the preformed oil drops in the same pattern but with a 30 μm nozzle. The ejected droplets carrying reagent A penetrate the oil droplets at high velocity, overcoming their surface tension and viscosity. Subsequently, the aqueous droplets sink to the bottom of the less-dense mineral oil droplets, forming stable droplet-in-oil structures. (3) During the second-pass printing of aqueous reagents, reagent B is inkjet-printed on the just formed droplet-in-oil array of reagent A. Thus, the dispensed reagent B droplet also penetrates the oil drop due to its high velocity and immediately merges with the preformed droplet A inside the same oil drop.
In
An embodiment of
As noted above, embodiments of the method of the invention may include one or more washing steps wherein a wash solution is flowed or sprayed on a substrate comprising an array of reaction sites. Wash solution may comprise a variety of solvents including, but not limited to, water, acetonitrile, methanol, PBS or other buffered salt solutions, or the like. In some embodiments, a wash solution may include one or more proteases. e.g. proteinase K, for the purpose of removing any polymerases that may adhere to the reaction site. That is, the embodiments of
Although
In some embodiments, including those described above, the plurality of polynucleotides enzymatically synthesized (that is, the number of reaction sites) on a substrate with inkjet delivery of reagents is in the range of from 100 to 2 million, or in the range of from 100 to 1 million, or in the range of from 100 to 100 thousand, or in the range of from 100 to 500 thousand, or in the range of from 1000 to 1 million. In some embodiments, such pluralities are synthesized on a substrate having a surface area in the range of from 1 to 500 cm2, or from 1 to 256 cm2, 1 to 30 cm2, or having a surface area in the range of from 1 to 15 cm2, or having a surface area in the range of from 1 to 7 cm2, or having a surface area in the range of from 7 to 20 cm2. In some embodiments, substrates may be prepare and undergo surface treatment after which it is cut, or diced, into smaller pieces for use. In some embodiments, the lengths of the polynucleotides synthesized in accordance with the invention are in the range of from 10 to 500 nucleotides, or in the range of from 50 to 500 nucleotides, or in the range of from 100 to 400 nucleotides, or in the range of from 100 to 500 nucleotides. In some embodiments, the per cycle coupling efficiency in the synthesis of polynucleotides in these length ranges is at least 98%, or is at least 99%, or is at least 99.5%, or is at least 99.8%, or is at least 99.9%. In some embodiments, the coupling cycle time in the synthesis of polynucleotides in these length ranges is less than 15 min per cycle, or less than 10 min per cycle, or less than 7 min per cycle, or less than 5 min per cycle.
In some embodiments, inkjet delivery of droplets may be directed to features on a substrate which have a dimension directly related to its size or area, such as a width of a square reaction site or a diameter of a round reaction site. Thus, in some embodiments, reaction sites have a width or diameter in the range from about 10 μm to about 1.0 cm. In some embodiments droplets can be deposited to reaction sites whose widths, or diameters, are in the range of from about 1.0 μm to about 1.0 mm, usually about 5.0 m to 500 μm, more usually about 10 μm to 200 μm, and still more usually from about 20 μm to about 100 μm.
In some embodiments, the volume of reagent ink delivered to a reaction site is in the range of 0.1 to 1000 μL, or in the range of from 0.5 to 500 μL. or in the range of from 1.0 to 250 pL, or in the range of from 1.0 to 100 μL. or in the range of from 2 to 50 pL. In some embodiments, reagent ink is delivered to each reaction site in a predetermined number of droplets, or “pulses,” generated by a print head wherein, for example, each pulse has about a 2.4 picoliter volume.
Delivering fluids by inkjets is a mature technology that has been available for several decades so that extensive literature is available describing it and providing guidance for adapting it to novel applications, as in the present invention. Exemplary references providing guidance for constructing inkjet delivery systems: Lausted et al, Genome Biology, 5: R58 (2004); Le, Recent Progress in Ink Jet Technologies II, chapter 1, pgs, 1-14 (1999); Derby (2010, cited above); Zapka, editor, “Handbook of Industrial Inkjet Printing.” (Wiley-VCH. Weinheim, Germany); U.S. Pat. Nos. 5,474,796; 10384189; 10669304; 6306; 6323043; 5847105; and the like. As noted by Le (1999) inkjet pumps may be classified as “continuous” and “drop-on-demand” (DOD). In some embodiments. DOD inkjet pumps are employed with apparatus of the invention, and in particular, of the various DOD inkjets, piezoelectric inkjet pumps are of interest. For example, droplet formation in DOD inkjets is described in Dong et al. Physics of Fluids, 18: 072102 (2006). Such varieties of inkjet pumps are available banks or assemblies of large numbers of inkjets (e.g. from 10's to 100's) that may be individually programmed for actuation and delivery of droplets. Such inkjets and inkjet assemblies (referred to herein as “inkjet heads”) are commercially available from many manufacturers including Epson. Xaar. Fujifilm, and the like. As used herein, “inkjet pump” means a device capable of generating and ejecting droplets of a fluid. In some embodiments, an inkjet pump is a device capable of generating and ejecting droplets of a fluid at a predetermined rate and of a predetermined uniform size. In some embodiments, an inkjet pump is capable of ejecting droplets each having approximately the same size in the range of from 0.1 μL to 5.0 nL, or the same size in the range of from 0.5 μL to 1.0 nL. In some embodiments, an inkjet pump is capable of ejecting droplets at a rate in the range of from 1 to 100 kilohertz.
In some embodiments, components of an inkjet apparatus of the invention may be arranged according to whether they may be moved relative to one another or whether they are fixed, as illustrated in
In some embodiments, cameras or microscopes may be used to capture images of the spots (i.e. reaction sites) and identify missing spots, determine spot size and spot placement. Lighting for image capture may be from above, from the side, from below or integrated into a substrate holder, whichever gives the best contrast in the absence or presence of dye in the inks. Where a dye is used (as described below), it is selected so that it would not interfere with the enzymatic reaction, would not react with the protecting group of the nucleotide and would be compatible with the enzyme and deprotection buffers. In some embodiments, each monomer would have a different distinguishable dye, covering a different part of the visible spectrum. In some embodiments, imaging of an array of reaction site is carried out during incubation (30 s-10 min) of the elongation reactions and using high enough magnification to see individual spots but not so high that an inordinate length of time would be needed to scan the array. The number of images taken in an imaging step may be 20 to 100 for a standard microscope slide. Images may be captured seamlessly in a video stream by scanning the substrate or captured in a move-stop process. The images captured may be stitched using algorithms and aided by the presence of fiducial markings on the slide. Fiducial markings also help determine whether the slide has moved in the slide holder and help determine spot positions. In some embodiments, real time image analysis allowing the identification of missing spots or poor spot placement could be accompanied by the automatic generation of a new image and an additional print or prints.
An exemplary inkjet apparatus for implementing various embodiments of the invention (e.g. those of
Typically, the distance between the inkjet nozzles and the substrate surface may be in the range of from about 10 μm to 10 mm, or in the range of from about 100 μm to 2 mm, or in the range of from about 200 μm to 1 mm, or in the range of from 500 μm to 3 mm. Droplet velocities may be in the range 1-10 meters/sec. Print head movement may be in the range of from 1-30 cm/sec, or 5-30 cm/sec. or 20-30 cm/sec. As described more fully below, print heads may have different droplet delivery modes, for example, single-pass mode, multiple pass mode, and move-stop mode.
As mentioned above, in some embodiments, nozzle diameters for use with the invention may be in the range of from 10 μm to 100 μm. In other embodiments, inkjet nozzle size may be in the range of from 20-30 μm for generating droplet sizes in the range of from 10-20 μL. In some embodiments, nozzle diameter, synthesis reagent density, surface tension and viscosity are selected to dispense droplets to reaction sites having a volume in the range of from 2 μL to 5 nL, or in the range of from 2 μL to 1 nL, or in the range from 2 μL to 500 μL, or in the range from 2 μL to 100 μL. In some embodiments, inkjet pumps are DOD inkjet pump and have a droplet generation rate in the range of from 1 to 100 kHz.
In some embodiments, inkjet-based synthesizers include droplet detection components to monitor and record any anomalies in droplet formation and delivery by the inkjet nozzles. In some embodiments, such droplet monitoring may comprise a laser diode mounted orthogonally to the direction of print-head motion such that the droplet stream of each bank of nozzles intersects the beam, causing the light to scatter if a droplet is present. Before each round of printing, nozzles may be fired in series through the beam and the forward scattering of each droplet is detected by a photodiode. Nozzles failing to fire may be taken off-line during synthesis. Apparatus of the invention may also be equipped with commercially available droplet monitors, such as, a Meteor dropwatcher, available from Meteor Inkjet Ltd, (Cambridge. UK) as well as a camera to image the solid support and array of reaction sites. The latter permits the array of reaction sites to be monitored to detect accuracy in droplet deposition, size and geometry of reaction sites, coalescence of reaction sites, and the like. In some embodiments, software may be provided to provide a full image of an array on a slide or solid support by patching together tiles comprising smaller images, e.g. S. Preibisch, S. Saalfeld, P. Tomancak, Bioinformatics, 2009, 25(11), 1463-1465.
In certain embodiments, it may be desirable to prevent evaporation of the synthesis reagents and reaction mixtures following deposition. Evaporation may be prevented in a number of different ways. In some embodiments, synthesis cycles may be carried out in a high humidity environment, such as a relative humidity in the range of from 75-85%. Alternatively or in addition to, one may employ reagents with an evaporation retarding agent or humectant. e.g. glycerol, polyethylene glycol, carboxymethyl cellulose, hydroxyethyl cellulose, and the like.
In some embodiments, recirculating ink print heads are employed because problems of drying and/or clogging of nozzles by enzymes is reduced. Recirculating ink print heads are commercially available, for example, from Fujifilm and are described in U.S. Pat. No. 8,820,899; 8534807; 8752946; 9144993; 9511598; 9457579, which are incorporated herein by reference.
In some embodiments, substrates for synthesis comprise surfaces that have been patterned with hydrophobic and hydrophilic regions wherein discrete hydrophilic reaction sites are formed. These allow the formation of droplets on hydrophilic reaction sites, for example, after flowing aqueous reagents or reactants of the entire surface. That is, in some embodiments, substrates for synthesis comprise so-called “droplet microarrays,” e.g. as disclosed in the following exemplary references, which are incorporated by reference: Brennan, U.S. Pat. No. 5,474,796: Chrisey et al. Nucleic Acids Research, 24(15): 3040-3047 (1996); Fixe et al, Materials Research Society Symposium Proceedings. Volume 723, Molecularly Imprinted Materials—Sensors and Other Devices. Symposia (San Francisco. California on Apr. 2-5, 2002); Goldfarb. U.S. patent publication 2008/0166667; Gopinath et al, ACS Nano, 8(12): 12030-12040 (2014); Hong et al, Microfluid. Nanofluid., 10: 991-997 (2011); Kumar et al, Nucleic Acids Research, 28(14): e71 (2000); Peck et al, U.S. patent Ser. No. 10/384,189; Indermuhle et al, U.S. patent Ser. No. 10/669,304; Wu et al, Thin Solid Films, 515: 4203-4208 (2007); Zhang et al, J. Phys. Chem., 111: 14521-14529 (2007): and like references. As used herein, the term “droplet microarray” refers to a planar substrate whose surface has been treated to create a plurality of discrete hydrophilic regions, which may serve as reaction sites either directly or with further treatment. e.g. attaching initiators. In some embodiments, each of the plurality of discrete hydrophilic regions are surrounded by hydrophobic regions. The discrete hydrophilic regions may have a variety of shapes, but are usually circular or rectangular or square for manufacturing convenience. In some embodiments, reaction sites have areas and capacities to hold an aqueous reaction mixture as described above. Although synthesis substrates of some embodiments may comprise droplet microarrays, in a synthesis process such arrays may undergo a drying step which removes liquid from reaction sites. That is, in some embodiments, a synthesis substrate comprising a droplet microarray may be devoid of droplets from time to time, for example, after an elongation cycle ending in a drying step. The hydrophilic-hydrophobic configurations permit the formation of droplets on the surface of a droplet microarray either after inkjet delivery of a synthesis reagent to the hydrophilic regions or by flowing a “bulk” aqueous solution, such as a synthesis reagent or wash solution, over the substrate. As disclosed in the above references, the droplets retained by the hydrophilic regions may serve as reaction chambers or vessels. Such a process is illustrated in
Preparation of substrates with discrete reaction sites can be accomplished by known methods. For example, such methods can involve the creation of hydrophilic reaction sites by first applying a protectant, or resist, over selected areas over the surface of a substrate, such as a silicon oxide, or like material. The unprotected areas are then coated with a hydrophobic agent to yield an unreactive surface. For example, a hydrophobic coating can be created by chemical vapor deposition of (tridecafluorotetrahydrooctyl)-triethoxysilane onto the exposed oxide surrounding the protected circles. Finally, the protectant, or resist, is removed exposing the well regions of the array for further modification and nucleoside synthesis using the high surface tension solvents described herein and procedures known in the art such as those described by Maskos & Southern, Nucl. Acids Res. 20:1679-1684 (1992). Alternatively, the entire surface of a glass plate substrate can be coated with hydrophobic material, such as 3-(1,1-dihydroperfluoroctyloxy)propyltriethoxysilane, which is ablated at desired loci to expose the underlying silicon dioxide glass. The substrate is then coated with glycidyloxypropyl trimethoxysilane, which reacts only with the glass, and which is subsequently “treated” with hexaethylene glycol and sulfuric acid to form an hydroxyl group-bearing linker upon which chemical species can be synthesized (Brennan, U.S. Pat. No. 5,474,796). Arrays produced in such a manner can localize small volumes of solvent within the reaction site by virtue of surface tension effects (Lopez et al., Science 260:647-649 (1993)).
In some embodiments, reaction sites may be formed on a substrate following the photolithographic methods of Brennan, U.S. Pat. No. 5,474,796; Peck et al, U.S. patent Ser. No. 10/384,189; Indermuhle et al, U.S. patent Ser. No. 10/669,304; Fixe et al (cited above); or like references cited above. In accordance with these methods, a set of hydrophilic molecules comprising an aminosilane is attached to the surface of a substrate to form reaction sites. Such hydrophilic molecules may comprise N-(3-triethoxysilylpropyl)-4-hydroxybutyramide (HAPS), 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane, 3-glycidoxypropyltrimethoxysilane (GOPS), or 3-iodo-propyltrimethoxysilane. A set of hydrophobic molecules comprising a fluorosilane is attached to the surface of the substrate in regions outside of the reaction sites. Such hydrophobic molecules may comprise perfluorooctyltrichlorosilane octylchlorosilane, octadecyltrichlorosilane. (tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane, or tridecafluoro-1,1,2,2-tetrahydrooctyl)trimethoxysilane. After such attachment, a substrate is prepared for polynucleotide synthesis by coupling initiators to the aminosilanes at the reaction sites. Such coupling may be accomplished using any number of available homo- or heterobifunctional linkers to form covalent bonds between amino groups on the substrate and 5′-thiol groups or 5′-amino groups on the initiators. Such linkers are, for example, available from Sigma-Aldrich (St. Louis, MO) and are described in treatises such as, Hermanson. Bioconjugate Techniques, 3rd Edition (Academic Press, 2013). Synthesis of oligonucleotides having 5′-thiol or 5′-amino groups is well-know and is described in Kupihar et al, Nucleosides Nucleotides & Nucleic Acids, 22(5-8): 1297-1299 (2003); Fung et al, U.S. Pat. No. 4,757,141; and like references.
In some embodiments, an array of reaction sites may be formed using click chemistry by depositing under coupling conditions droplets of 5′-DBCO (dibenzocyclooctyl) labeled initiators (e.g. Glen Reseach) on a planar substrate comprising an azide layer (e.g. PolyAn 2D azide glass slide). In some embodiments, such reactions may be carried out as a copper-free click reaction which is less damaging to the DNA. e.g. Donnerholt et al. Top. Curr. Chem. (Z) 374: 16 (2016).
A wide variety of substrates may be employed for creating arrays of reaction sites for enzymatic synthesis of polynucleotides. Substrates may be a rigid material including, without limitation, glass; fused silica; silicon such as silicon dioxide or silicon nitride; metals such as gold or platinum; plastics such as polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, and any combination thereof. A rigid surface can be fabricated from a material selected from the group consisting of silicon, polystyrene, agarose, dextran, cellulosic polymers, polyacrylamides, polydimethylsiloxane (PDMS), and glass. Substrates may also comprise flexible materials, which is capable of being bent, folded or similarly manipulated without breakage. Exemplary flexible materials include, without limitation, nylon (unmodified nylon, modified nylon, clear nylon), nitrocellulose, polypropylene, polycarbonate, polyethylene, polyurethane, polystyrene, acetal, acrylic, acrylonitrile, butadiene styrene (ABS), polyester films such as polyethylene terephthalate, polymethyl methacrylate or other acrylics, polyvinyl chloride or other vinyl resin, transparent PVC foil, transparent foil for printers. Poly(methyl methacrylate) (PMMA), methacrylate copolymers, styrenic polymers, high refractive index polymers, fluorine-containing polymers, polyethersulfone, polyimides containing an alicyclic structure, rubber, fabric, metal foils, and any combination thereof.
In some embodiments, patterned surfaces of superhydrophobic and superhydrophilic regions may be formed on a substrate. Guidance for forming droplet microarrays with such patterned surfaces are described in the following references, which are incorporated by reference: Feng et al, Adv. Mater. Interfaces, 1400269 (2014); Zhan et al. Trends Anal. Chem., 108: 183-194 (2018); Neto et al, Adv. Functional Mater., 201400503 (2014);
Achieving accurate alignment of droplet delivery to reaction sites of a prefabricated droplet microarray is an important aspect of inkjet-assisted synthesis of polynucleotides. In some embodiments, such alignment tasks may be minimized or avoided by creating immediately prior to synthesis an array of reaction sites by depositing droplets of synthesis reagents onto a layer of initiator oligonucleotides on a substrate in order to define the locations of reaction sites. Following this initial deposit of droplets, the initiator layer outside of the droplet-defined sites are treated to render them inert to subsequent extension or to render them inert to extension as well as hydrophobic. After such an initial surface treatment to create reaction sites, further or subsequent inkjet delivery of droplets to the same reaction sites will be accurate because the same inkjet head and pumps that were used to define the locations of the reaction sites will be used to deliver subsequent droplets during synthesis of the polynucleotides. In some embodiments, the synthesis reagents delivered to the initiator layer comprise a mixture of a template-free polymerase and a 3′-O-protected-dNTP. These reagents extend the initiators to define reaction sites or regions on the oligonucleotide layer which is populated by extended fragments having 3′-O-protected ends. The areas outside of these regions are then treated to render them inert to extensions. In some embodiments, after the initial coupling step defining reaction sites, the entire substrate is exposed to a template-free polymerase and a terminator, such as a dideoxynucleoside triphosphate (ddNTP), or like reagent. In some embodiments, such ddNTP could be, for example, a ddNTP conjugated to a hydrophobic moiety, thereby rendering the coating outside of the reaction sites hydrophobic. Such a hydrophobic moiety may be, for example, a dye or quencher molecule, such as, a Black Hole Quencher® molecule. A variety of terminators may be employed for this purpose. In particular, terminators include nucleoside triphosphates that lack a 3′-hydroxyl substituent and include 2′,3′-dideoxyribose, 2′,3′-didehydroribose, and 2′,3′-dideoxy-3′-haloribose, e.g. 3′-deoxy-3′-fluoro-ribose or 2′,3′-dideoxy-3′-fluororibose nucleosides. Alternatively, a ribofuranose analog can be used in terminators, such as 2′,3′-dideoxy-p-D-ribofuranosyl, β-D-arabinofuranosyl, 3′-deoxy-β-D-arabinofuranosyl, or the like. Further terminators are disclosed in the following references: Chidgcavadze et al., Nucleic Acids Res., 12: 1671-1686 (1984); Chidgcavadze et al., FEBS Lett., 183: 275-278 (1985); Izuta et al, Nucleosides & Nucleotides, 15: 683-692 (1996); and Krayevsky et al, Nucleosides & Nucleotides, 7: 613-617 (1988). Nucleotide terminators also include reversible nucleotide terminators, e.g. Metzker et al. Nucleic Acids Res., 22(20):4259 (1994).
Thus, in such embodiments, a starting material for a synthesis operation is a surface coated with a layer of initiator oligonucleotides. An exemplary fabrication of reaction site on such starting material is illustrated in
An embodiment of the invention for synthesizing a plurality of polynucleotides employing reaction site formation as described in
An aspect of the invention is a method for preparing an array of reaction sites for template-free enzymatic synthesis of a plurality of polynucleotides. In some embodiments, such method of array preparation may be carried out by the steps of (a) providing a surface with initiators attached. (b) delivering with one or more inkjet pumps droplets to a plurality of distinct locations on the surface to form a plurality reaction sites, the droplets containing a synthesis reagent that reacts with initiators in the reaction sites to remove 3′-O-protecting groups or to elongate such initiators by addition of a 3′-O-protected nucleoside triphosphate, and (c) capping initiators on the surface outside of the reaction sites. In some embodiments, initiators on the surface of step (a) have free 3′-hydroxyls and the synthesis reagent delivered in step (b) comprises a template-free polymerase and a 3′-O-protected nucleoside triphosphate, so that the template-free polymerase catalyzes the addition of the 3′-O-protected nucleoside triphosphate to produce 3′-O-protected elongated fragments within the reaction sites. Thus, initiators outside of the reaction sites may be capped by immersion of the surface in a capping reagent (such as a mixture containing a dideoxynucleoside triphosphate and template-free polymerase). In some embodiments, initiators on the surface may have 3′-O-protection groups and the synthesis reagent delivered by droplets may contain a deprotection agent that removes the 3′-O-protection groups from initiators to form reaction sites. In the newly formed reaction sites, a reagent is delivered which contains 3′-O-protected nucleoside triphosphates and a template-free polymerase, wherein the protection group of the delivered nucleoside triphosphate is orthogonal to that of the initiators of the surface. Exemplary orthogonal 3′-O-protection groups are described below. For example, such orthogonal protection groups may be azidomethyl and amino.
One of ordinary skill would appreciate that similar reaction site formation can be implemented for other embodiments, such as those described in
In another embodiment, a beginning layer of initiator oligonucleotides all have 3′-O-amino-protected or 3′-O-azidomethyl-protected ends. The process steps in this embodiment are similar to those of
In another embodiment, a buffer comprising a template-free polymerase/3′-O-protected-dNTP mixture is printed on the initiator oligonucleotide layer with free 3′-hydroxyls as described above to define reaction sites having extended initiators with 3′-O-protected ends. The surface outside these defined sites is then treated with template-free polymerase and azide or alkyne derivatized ddNTP to block further 3′ extensions. A hydrophobic molecule with a complementary click chemistry group (e.g. DBCO, benzyl-azide) may then be reacted with the ddNTP terminator to render the surface outside of the reaction sites hydrophobic. Exemplary click chemistry pairs are described in Feng et al. Adv. Mater. Interfaces, 1400269 (2014).
In still another embodiment, to a substrate surface without a layer of initiator oligonucleotides, a buffer comprising initiator oligonucleotides having 5′ linker groups is inkjet printed on the surface derivatized with a complementary reactive group (e.g. epoxy, azide/alkyne so that the initiators are attached to the surface by their 5′-ends. To these attached initiators, cycles of coupling reactions can take place in accordance with the invention. Also, unreacted complementary reactive groups may be quenched by reacting them with an inert group (e.g. ethanolamine for epoxy) and the inert group may be selected to have a hydrophobic character.
In some embodiments, substrates for synthesis may include surface-bound cDNAs copied from messenger RNA extracted from a fixed or non-fixed tissue slice. Procedures for placing tissue slices on a planar array of oligonucleotides, identifying and imaging tissue features (such as cell boundaries), permeablizing cells of tissues, implementing reverse transcriptase reactions to produce a cDNA library attached to a planar array are disclosed in Stahl et al, Science, 353: 78-82 (2016); and Frisen et al, U.S. Pat. No. 9,593,365 and 10030261; and like references, which are incorporated herein by reference. Briefly, referring to
The image information is used to define regions on array (564) within which common position tags are synthesized on cDNAs. Treatments may include staining with tissue-specific or biomolecule-specific compounds or dyes. The position tags allow cDNAs to be harvested and sequenced in bulk, yet be related to specific regions by their position tags. After the above steps (i) and (ii), reagents for a reverse transcriptase reaction are applied in order to synthesize cDNAs (571) using captured mRNAs (570) as templates to produce a spatial cDNA library array. Tissue slice (581) is then removed leaving array (564) with a pattern of different cDNAs attached to its surface. The different cDNAs at the different positions may be identified and quantified by attaching position tags to samples of cDNAs from a plurality of locations by inkjet delivery of synthesis reagents for the tags, which is illustrated in
In some embodiments, this application of the invention may be carried out with the following steps: (a) providing an array comprising a uniform coating of capture probes each comprising a capture segment; (b) contacting a tissue sample with the array and allowing the nucleic acid of the tissue sample to interact with the capture domain of the capture probe so that the nucleic acid is captured; (c) treating the tissue sample to identify different regions of the tissue sample; (d) generating a nucleic acid molecule from the nucleic acid that interacts with the capture domain; (e) enzymatically synthesizing position tags onto the nucleic acid molecules; (f) determining the region that is associated with the nucleic acid that interacts with the capture domain; and (e) correlating the determined regions to the cDNAs. In some embodiments, the nucleic acid molecules from the tissue sample is RNA. In other embodiments, the nucleic acid molecules from the tissue sample may be genomic DNA. In other embodiments, the nucleic acid molecules from the tissue sample may be mRNA. In some embodiments, the step of enzymatically synthesizing position tags onto the nucleic acid molecules is carried out by inkjet delivery of synthesis reagents to the locations of the position tags in accordance with methods of the present invention.
In typical inkjet synthesis applications, an array of distinct non-overlapping reaction sites is defined by repeated deposition of reagents. Usually, such reaction sites are roughly circular regions having diameters in the range of from about 20-50 μm. Thus, for some applications, such as those described in
The formation of arrays of reaction sites with predetermined locations is illustrated in
An example of how overwriting may increase the density of spatial barcoding is illustrated in
The translation of the positions of a subsequent array relative to a previous array may be carried out using two XY-stages, one to move the print head to generate an array of oligonucleotides, and another to provide the offset or new position of the reference reaction site relative to the reference reaction site of a previously synthesized army. The two XY stages may be used in tandem. e.g. one mounted on top of the other, or one may be used to move the print head, which the other moves the mounting stage holding the substrate. In other embodiments, a single XY stage that can be programmed to generate the desired offsets may be used. For example. Offsets can also be introduced by using high dpi print heads and choosing whether to switch on certain nozzles in the y direction that were not previously being used.
As illustrated in
As illustrated in
In some embodiments, the invention is directed to a method of enzymatically synthesizing a plurality of oligonucleotide barcodes each having a predetermined sequence at distinct predetermined regions of substrate, such method comprising the steps of: (a) providing a substrate with a surface comprising a coating of initiators, wherein each initiator has a free 3-hydroxyl; (b) determining a position of a reference reaction site of an array of a plurality of reaction sites, the position of each reference reaction site after the first reference site position is selected so that at least one reaction site of the array overlaps a reaction site of the previous array; (c) synthesizing an oligonucleotide in each reaction site of the array by (i) dispensing through one or more inkjet pumps at least one droplet of at least one synthesis reagent to each reaction site of the plurality to perform a reaction cycle comprising the steps of (A) reacting under elongation conditions the initiator or elongated fragments having free 3′-O-hydroxyls with a 3′-O-protected nucleoside triphosphate and a template-free DNA polymerase so that the initiator or elongated fragments are elongated by incorporation of a 3′-O-protected nucleoside triphosphate to form 3′-O-protected elongated fragments, and (B) deprotecting the elongated fragments to form elongated fragments having free 3′-hydroxyls, wherein the synthesis reagent comprises a template-free polymerase, a 3′-O-protected nucleoside triphosphate, a mixture of a template-free polymerase and a 3′-O-protected nucleoside triphosphate, or a deprotection solution; and (ii) repeating a predetermined number of times step (i) until the plurality of oligonucleotides is synthesized; (c) repeating steps (b) and (c) until the plurality of oligonucleotide barcodes are synthesized each having a predetermined sequence at predetermined regions of the substrate comprising overlapping and non-overlapping reaction sites. In some embodiments, each array of step (b) is the same except for the location of its reference reaction site. In some embodiments, each array of step (b) has the same plurality of reaction sites and the same pattern and inter-reaction site distances. In some embodiments, arrays of reaction sites are identical rectilinear arrays (such as illustrated in
Generally, methods of template-free (or equivalently. “template-independent”) enzymatic polynucleotide synthesis comprise repeated cycles of steps, such as are illustrated in
In the present invention synthesis reagents delivered by inkjet pumps must be formulated to satisfy at least two constraints: (i) the need to preserve the elongation activity of the template-free polymerase (in the case of template-free polymerase inks), and (ii) the need to meet the rheological requirements for droplet formation. The key solution parameters affecting droplet formation by inkjets are viscosity, surface tension, liquid density and the diameter of the inkjet nozzle. For particular embodiments of the invention, synthesis reagents prepared for non-droplet delivery to a reaction mixture may be reformulated by adding viscosity modifiers, surface tension modifiers and density modifiers, and the like, in order to form “printable inks” that may be delivered in droplets generated by inkjet pumps. “Printable” in reference to a reagent ink means repeatable droplets are able to be ejected from the nozzle, with uniform velocities and volumes and without satellite droplets.
As illustrated in
If the elongated initiator polynucleotide is not a completed sequence (i.e. the end product), then the 3′-O-protection groups are removed to expose free 3′-hydroxyls (130) and the elongated initiator polynucleotides are subjected to another cycle of nucleotide addition and deprotection.
As used herein, an “initiator” (or equivalent terms, such as “initiating fragment,” “initiator nucleic acid.” “initiator oligonucleotide.” or the like) usually refers to a short oligonucleotide sequence with a free 3′-hydroxyl at its end, which can be further elongated by a template-free polymerase, such as TdT. In one embodiment, the initiating fragment is a DNA initiating fragment. In an alternative embodiment, the initiating fragment is an RNA initiating fragment. In some embodiments, an initiating fragment possesses between 3 and 100 nucleotides, in particular between 3 and 20 nucleotides. In some embodiments, the initiating fragment is single-stranded. In alternative embodiments, the initiating fragment may be double-stranded. In some embodiments, an initiator oligonucleotide may be attached to a synthesis support by its 5′end; and in other embodiments, an initiator oligonucleotide may be attached indirectly to a synthesis support by forming a duplex with a complementary oligonucleotide that is directly attached to the synthesis support, e.g. through a covalent bond. In some embodiments a synthesis support is a solid support which may be a discrete region of a solid planar solid, or may be a bead.
In some embodiments, an initiator may comprise a non-nucleic acid compound having a free hydroxyl to which a TdT may couple a 3′-O-protected dNTP, e.g. Baiga. U.S. patent publications US201910078065 and US2019/0078126.
After synthesis is completed polynucleotides with the desired nucleotide sequence may be released from initiators and the synthesis supports by cleavage.
A wide variety of cleavable linkages or cleavable nucleotides may be used for this purpose. In some embodiments, cleaving the desired polynucleotide leaves a natural free 5′-hydroxyl on a cleaved strand; however, in alternative embodiments, a cleaving step may leave a moiety, e.g. a 5′-phosphate, that may be removed in a subsequent step, e.g. by phosphatase treatment. Cleaving steps may be carried out chemically, thermally, enzymatically or by photochemical methods. In some embodiments, cleavable nucleotides may be nucleotide analogs such as deoxyuridine or 8-oxo-deoxyguanosine that are recognized by specific glycosylases (e.g. uracil deoxyglycosylase followed by endonuclease VIII, and 8-oxoguanine DNA glycosylase, respectively). In some embodiments, cleavage may be accomplished by providing initiators with a deoxyinosine as the penultimate 3′ nucleotide, which may be cleaved by endonuclease V at the 3′ end of the initiator leaving a 5′-phosphate on the released polynucleotide. In some embodiments, an initiator may contain a terminal uridine so that after synthesis the desired polynucleotide may be cleaved from the initiator by treatment with KOH, or like base. Further methods for cleaving single stranded polynucleotides are disclosed in the following references, which are incorporated by reference: U.S. Pat. Nos. 5,739,386, 5,700,642 and 5,830,655; and U.S. Patent Publication Nos. 2003/0186226 and 2004/0106728; and in Urdea and Horn, U.S. Pat. No. 5,367,066.
Returning to
In some embodiments, after the synthesis cycles of
When the predetermined sequences of polynucleotides on a synthesis support includes reverse complementary subsequences, secondary intra-molecular or cross-molecular structures may be created by the formation of hydrogen bonds between the reverse complementary regions. In some embodiments, base protecting moieties for exocyclic amines are selected so that hydrogens of the protected nitrogens cannot participate in hydrogen bonding, thereby preventing the formation of such secondary structures. That is, base protecting moieties may be employed to prevent the formation of hydrogen bonds, such as are formed in normal base pairing, for example, between nucleosides A and T and between G and C. At the end of a synthesis, the base protecting moieties may be removed and the polynucleotide product may be cleaved from the solid support, for example, by cleaving it from its initiator.
In addition to providing 3′-O-blocked dNTP monomers with base protection groups, elongation reactions may be performed at higher temperatures using thermal stable template-free polymerases. For example, a thermal stable template-free polymerase having activity above 40° C. may be employed; or, in some embodiments, a thermal stable template-free polymerase having activity in the range of from 40-85° C. may be employed; or, in some embodiments, a thermal stable template-free polymerase having activity in the range of from 40-65° C. may be employed.
In some embodiments, elongation conditions may include adding solvents to an elongation reaction mixture that inhibit hydrogen bonding or base stacking. Such solvents include water miscible solvents with low dielectric constants, such as dimethyl sulfoxide (DMSO), methanol, and the like. Likewise, in some embodiments, elongation conditions may include the provision of chaotropic agents that include, but are not limited to, n-butanol, ethanol, guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, urea, and the like. In some embodiments, elongation conditions include the presence of a secondary-structure-suppressing amount of DMSO. In some embodiments, elongation conditions may include the provision of DNA binding proteins that inhibit the formation of secondary structures, wherein such proteins include, but are not limited to, single-stranded binding proteins, helicases. DNA glycolases, and the like.
When base-protected dNTPs are employed, the above method of
The above method may also include capping step(s) as well as washing steps after the reacting, or extending, step, as well as after the deprotecting step. As mentioned above, in some embodiments, capping steps may be included in which non-extended free 3′-hydroxyls are reacted with compounds that prevents any further extensions of the capped strand. In some embodiments, such compound may be a dideoxynucleoside triphosphate. In other embodiments, non-extended strands with free 3′-hydroxyls may be degraded by treating them with a 3′-exonuclease activity, e.g. Exo I. For example, see Hyman, U.S. Pat. No. 5,436,143. Likewise, in some embodiments, strands that fail to be deblocked may be treated to either remove the strand or render it inert to further extensions. When a capping agent, such as ddNTPs, are used, the buffer or synthesis reagents containing such agents may be delivered by flowing or spraying such reagent over substrate containing the reaction sites.
In some embodiments, reaction conditions for an elongation step (also sometimes referred to as an extension step or a coupling step) may comprising the following: 2.0 μM purified TdT; 125-600 μM 3′-O-blocked dNTP (e.g. 3′-O—NH2-blocked dNTP); about 10 to about 500 mM potassium cacodylate buffer (pH between 6.5 and 7.5) and from about 0.01 to about 10 mM of a divalent cation (e.g. CoCl2 or MnCl2), where the elongation reaction may be carried out at a temperature within the range RT to 45° C., for 3 minutes. It is understood that whenever the foregoing coupling reagent is delivered by inkjet-produced droplets its viscosity, density and surface tension must be adjusted so that it becomes a printable ink. In this connection, the invention in part includes the recognition and appreciation that an ink for delivering TdT to a reaction site may have its viscosity modified for droplet formation and activity preserved by selection of a viscosity modifier, such as, when carboxymethyl cellulose is selected as the viscosity modifying agent.
In embodiments, in which the 3′-O-blocked dNTPs are 3′-O—NH2-blocked dNTPs, reaction conditions for a deblocking step may comprise the following: 700 mM NaNO2; 1 M sodium acetate (adjusted with acetic acid to pH in the range of 4.8-6.5), where the deblocking reaction may be carried out at a temperature within the range of RT to 45° C. for 30 seconds to several minutes. Washes may be performed with the cacodylate buffer without the components of the coupling reaction (e.g. enzyme, monomer, divalent cations). If the above reagent compositions are delivered to reaction sites by inkjet delivery, it is understood that the compositions would be altered to meet the rheological requirements for droplet formation by the nozzles of the inkjet print heads used.
In some embodiments, RNA synthesis may be accomplished by similar steps as described above but with template-free polymerases and monomers specifically selected for RNA synthesis, such as, polyA polymerase (PAP), polyU polymerase (PUP), or the like, e.g. International patent publication WO2020/077227. For example, systems, apparatus and kits of the invention may implement methods of synthesizing a polyribonucleotide having a predetermined sequence comprising the steps of: a) providing an initiator having a 3′-terminal nucleotide having a free 3′-hydroxyl; and b) repeating, until the polyribonucleotide is formed, cycles of (i) contacting under elongation conditions the initiator or elongated fragments having free 3′-hydroxyls with a 3′-O-blocked-nucleoside triphosphate and a template-free polymerase so that the initiator or elongated fragments are elongated by incorporation of a 3′-O-blocked-nucleoside triphosphate to form 3′-O-blocked-elongated fragments, and (ii) deblocking the elongated fragments to form elongated fragments having free 3′-hydroxyls; wherein the template-free polymerase is a poly(A) polymerase (PAP) or a poly(U) polymerase. In further embodiments, the initiator may be attached to a support by a 5′ end, the support may be a solid support, and the above method may include a step of cleaving the polynucleotide from the initiator. In some embodiments, reaction conditions for an extension or elongation step using PAP or PUP may comprising the following: Reaction conditions 1 (for primer+AM-rATP): 250 uM AM-rATP, 0.1 uM ATTO488-(rA)5, 1 uM PAP, 1×ATP buffer (20 mM Tris-HCl, 0.6 mM MnCl2, 0.02 mM EDTA, 0.1% BSA, 10% glycerol, 100 mM imidazole, pH 7-8), 37 C, 30 min. Reaction condition 2 (for primer+AM-rGTP): 250 uM rGTP, 0.1 uM ATT0488-(rA)5, 1 uM PAP, 1×GTP buffer (0.6 mM MnCl2, 0.1% BSA, 10 mM imidazole, pH 6), 37 C, 30 min. In the foregoing, “AM-rNTP” refers to 3′-azidomethyl-O-ribonucleoside triphosphate. Many of the 3′-O-blocked rNTPs employed in the invention may be purchased from commercial vendors (e.g. Jena Bioscience, MyChemlabs, or the like) or synthesized using published techniques, e.g. U.S. Pat. No. 7,057,026; International patent publications WO2004/005667, WO91/06678; Canard et al. Gene (cited above); Metzker et al, Nucleic Acids Research, 22: 4259-4267 (1994); Meng et al, J. Org. Chem., 14: 3248-3252 (3006): U.S. patent publication 2005/037991; Zavgorodny et al. Tetrahedron Letters, 32(51): 7593-7596 (1991). In a further particular embodiments, the 3′-blocked nucleotide triphosphate is blocked by either 3′-O-propargyl, a 3′-O-azidomethyl, 3′-O—NH2 or 3′-O-allyl group. In still other embodiments, 3′-O-blocking groups of the invention include 3′-O-methyl, 3′-O-(2-nitrobenzyl)#3′-O-allyl, 3′-O-amine, 3′-O-azidomethyl, 3′-O-tert-butoxy ethoxy, 3′-O-(2-cyanoethyl), and 3′-O-propargyl. As above, if the above reagent compositions are delivered to reaction sites by inkjet delivery, it is understood that the compositions would be altered to meet the rheological requirements for droplet formation by the nozzles of the inkjet print heads used.
Depending on particular applications, the steps of deblocking and/or cleaving may include a variety of chemical or physical conditions. e.g. light, heat. pH, presence of specific reagents, such as enzymes, which are able to cleave a specified chemical bond. Guidance in selecting 3′-O-blocking groups and corresponding de-blocking conditions may be found in the following references, which are incorporated by reference: Benner, U.S. Pat. Nos. 7,544,794 and 8,212,020; 5,808,045; 8,808,988; International patent publication WO91/06678; and references cited below. In some embodiments, the cleaving agent (also sometimes referred to as a de-blocking reagent or agent) is a chemical cleaving agent, such as, for example, dithiothreitol (DTT). In alternative embodiments, a cleaving agent may be an enzymatic cleaving agent, such as, for example, a phosphatase, which may cleave a 3′-phosphate blocking group. It will be understood by the person skilled in the art that the selection of deblocking agent depends on the type of 3′-nucleotide blocking group used, whether one or multiple blocking groups are being used, whether initiators are attached to living cells or organisms or to solid supports, and the like, that necessitate mild treatment. For example, a phosphine, such as tris(2-carboxyethyl)phosphine (TCEP) can be used to cleave a 3′O-azidomethyl groups, palladium complexes can be used to cleave a 3′O-allyl groups, or sodium nitrite can be used to cleave a 3′O-amino group. In particular embodiments, the cleaving reaction involves TCEP, a palladium complex or sodium nitrite.
As noted above, in some embodiments it is desirable to employ two or more blocking groups that may be removed using orthogonal de-blocking conditions. The following exemplary pairs of blocking groups may be used in parallel synthesis embodiments. It is understood that other blocking group pairs, or groups containing more than two, may be available for use in these embodiments of the invention.
In some embodiments, specific enzymatically removable blocking groups are require specific enzymes for their removal. For example, ester- or acyl-based blocking groups may be removed with an esterase, such as acetylesterase, or like enzyme, and a phosphate blocking group may be removed with a 3′ phosphatase, such as T4 polynucleotide kinase. By way of example, 3′-O-phosphates may be removed by treatment with as solution of 100 mM Tris-HCl (pH 6.5) 10 mM MgCl2, 5 mM 2-mercaptoethanol, and one Unit T4 polynucleotide kinase. The reaction proceeds for one minute at a temperature of 37° C. As above, if the foregoing compositions are delivered to reaction sites by inkjet delivery, it is understood that the compositions would be altered to meet the rheological requirements for droplet formation by the nozzles of the inkjet print heads used.
Further examples of synthesis and enzymatic deprotection of 3′-O-ester-protected dNTPs or 3′-O-phosphate-protected dNTPs are described in the following references: Canard et al. Proc. Natl. Acad. Sci., 92:10859-10863 (1995); Canard et al, Gene, 148: 1-6 (1994); Cameron et al, Biochemistry, 16(23): 5120-5126 (1977); Rasolonjatovo et al, Nucleosides & Nucleotides, 18(4&5): 1021-1022 (1999): Ferrero et al. Monatshefte fur Chemie, 131: 585-616 (2000); Taunton-Rigby et al, J. Org. Chem., 38(5): 977-985 (1973): Uemura et al, Tetrahedron Lett., 30(29): 3819-3820 (1989); Becker et al, J. Biol. Chem., 242(5): 936-950 (1967); Tsien, International patent publication WO1991/006678.
In some embodiments, the modified nucleotides comprise a modified nucleotide or nucleoside molecule comprising a purine or pyrimidine base and a ribose or deoxyribose sugar moiety having a removable 3′-OH blocking group covalently attached thereto, such that the 3′ carbon atom has attached a group of the structure:
—O—Z
wherein —Z is any of —C(R′)2—O—R″, —C(R′)2—N(R′)2, —C(R′)2—N(H)R″, —C(R′)2—S—R″ and —C(R′)2—F, wherein each R″ is or is part of a removable protecting group; each R′ is independently a hydrogen atom, an alkyl, substituted alkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclic, acyl, cyano, alkoxy, aryloxy, heteroaryloxy or amido group, or a detectable label attached through a linking group; with the proviso that in some embodiments such substituents have up to 10 carbon atoms and/or up to 5 oxygen or nitrogen heteroatoms; or (R′)2 represents a group of formula ═(R′″)2 wherein each R′″ may be the same or different and is selected from the group comprising hydrogen and halogen atoms and alkyl groups, with the proviso that in some embodiments the alkyl of each R′″ has from 1 to 3 carbon atoms; and wherein the molecule may be reacted to yield an intermediate in which each R″ is exchanged for H or, where Z is —(R′)2—F, the F is exchanged for OH, SH or NH2, preferably OH, which intermediate dissociates under aqueous conditions to afford a molecule with a free 3′-OH; with the proviso that where Z is —C(R′)2—S—R″, both R′ groups are not H. In certain embodiments. R′ of the modified nucleotide or nucleoside is an alkyl or substituted alkyl, with the proviso that such alkyl or substituted alkyl has from 1 to 10 carbon atoms and from 0 to 4 oxygen or nitrogen heteroatoms. In certain embodiments, −Z of the modified nucleotide or nucleoside is of formula —C(R′)2-N3. In certain embodiments, Z is an azidomethyl group.
In some embodiments. Z is a cleavable organic moiety with or without heteroatoms having a molecular weight of 200 or less. In other embodiments. Z is a cleavable organic moiety with or without heteroatoms having a molecular weight of 100 or less. In other embodiments. Z is a cleavable organic moiety with or without heteroatoms having a molecular weight of 50 or less. In some embodiments, Z is an enzymatically cleavable organic moiety with or without heteroatoms having a molecular weight of 200 or less. In other embodiments, Z is an enzymatically cleavable organic moiety with or without heteroatoms having a molecular weight of 100 or less. In other embodiments, Z is an enzymatically cleavable organic moiety with or without heteroatoms having a molecular weight of 50 or less. In other embodiments. Z is an enzymatically cleavable ester group having a molecular weight of 200 or less. In other embodiments. Z is a phosphate group removable by a 3′-phosphatase. In some embodiments, one or more of the following 3-phosphatases may be used with the manufacturer's recommended protocols: T4 polynucleotide kinase, calf intestinal alkaline phosphatase, recombinant shrimp alkaline phosphatase (e.g. available from New England Biolabs, Beverly, MA)
In a further embodiments, the 3′-blocked nucleotide triphosphate is blocked by either a 3′-O-azidomethyl, 3′-O—NH2 or 3′-O-allyl group.
In still other embodiments, 3′-O-blocking groups of the invention include 3′-O-methyl, 3′-O-(2-nitrobenzyl), 3′-O-allyl, 3′-O-amine, 3′-O-azidomethyl, 3′-O-tert-butoxy ethoxy, 3′-O-(2-cyanoethyl), and 3′-O-propargyl.
3′-O-blocked dNTPs without base protection may be purchased from commercial vendors or synthesized using published techniques. e.g. U.S. Pat. No. 7,057,026; Guo et al. Proc. Natl. Acad. Sci., 105(27): 9145-9150 (2008); Benner. U.S. Pat. Nos. 7,544,794 and 8,212,020; International patent publications WO2004/005667, WO91/06678; Canard et al. Gene (cited herein); Metzker ct al, Nucleic Acids Research, 22: 4259-4267 (1994); Meng et al, J. Org. Chem., 14: 3248-3252 (2006); U.S. patent publication 2005/037991. 3′-O-blocked dNTPs with base protection may be synthesized as described below.
A variety of different template-free polymerases are available for use in methods of the invention. Template-free polymerases include, but are not limited to, polX family polymerases (including DNA polymerases β, λ and μ), poly(A) polymerases (PAPs), poly(U) polymerases (PUPs), DNA polymerase θ, and the like, for example, described in the following references: Ybert et al. international patent publication WO2017/216472; Champion et al, U.S. patent Ser. No. 10/435,676; Champion et al, International patent publication WO2020/099451; Heinisch et al. International patent publication WO2021/018919. In particular, terminal deoxynucleotidyltransferases (TdTs) and variants thereof are useful in template-free DNA synthesis.
In some embodiments, TdT variants are employed with the invention which display increased incorporation activity with respect to 3′-O-amino nucleoside triphosphates. For example, such TdT variants may be produced using techniques described in Champion et al. U.S. patent Ser. No. 10/435,676, which is incorporated herein by reference. In some embodiments, a TdT variant is employed having (a) an amino acid sequence at least 80 percent identical to a TdT having an amino acid sequence of any of SEQ ID NOs 7 through 20, inclusive, and 24 through 39, inclusive, and (b) one or more of the substitutions listed in Table 1, wherein the TdT variant (i) is capable of synthesizing a nucleic acid fragment without a template and (ii) is capable of incorporating a 3′-O-modified nucleotide onto a free 3′-hydroxyl of a nucleic acid fragment. In some embodiments, the above TdT variants include a substitution at every position listed in Table 1. In some embodiments, the above percent identity value is at least 85 percent identity with the indicated SEQ ID NOs; in some embodiments, the above percent identity value is at least 90 percent identity with the indicated SEQ ID) NOs; in some embodiments, the above percent identity value is at least 95 percent identity with the indicated SEQ ID NOs; in some embodiments, the above percent identity value is at least 97 percent identity; in some embodiments, the above percent identity value is at least 98 percent identity; in some embodiments, the above percent identity value is at least 99 percent identity. As used herein, the percent identity values used to compare a reference sequence to a variant sequence do not include the expressly specified amino acid positions containing substitutions of the variant sequence, that is, the percent identity relationship is between sequences of a reference protein and sequences of a variant protein outside of the expressly specified positions containing substitutions in the variant.
In some embodiments, a TdT variant of the invention is derived from a TdT comprising an amino acid sequence at least 80 percent identical to an amino acid sequence selected from SEQ ID NOs 40 through 75, inclusive, and one or more of the substitutions listed in Table 2, wherein the TdT variant (i) is capable of synthesizing a nucleic acid fragment without a template and (ii) is capable of incorporating a 3′-O-modified nucleotide onto a free 3′-hydroxyl of a nucleic acid fragment. In some embodiments, the above TdT variants include a substitution at every position listed in Table 2. In some embodiments, the above percent identity value is at least 85 percent identity with the indicated SEQ ID NOs; in some embodiments, the above percent identity value is at least 90 percent identity with the indicated SEQ ID NOs; in some embodiments, the above percent identity value is at least 95 percent identity with the indicated SEQ ID NOs; in some embodiments, the above percent identity value is at least 97 percent identity; in some embodiments, the above percent identity value is at least 98 percent identity; in some embodiments, the above percent identity value is at least 99 percent identity. As above, the percent identity values used to compare a reference sequence to a variant sequence do not include the expressly specified amino acid positions containing substitutions of the variant sequence; that is, the percent identity relationship is between sequences of a reference protein and sequences of a variant protein outside of the expressly specified positions containing substitutions in the variant.
TdT variants of SEQ ID NOs 40 through 54, inclusive, 56, 59, 61, 63, 65, 67, 69, 70, 73 and 74 includes substitutions at one or more of the indicated amino acid positions as listed in Table 2 in addition to a stabilizing substitution of the glutamine at position 4 (or a functionally equivalent position). In other embodiments, TdT variants of the invention are derived from natural TdTs such as those listed in Table 2 with a substitution at every one of the indicated amino acid positions in addition to the stabilizing substitution of the glutamine at position 4. In some embodiments, such stabilizing amino acid substituted for glutamine is selected from the group consisting of E, S. D and N. In other embodiments, the stabilizing amino acid is E.
In some embodiments, further TdT variants for use with methods of the invention include one or more of the substitutions of methionine, cysteine, arginine (first position), arginine (second position) or glutamic acid, as shown in Table 2.
In some embodiments, a TdT variant comprising an amino acid sequence at least ninety percent identical to an amino acid sequence of SEQ ID NOs 55, 57, 58, 60, 62, 64, 66, 68, 71, 72, and 75 through 112, inclusive, may also be used with the present invention.
TdT, PAP and PUP variants for use with the invention each comprise an amino acid sequence having a percent sequence identity with a specified SEQ ID NO, subject to the presence of indicated substitutions. In some embodiments, the number and type of sequence differences between a variant of the invention described in this manner and the specified SEQ ID NO may be due to substitutions, deletion and/or insertions, and the amino acids substituted, deleted and/or inserted may comprise any amino acid. In some embodiments, such deletions, substitutions and/or insertions comprise only naturally occurring amino acids. In some embodiments, substitutions comprise only conservative, or synonymous, amino acid changes, as described in Grantham, Science, 185: 862-864 (1974). That is, a substitution of an amino acid can occur only among members of its set of synonymous amino acids. In some embodiments, sets of synonymous amino acids that may be employed are set forth in Table 3A.
In some embodiments, sets of synonymous amino acids that may be employed are set forth in Table 3B.
TdT, PAP and PUP variants for use with the invention are produced by conventional biotechnology techniques and may include an affinity tag for purification, which may be attached to the N-terminus. C-terminus or at an interior position of the template-free polymerase. In some embodiments, affinity tags are cleaved before the template-free polymerase is used. In other embodiments, affinity tags are not cleaved before use. In some embodiments, a peptide affinity tag is inserted into a loop 2 region of a TdT variant. An exemplary N-terminal His-tag for use with TdT variants of the invention is MASSHHHHHHSSGSENLYFQTGSSG-(SEQ ID NO: 6)). Guidance for selecting a peptide affinity tag is described in the following references: Terpe. Appl. Microbiol. Biotechnol., 60: 523-533 (2003); Arnau et al. Protein Expression and Purification, 48: 1-13 (2006); Kimple et al. Curr. Protoc. Protein Sci., 73: Unit-9.9 (2015); Kimple et al, U.S. Pat. No. 7,309,575; Lichty et al, Protein Expression and Purification, 41: 98-105 (2005); and the like. Guidance for selecting a peptide affinity tag is described in the following references: Terpe. Appl. Microbiol. Biotechnol., 60: 523-533 (2003); Arnau et al, Protein Expression and Purification, 48: 1-13 (2006); Kimple et al. Curr. Protoc. Protein Sci., 73: Unit-9.9 (2015); Kimple et al. U.S. Pat. No. 7,309,575; Lichty et al, Protein Expression and Purification, 41: 98-105 (2005); and the like.
The efficiency of nucleotide incorporation by variants used with the invention may be measured by an extension, or elongation, assay, e.g. as described in Boule et al (cited below); Bentolila et al (cited below); and Hiatt ct al, U.S. Pat. No. 5,808,045, the latter of which is incorporated herein by reference. Briefly, in one form of such an assay, a fluorescently labeled oligonucleotide having a free 3-hydroxyl is reacted with a template-free polymerase, such as a TdT, under extension conditions for a predetermined duration in the presence of a reversibly blocked nucleoside triphosphate, after which the extension reaction is stopped and the amounts of extension products and unextended oligonucleotide are quantified after separation by gel electrophoresis. By such assays, the incorporation efficiency of a variant template-free polymerase may be readily compared to the efficiencies of other variants or to that of wild type or reference polymerases. In some embodiments, a measure of template-free polymerase efficiency may be a ratio (given as a percentage) of amount of extended product using the variant template-free polymerase over the amount of extended product using wild type template-free polymerase, or reference polymerase, in an equivalent assay.
In some embodiments, the following particular extension assay may be used to measure incorporation efficiencies of TdTs: The primer used is the following:
The Activity buffer comprises, for example, TdT reaction buffer (available from New England Biolabs) supplemented with CoCl2.
The product of the assay is analyzed by conventional polyacrylamide gel electrophoresis. For example, products of the above assay may be analyzed in a 16 percent polyacrylamide denaturing gel (Bio-Rad). Gels are made just before the analysis by pouring polyacrylamide inside glass plates and let it polymerize. The gel inside the glass plates is mounted on an adapted tank filed with TBE buffer (Sigma) for the electrophoresis step. The samples to be analyzed are loaded on the top of the gel. A voltage of 500 to 2,000V is applied between the top and bottom of the gel for 3 to 6 h at room temperature. After separation, gel fluorescence is scanned using, for example, a Typhoon scanner (GE Life Sciences). The gel image is analyzed using ImageJ software (imagej.nih.gov/ij/), or its equivalent, to calculate the percentage of incorporation of the modified nucleotides.
The elongation efficiency of a template-free polymerase may also be measured in the following hairpin completion assay. In such assay, a test polynucleotide is provided with a free 3′ hydroxyl such that under reaction conditions it is substantially only single stranded, but that upon extension with a polymerase, such as a TdT variant, it forms a stable hairpin structure comprising a single stranded loop and a double stranded stem. This allows the detection of an extension of the 3′ end by the presence of the double stranded polynucleotide. The double stranded structure may be detected in a variety of ways including, but not limited to, (i) fluorescent dyes that preferentially fluoresce upon intercalation into the double stranded structure, (ii) fluorescent resonance energy transfer (FRET) between an acceptor (or donor) on the extended polynucleotide and a donor (or acceptor) on an oligonucleotide that forms a triplex with the newly formed hairpin stem, (iii) FRET acceptors and donors that are both attached to the test polynucleotide and that are brought into FRET proximity upon formation of a hairpin, or the like. In some embodiments, a stem portion of a test polynucleotide after extension by a single nucleotide is in the range of 4 to 6 basepairs in length; in other embodiments, such stem portion is 4 to 5 basepairs in length; and in still other embodiments, such stem portion is 4 basepairs in length. In some embodiments, a test polynucleotide has a length in the range of from 10 to 20 nucleotides; in other embodiments, a test polynucleotide has a length in the range of from 12 to 15 nucleotides. In some embodiments, it is advantageous or convenient to extend the test polynucleotide with a nucleotide that maximizes the difference between the melting temperatures of the stem without extension and the stem with extension; thus, in some embodiments, a test polynucleotide is extended with a dC or dG (and accordingly the test polynucleotide is selected to have an appropriate complementary nucleotide for stem formation).
Exemplary test polynucleotides for hairpin completion assays include p875 (5′-CAGTTAAAAACT) (SEQ ID NO: 2) which is completed by extending with a dGTP; p876 (5′-GAGTTAAAACT) (SEQ ID NO: 3) which is completed by extending with a dCTP; and p877 (5′-CAGCAAGGCT) (SEQ ID NO: 4) which is completed by extending with a dGTP. Exemplary reaction conditions for such test polynucleotides may comprise: 2.5-5 μM of test polynucleotide, 1:4000 dilution of GelRed* (intercalating dye from Biotium, Inc., Fremont. CA), 200 mM Cacodylate KOH pH 6.8, 1 mM CoCl2, 0-20% of DMSO and 3′-ONH2 dGTP and TdT at desired concentrations. Completion of the hairpin may be monitored by an increase in fluorescence of GelRed® dye using a conventional fluorimeter, such as a TECAN reader at a reaction temperature of 28-38° C. using an excitation filter set to 360 nm and an emission filter set to 635 nm.
As mentioned above, synthesis reagents delivered by inkjet pumps must be formulated so as to preserve activity of synthesis reagents and to meet the rheological requirements for droplet formation. Such formulations are referred to herein as “inks.” For example, satisfying the first constraint (activity) may require that a template-free polymerase be present in a reaction mixture at a certain minimal concentration. However, because of high protein viscosity, the concentration for the desired activity may prevent satisfaction of the second constraint, i.e. capability of droplet formation. In such cases, embodiments of the invention may call for delivery of a plurality of droplets each with lower concentrations of polymerase, which coupled with evaporation permit the build-up of polymerase concentration for a desired level of activity. Such an embodiment is illustrated in
As also mentioned above, the key solution parameters affecting droplet formation by inkjets are viscosity, surface tension, density and diameter of the inkjet nozzle, which are related through the formula: Z=[(ργa){circumflex over ( )}(0.5)]/η, where ρ is the density of the fluid, γ is surface tension, η is viscosity, a is the radius of the inkjet pump nozzle and Z is in the range of from 1 to 10 for reliable droplet formation. e.g. Derby, J. Mater. Chem., 18: 5717-5721 (2008). This relationship applies to any of the synthesis reagents delivered by inkjet-generated droplets, including (i) template-free polymerase in its coupling buffer, (ii) a mixture of template-free polymerase in its coupling buffer and a 3′-O-protected-dNTP, (iii) a 3′-O-protected-dNTP in a buffer, (iv) a deprotection buffer, and (v) a buffer containing a 5′-linker-derivatized initiator. Applying this relationship to determine ink compositions that are capable of forming desired droplets for particular embodiments may be carried out by one of ordinary skill in the art by adjusting densities of reactants, viscosity modifiers, surface tension modifiers, and the like.
In some embodiments, an elongation reaction buffer is an acetate buffer, for example, 0.1 M acetate, 0.5 M NaCl, pH 4.5.
Thus, for particular embodiments of the invention, synthesis reagents prepared for non-droplet delivery to a reaction mixture may be reformulated by adding viscosity modifiers, surface tension modifiers and density modifiers, and the like, in order to form “printable inks” that may be delivered in droplets generated by inkjet pumps. “Printable” in reference to a reagent ink means repeatable droplets are able to be ejected from the nozzle, with uniform velocities and volumes and no satellite droplets.
In some embodiments, if the specific activity of a template-free polymerase is relatively low, so that a relatively large amount of protein must be delivered to reaction sites to complete a coupling step, then the delivery of the polymerase may be carried out by dispensing a plurality of droplets in each coupling cycle together with allowing a controlled amount of evaporation to maintain a reaction volume within a prescribed range. e.g. 10-100 μL. In some embodiments, the plurality of droplets delivered is in the range of from 2 to 10, or in the range of from 2 to 5, or in the range of from 2 to 3. In other embodiments, the plurality of droplets may be in the range of from 2 to 150, or in the range of from 10 to 120. In some embodiments, whenever the template-free polymerase is a TdT, the plurality of droplets is the number required to bring the concentration of TdT in the reaction mixture at a reaction site to a value in the range of from 1 μM to 30 μM, or in the range of from 2 μM to 20 μM. In some embodiments, a concentration of TdT in an ink is the concentration that produces an approximate 1:1 stoichiometry between TdT molecules and polynucleotides at a reaction site. In other embodiments, a concentration of TdT in an ink is a concentration that produces a stoichiometry between TdT molecules and polynucleotides at a reaction site that is 1:1 or greater.
In some embodiments, the invention includes a printable ink comprising a TdT variant and a viscosity modifier. In some embodiments, such TdT in a concentration in the range of from 1 μM to 20 μM/mg in a buffer suitable for coupling activity. In some embodiments, such buffer comprises about 10 to about 500 mM potassium cacodylate buffer (pH between 6.5 and 7.5) and from about 0.01 to about 10 mM of a divalent cation (e.g. CoCl2 or MnCl2). In some embodiments, TdT inks are characterized by a viscosity of about 1 centipoise (0.001 Pa-sec) to about 20 centipoise (0.02 Pa-sec) when viscosity is measured at room temperature; and a surface tension of about 15 dynes/cm and about 50 dynes/cm when measured at room temperature. In some embodiments, the viscosity modifier is selected from the group consisting of glycerol, ethylene glycol, polyethylene glycol of different molecular weights, glycerol, poly(vinyl alcohol), carboxymethyl cellulose and hydroxyethyl cellulose. In some embodiments, a template-free polymerase ink, such as a TdT ink, comprises glycerol at a concentration in the range of from 5 percent (w/w) to 55 percent (w/w); in other embodiments, a template-free polymerase ink, such as a TdT ink, comprises glycerol at a concentration in the range of from 5 percent (w/w) to 40 percent (w/w); in other embodiments, a template-free polymerase ink, such as a TdT ink, comprises glycerol at a concentration in the range of from 5 percent (w/w) to 25 percent (w/w); in other embodiments, a TdT ink comprises glycerol at a concentration in the range of from 8 percent (w/w) to 40 percent (w/w); in other embodiments, a TdT ink comprises glycerol at a concentration in the range of from 8 percent (w/w) to 25 percent (w/w); in other embodiments, a TdT ink comprises glycerol at a concentration in the range of from 10 percent (w/w) to 40 percent (w/w). It is understood by those of ordinary skill that the viscosity ranges achieved by the above glycerol concentration ranges also may be achieved by equivalent concentration ranges of other viscosity modifiers. Thus, in some embodiments, a TdT ink comprises a concentration of a viscosity modifier that produces an equivalent viscosity as glycerol at a concentration in the range of from 10 percent (W/W) to 40 percent (w/w). In addition to glycerol, of particular interest are the viscosity modifiers carboxymethyl cellulose and hydroxyethyl cellulose which have minimal effect on template-free polymerase coupling activity, such as TdT coupling activity. In some embodiments, besides a viscosity modifier, the printable template-free polymerase ink, such as a TdT ink, comprises a surface tension modifier. Such surface tension modifier may be a detergent. Such detergent may be selected from Tween 20, Triton X-100, CHAPS, NP-40, octyl thioglycoside, octyl glucoside or dodecyl maltoside. Of particular interest is Triton X-100. Also of particular interest is Tween 20. Additional surface tension modifiers (i.e. surfactants) are disclosed in Buret, LabChip, 12: 422-433 (2012).
In some embodiments, the invention includes a printable ink comprising a TdT variant, a 3′-O-protected-dNTP and a viscosity modifier. In some embodiments, such TdT is in a concentration in the range of from 1 μM to 50 μM/mg, or 1 μM to 20 μM/mg, in a buffer suitable for coupling activity. In some embodiments, such buffer comprises about 10 to about 500 mM potassium cacodylate buffer (pH between 6.5 and 7.5) and from about 0.01 to about 10 mM of a divalent cation (e.g. COCl2 or MnCl2); such 3′-O-protected-dNTP is in a concentration in the range of 125-600 μM. In some embodiments, the viscosity modifier is selected from the group consisting of glycerol, ethylene glycol, polyethylene glycol of different molecular weights, glycerol, poly(vinyl alcohol), carboxymethyl cellulose and hydroxyethyl cellulose. In some embodiments, besides a viscosity modifier, the printable TdT ink comprises a surface tension modifier. Such surface tension modifier may be a detergent. Such detergent may be selected from Triton X-100, CHAPS, NP-40, octyl thioglycoside, octyl glucoside or dodecyl maltoside. Of particular interest are Tween 20 and Triton X-100.
In each of the above embodiments of TdT inks, a TdT has an amino acid sequence that is at least 80 percent identical to a TdT variant selected disclosed herein. In each of the above embodiments of TdT inks, a TdT has an amino acid sequence that is at least 80 percent identical to a TdT variant selected from Table 1 or Table 2. In some embodiments, a TdT ink of the invention comprises a TdT with an amino acid sequence that is at least 90 percent identical to a TdT variant disclosed herein. In some embodiments, a TdT ink of the invention comprises a TdT with an amino acid sequence that is at least 90 percent identical to a TdT variant selected from Table 1 or Table 2.
In some embodiments, inks of synthesis reagents may be formulated as emulsions. In particular. TdT inks of the invention may be emulsions.
In some embodiments, a printable ink comprising a template-free polymerase, such as a TdT variant, includes a humectant for reducing droplet evaporation. Suitable humectants include, but are not limited to, glycerol, alcohol sugars, ethylhexylglycerin, panthenol, sorbitol, xylitol, maltitol, propylene glycol, hexylene glycol, butylene glycol, sodium lactate, hyaluronic acid, and polydextrose,
In some embodiments, a TdT ink of the invention is delivered in a droplet in range of from 1 μL to 200 μL, or from 1 μL to 100 μL, or from 1 μL-50 μL.
In some embodiments, the invention is directed to a terminal deoxynucleotidyl transferase (TdT) composition comprising a droplet of an aqueous solution having a volume in the range of from 2 μL to 5 nL and comprising (i) a TdT or variant thereof in a concentration in the range of from 1.0 μM to 30 μM, or in the range of from 2.0 μM to 20 μM, a divalent cation in a concentration in the range of from 0.01 to 10 mM, and a viscosity modifier. In some embodiments, the divalent cation is cobalt or manganese and such composition further comprises a surface tension modifier. In some embodiments, such viscosity modifier is selected from the group consisting of glycerol, ethylene glycol, polyethylene glycol, poly(vinyl alcohol), carboxymethyl cellulose and hydroxyethyl cellulose. In some embodiments, any of the above compositions may further comprise an aldehyde scavenger (described more fully below) whenever the printable template-free polymerase ink, such as a printable TdT ink, comprises a 3′O-amino-nucleotide. In some embodiments, any of the above compositions may further comprise a 3′-O-protected-2′-deoxynucleoside triphosphate monomer in a concentration in the range of from 100-1000 μM, or from 125-600 μM.
In some embodiments, the invention is directed to a 3′-O-protected-2′-deoxynucleoside triphosphate composition comprising a droplet of an aqueous solution having a volume in the range of from 2 μL to 5 nL and comprising (i) a 3′-O-protected-2′-deoxynucleoside triphosphate in a concentration in the range of from 125-600 μM. and a viscosity modifier. In some embodiments, the foregoing 3′-O-protected-2′-deoxynucleoside triphosphate composition further comprising a surface tension modifier. In some embodiments of the foregoing compositions, the viscosity modifier is selected from the group consisting of glycerol, ethylene glycol, polyethylene glycol, poly(vinyl alcohol), carboxymethyl cellulose and hydroxyethyl cellulose. In some embodiments, the 3′-O-protected-2′-deoxynucleoside triphosphate of the foregoing composition is a 3′-O-protected-2′-deoxyadenosine triphosphate, a 3′-O-protected-2′-deoxyguanosine triphosphate, a 3′-O-protected-2′-deoxycytidine triphosphate, or a 3′-O-protected-2′-deoxythymidine triphosphate. In some embodiments of the foregoing composition, the 3′-O-protection group is selected from the group consisting of 3′-O-methyl, 3′-O-(2-nitrobenzyl), 3′-O-allyl, 3′-O-amine, 3′-O-azidomethyl, 3′-O-tert-butoxy ethoxy, 3′-O-(2-cyanoethyl), and 3′-O-propargyl.
In some embodiments, the invention is directed to a printable 3′-O-protected-2′-deoxynucleoside triphosphate ink, comprising: an aqueous solution of a 3′-O-protected-2′-deoxynucleoside triphosphate having a concentration in a range of from 125 μM to 600 μM; wherein whenever the ink is printed to a substrate, printed droplets each have a volume in the range of 0.1 μL to 5 nL of the aqueous 3′-O-protected-2′-deoxynucleoside triphosphate solution and is characterized by a viscosity of about 1 centipoise to about 20 centipoise when viscosity is measured at room temperature; and a surface tension of about 15 dynes/cm and about 50 dynes/cm when measured at room temperature.
As mentioned above, in some embodiments of the invention where 3′-O-amino-dNTP monomers are employed, the presence of an aldehyde scavenger in the template-free polymerase inks reduces spurious capping of the 3′-amines by reaction with adventitious aldehydes or ketones, such as formaldehyde, which are pervasive in the environment. This is a special problem with inkjet synthesis because droplets of ink have very high surface-to-volume ratios that enhances absorption of environmental aldehydes. Thus, in embodiments of the invention employing 3′-O-amino-dNTP monomers, template-free polymerase inks as described above further include an effective amount of at least one aldehyde scavenger. As used herein, “effective amount” in reference to an aldehyde scavenger means an amount (or concentration) sufficient to produce a measurable decrease in spuriously capped polynucleotides in a product. Such measurements may be made readily using conventional techniques. e.g. DNA sequence analysis of a sample of a product, gel electrophoresis, or the like. As used herein, the term “aldehyde scavenger” includes ketone scavengers. In some embodiments, aldehyde scavengers are agents that react with compounds having chemical groups of the formula R—C(═O)H or R1—C(═O)—R2, where R, R1 and R2 are typically alkyl or aryl. More particularly, in some embodiments, aldehyde scavengers are agents that react with R—C(═O)H or R1—C(═O)—R2 groups on compounds at a sufficiently high rate that such compounds do not react with (or react only negligibly with) the 3′-amine group of 3′-O-amino-nucleotides. As used herein, the term “scavenger” means a chemical substance added to a mixture in order to remove or de-activate impurities or compounds that lead unwanted reaction products. In various embodiments, aldehyde scavengers may be in solution, immobilized on the materials used for storage or synthesis or coupled to reagents employed in method of the invention, for example, template-free polymerases, such as TdTs.
As noted above, enzymatic synthesis may be carried out using a variety of reagents (referred to herein as “synthesis reagents”) that may contain or consist of reactants, wash solutions, deprotection buffers, enzymes, and the like. (The term “synthesis reagent” means any reagent used in a synthesis cycle to couple a monomer, particularly a 3′-O-amino-nucleoside triphosphate, to an initiator or elongated fragment, such as, buffers comprising a template-free polymerase, buffers comprising 3′-O-protected-nucleotide monomers, deprotection (or deblocking) buffers, and the like.) In various embodiments, an aldehyde scavenger may be a component of one or more of the synthesis reagents. In some embodiments, an aldehyde scavenger may be added to a reaction mixture as a separate synthesis reagent (without other reactants, wash buffers or enzymes). In some embodiments, an aldehyde scavenger is added to a reaction mixture as a component of a synthesis reagent comprising a template-free polymerase.
In some embodiments. e.g. employing aldehyde scavengers disclosed by Sudo et al. U.S. patent publication US2020/0061225 or listed in
In some embodiments, aldehyde scavengers employed in the invention comprise O-substituted hydroxylamines or polyhydroxylamines. In some embodiments. O-substituted hydroxylamines used in the invention are defined by the formula:
R1—ONH2
such as disclosed by Sudo et al, U.S. patent publication US2020/0061225, or Kitasaka et al. U.S. Pat. No. 7,241,625, which are incorporated herein by reference. In some embodiments, R1 is a C1-18 linear, branched or cyclic alkyl group which may be substituted by at least one substituent selected from the group consisting of a halogen atom: a C1-6 alkyloxy group; a C1-6 haloalkyl group; a C1-6 haloalkyloxy group; a carboxy group; a hydroxy group; a mercapto group; a cyano group: a nitro group: a C6-14 aryl group which may be substituted by a halogen atom, a C1-6 alkyl group, a C1-6 alkyloxy group, a C1-6 haloalkyl group, a C1-6 haloalkyloxy group, a carboxy group, a hydroxy group, a mercapto group, a cyano group or a nitro group; a C4-14 heteroaryl group which may be substituted by a halogen atom, a C1-6 alkyl group, a C1-6alkyloxy group, a C1-6 haloalkyl group, a C1-6 haloalkyloxy group, a carboxy group, a hydroxy group, a mercapto group, a cyano group or a nitro group; an alkoxycarbonyl group represented by the following formula:
—(C═O)—O—R2
and a carbamoyl group represented by the following formula:
—(C═O)—NR3(R3)
wherein R2 is a C1-18 linear, branched or cyclic alkyl group which may be substituted, at a chemically acceptable optional position, by at least one substituent selected from the group consisting of a carboxy group; a hydroxy group; a mercapto group; a halogen atom; a C1-6 alkyloxy group; a C1-6 haloalkyloxy group; a C6-14 aryl group; and a C4-14 heteroaryl group; and wherein each R3 may be the same or different and each independently a C1-18 linear, branched or cyclic alkyl group which may be substituted by at least one substituent selected from the group consisting of a carboxy group; a hydroxy group; a mercapto group; a halogen atom; a C1-6 alkyloxy group; a C1-6 haloalkyloxy group; a C6-14 aryl group; and a C4-14 heteroaryl group; a C6-14 aryl group, a C4-14 heteroaryl group, or a hydrogen atom.
In particular, exemplary O-substituted hydroxylamines or polyhydroxylamines which may be used in the invention are shown as compounds (1)-(14) in
In some embodiments aldehyde scavengers comprise carbonyl compounds disclosed by Pacifici, U.S. Pat. No. 5,446,195 or Burdeniuc et al. U.S. patent publication, US20160369035; which are incorporated herein by reference, and are defined by the formula:
wherein R and R′ are CH3 or H[O(CH2)m]nO- and wherein m and n are selected from the group of combinations of m and n consisting of: m=1 and n=1, 3-19; m=2 and n=2-19; or m=3 and n=1-19, Y is —CH2— or —CH2—CO—CH2—.
In some embodiments of the invention, template-free polymerase inks as described above further include a dye to permit monitoring of the location, size, shape and possible overlap of reaction sites, either at an initial dispensing of reagents to define the reaction sites or at subsequent droplet dispensations during synthesis, particularly to monitor possible coalescence of reaction mixtures at adjacent sites. A large selection of fluorescent and non-fluorescent dyes are available for this purpose. The main criteria for use is that the dye (i) not adversely affect the performance of any reaction component, (ii) be bright or concentrated enough to make droplets or reaction sites readily detectable, (iii) be spectrally distinct if more than one is used, and (iv) not affect the rheological properties of the ink. In some embodiments, food dyes are used in inks of the invention. In other embodiments, pH indicator dyes are used in inks of the invention. In other embodiments, fluorescent dyes are used in inks of the invention. Exemplary dyes for use with template-free polymerase inks include Brilliant Blue FCF. Fast Green FCF, Ponceau 4R and Sunset Yellow FCF. In some embodiments, food dyes are used at a concentration in the range of from 1 to 20 mM, or at a concentration in the range of from 1 to 10 mM.
In this example, a test bed was created for evaluating different inkjet reaction conditions. Because the very small amounts of materials at individual reaction sites are difficult to analyze, slides were prepared that allowed regions containing large numbers of reaction sites (e.g. 10 or more) to be processed together and pooled for analysis by gel electrophoresis. The regions were created by depositing equal volumes of alkali- or photo-cleavable initiators in each of the regions. An exemplary slide (900) (which may be a PolyAn 3D-epoxy coated glass slide) is shown in
In this Example, a Fujifilm Dimatix Samba recirculating silicon MEMS print head was used to deliver in “move-stop” or “jetting” mode reagent droplets in 5 cycles of enzymatic synthesis. (Capping and deblocking steps were performed manually). The recirculating feature was employed to address problems due to ink drying on nozzles. The “move-stop” feature was employed to improve deposition accuracy which is especially important in the synthesis of high density arrays of polynucleotides. In the “move-stop” or “jetting” mode, the print head stops above each target reaction site (i.e. a reaction site programmed to receive a synthesis reagent), deposits a predetermined number of droplets (sometimes referred to herein as “pulses”) at the reaction site, then moves to the next target reaction site programmed to receive a deposition. Using this mode reaction site size (e.g. diameter) and the amount of synthesis reagent delivered can be determined by programming individual nozzles to deliver a predetermined number of droplets or pulses. Droplet sizes are predetermined and in the case of the Samba print head, droplet volumes are each about 2.4 μL. Different amounts of enzyme buffer (22% w/w glycerol, 20 mM BOX aldehyde scavenger, 20 μM TdT (M57), 500 μM dTTP-3′ONH2) were deposited onto a PolyAn 3D epoxy slide with 24 (8 rows of 3 spots) FAM-labelled photocleavable DNA spots (as described in Example 1) approximately 3 mm in diameter using the jetting mode of the Dimatix Samba print head, 1024 of the 2048 nozzles of the Samba print head were used; in synthesis cycle 1: 10-60 pulses of reagent were delivered depending on reaction site location; in synthesis cycles 2-5 60 pulses were delivered at each reaction site; the print head was operated in move-stop (i.e. jetting) mode with 5 kHz firing, tickle pulse non-printing waveform; 268, 38 and 355 inlet, return and meniscus pressure, respectively.
After printing enzyme buffer, the slide was removed from the print stage and incubated 10 minutes in a humidity chamber, 5 μL capping solution (500 μM ddATP, 10 μM TdT (M57 (SEQ ID NO: 102) in TdT elongation buffer) (a 0.09% Tween 20 in MQ-water solution was used as a diluent)) was then pipetted onto the 6 rows of DNA spots (i.e. rows 3 through 8 of
In this example, synthesis (5 cycles) was carried out as described above by move-stop-jetting onto a glass slide with 24 (8 rows×3 columns) large (ca. 4 mm) FAM-labelled, photocleavable DNA regions. Synthesis and cleaving were done as described above. The only thing different was the reaction site (i.e. “spot”) density within the 3-region rows, 60 pulses in cycle 1 resulted in enough elongated material to be detectable on a gel, move-stop-jetting tests to increase the density of spots were done with 60 pulses. Instead of moving the substrate 8 times vertically under the print head and each time jetting, in this example, the substrate was moved 15 times i.e. in 5 mm increments. The slide was then shifted laterally 232 μm and the move-stop-jetting process repeated. This was repeated a further time to increase the reaction site density from 8880 to 26640 reaction sites/slide.
The effect of TdT ink viscosity and volume of ink delivered to reaction sites was examined using the above experimental set-up. The volume of ink delivered to each reaction site determines the size of the reaction site and therefore an upper bound on the density of reaction sites. Successful elongation (for a given viscosity and delivered volume) was determined by the observation of bimodal distributions or bands on a gel (i.e., a band of successfully elongated product and a band of capped initiators from outside of the reaction sites, as illustrated in
The results show that for successful synthesis on small reaction sites (necessary for high densities) the viscosity of an ink must be low, e.g. less than 2 mPa·s for reaction sites formed using 2-3 μL droplets.
In this example, the benefit of a capping step is demonstrated. The synthesis was performed essentially as described in Example 2 for each of two slides (i.e. synthesis took place on eight rows of three regions each), with the exceptions that the enzyme buffers contained 11% glycerol and the no-capping slide 2 was incubated for 5 min post-synthesis. On slide 1 a capping step (performed as described in Example 2) was included and on slide 2 the capping step was omitted. The results are shown in
In this example, a 5-cycle synthesis was performed on two slides as described above, except that synthesis took place in rows 1-6 instead of rows 3-8. The syntheses on the two slides were identical except that one was incubated in a humidity chamber during elongation (slide 1) and the slide was left out on the lab bench during elongation (slide 2). Humidity and temperature were measured with a Testo 175H1 data logger. The temperature and average relative humidity for slide 1 were 21° C. and about 80%, respectively, and for slide 2 the values were 21° C. and about 36%, respectively. After synthesis, products were cleaved, pooled and loaded onto gels as described above. The results are shown in
In this example, various dyes were added to synthesis reagent inks to facilitate monitoring of reaction site geometry, including size, shape, overlap or coalescence with adjacent sites, and the like. The object of these experiments was to identify at least four different colored dyes that facilitated image processing while at the same time had no effect on synthesis. The following dyes were tested: Bromocresol Green, Bromocresol Purple. Chlorophenol Red, Methylene Blue and Xylenecyanol FF. The dyes were combined with the standard enzyme buffer (0.5 M cacodylate buffer pH 7.5, 1 mM CoCl2, 500 μM dTTP-ONH2, 20 μM4 H4 M57, 50 mM BOX aldehyde scavenger, 0.005% Tween 20) with 11 w % glycerol) with a final concentration of about 0.6 mg/mL (or between 1-2 mM depending on the dye). The enzyme buffers were spotted manually (10 uL droplets) on discrete regions of FAM labelled, photocleavable, DNA initiators (as described above). Each colored enzyme buffer was applied to 3 such regions, and enzyme buffer without dye was applied to 9 regions as controls. The slide was incubated for 5 min in a pipette tip box containing−50 mL of SSC buffer 1×. After incubation, the slide was washed for 3 min in deblocking buffer and 1 min in MQ H2O, 10 cycles of synthesis were then performed, after which synthesis products were cleaved (25 min incubation in PBS under UV illumination at 365 nm) and analyzed by gel electrophoresis. As shown in
Unless otherwise specifically defined herein, terms and symbols of nucleic acid chemistry, biochemistry, genetics, and molecular biology used herein follow those of standard treatises and texts in the field. e.g. Kornberg and Baker, DNA Replication. Second Edition (W.H. Freeman, New York, 1992); Lchninger. Biochemistry. Second Edition (Worth Publishers. New York, 1975); Strachan and Read, Human Molecular Genetics, Second Edition (Wiley-Liss, New York, 1999); Le. Recent Progress in Ink Jet Technologies II, chapter 1, pgs, 1-14 (1999); Zapka, editor, “Handbook of Industrial Inkjet Printing,” (Wiley-VCH, Weinheim. Germany).
“Functionally equivalent” in reference to amino acid positions in two or more different TdTs means (i) the amino acids at the respective positions play the same functional role in an activity of the TdTs, and (ii) the amino acids occur at homologous amino acid positions in the amino acid sequences of the respective TdTs. It is possible to identify positionally equivalent or homologous amino acid residues in the amino acid sequences of two or more different TdTs on the basis of sequence alignment and/or molecular modelling. In some embodiments, functionally equivalent amino acid positions belong to inefficiency motifs that are conserved among the amino acid sequences of TdTs of evolutionarily related species. e.g. genus, families, or the like. Examples of such conserved inefficiency motifs are described in Motca ct al, Biochim. Biophys. Acta. 1804(5): 1151-1166 (2010); Dclaruc ct al. EMBO J., 21: 427-439 (2002); and like references.
“Humectant” is any hygroscopic substance that attracts and retains moisture. Exemplary humectants include, but are not limited to, glycerol, alcohol sugars, ethylhexylglycerin, panthenol, sorbitol, xylitol, maltitol, propylene glycol, hexylene glycol, butylene glycol, sodium lactate, hyaluronic acid, polydextrose, or the like.
“Mutant” or “variant,” which are used interchangeably, refer to polypeptides derived from a natural or reference TdT polypeptide described herein, and comprising a modification or an alteration, i.e., a substitution, insertion, and/or deletion, at one or more positions. Variants may be obtained by various techniques well known in the art. In particular, examples of techniques for altering the DNA sequence encoding the wild-type protein, include, but are not limited to, site-directed mutagenesis, random mutagenesis, sequence shuffling and synthetic oligonucleotide construction. Mutagenesis activities consist in deleting, inserting or substituting one or several amino-acids in the sequence of a protein or in the case of the invention of a polymerase. The following terminology is used to designate a substitution: L238A denotes that amino acid residue (Leucine. L) at position 238 of a reference, or wild type, sequence is changed to an Alanine (A). A132V/I/M denotes that amino acid residue (Alanine, A) at position 132 of the parent sequence is substituted by one of the following amino acids: Valine (V), Isoleucine (I), or Methionine (M). The substitution can be a conservative or non-conservative substitution. Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine, asparagine and threonine), hydrophobic amino acids (methionine, leucine, isoleucine, cysteine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine and serine).
“Polynucleotide” or “oligonucleotide” are used interchangeably and each mean a linear polymer of nucleotide monomers or analogs thereof. Monomers making up polynucleotides and oligonucleotides are capable of specifically binding to a natural polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, base stacking, Hoogsteen or reverse Hoogsteen types of base pairing, or the like. Such monomers and their internucleosidic linkages may be naturally occurring or may be analogs thereof, e.g. naturally occurring or non-naturally occurring analogs. Non-naturally occurring analogs may include PNAs, phosphorothioate internucleosidic linkages, bases containing linking groups permitting the attachment of labels, such as fluorophores, or haptens, and the like. Whenever the use of an oligonucleotide or polynucleotide requires enzymatic processing, such as extension by a polymerase, ligation by a ligase, or the like, one of ordinary skill would understand that oligonucleotides or polynucleotides in those instances would not contain certain analogs of internucleosidic linkages, sugar moieties, or bases at any or some positions. Polynucleotides typically range in size from a few monomeric units, e.g. 5-40, when they are usually referred to as “oligonucleotides,” to several thousand monomeric units. Whenever a polynucleotide or oligonucleotide is represented by a sequence of letters (upper or lower case), such as “ATGCCTG,” it will be understood that the nucleotides are in 5′-+3′ order from left to right and that “A” denotes deoxyadenosine. “C” denotes deoxycytidine, “G” denotes deoxyguanosine, and “T” denotes thymidine, “I” denotes deoxyinosine, “U” denotes uridine, unless otherwise indicated or obvious from context. Unless otherwise noted the terminology and atom numbering conventions will follow those disclosed in Strachan and Read, Human Molecular Genetics 2 (Wiley-Liss, New York, 1999). Usually polynucleotides comprise the four natural nucleosides (e.g. deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine for DNA or their ribose counterparts for RNA) linked by phosphodiester linkages; however, they may also comprise non-natural nucleotide analogs. e.g. including modified bases, sugars, or internucleosidic linkages. It is clear to those skilled in the art that where an enzyme has specific oligonucleotide or polynucleotide substrate requirements for activity, e.g. single stranded DNA, RNA/DNA duplex, or the like, then selection of appropriate composition for the oligonucleotide or polynucleotide substrates is well within the knowledge of one of ordinary skill, especially with guidance from treatises, such as Sambrook et al. Molecular Cloning, Second Edition (Cold Spring Harbor Laboratory, New York, 1989), and like references. Likewise, the oligonucleotide and polynucleotide may refer to either a single stranded form or a double stranded form (i.e. duplexes of an oligonucleotide or polynucleotide and its respective complement). It will be clear to one of ordinary skill which form or whether both forms are intended from the context of the terms usage.
“Primer” means an oligonucleotide, either natural or synthetic that is capable, upon forming a duplex with a polynucleotide template, of acting as a point of initiation of nucleic acid synthesis and being extended from its 3′ end along the template so that an extended duplex is formed. Extension of a primer is usually carried out with a nucleic acid polymerase, such as a DNA or RNA polymerase. The sequence of nucleotides added in the extension process is determined by the sequence of the template polynucleotide. Usually primers are extended by a DNA polymerase. Primers usually have a length in the range of from 14 to 40 nucleotides, or in the range of from 18 to 36 nucleotides. Primers are employed in a variety of nucleic amplification reactions, for example, linear amplification reactions using a single primer, or polymerase chain reactions, employing two or more primers. Guidance for selecting the lengths and sequences of primers for particular applications is well known to those of ordinary skill in the art, as evidenced by the following references that are incorporated by reference: Dieffenbach, editor, PCR Primer: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Press, New York, 2003).
“Sequence identity” refers to the number (or fraction, usually expressed as a percentage) of matches (e.g., identical amino acid residues) between two sequences, such as two polypeptide sequences or two polynucleotide sequences. The sequence identity is determined by comparing the sequences when aligned so as to maximize overlap and identity while minimizing sequence gaps. In particular, sequence identity may be determined using any of a number of mathematical global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithm (e.g. Needleman and Wunsch algorithm; Needleman and Wunsch. 1970) which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g. Smith and Waterman algorithm (Smith and Waterman, 1981) or Altschul algorithm (Altschul et al., 1997; Altschul et al., 2005)). Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software available on internet web sites such as http://blast.ncbi.nlm.nih.gov/or ttp://www.ebi.ac.uk/Tools/emboss/. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, % amino acid sequence identity values refer to values generated using the pair wise sequence alignment program EMBOSS Needle, that creates an optimal global alignment of two sequences using the Needleman-Wunsch algorithm, wherein all search parameters are set to default values, i.e. Scoring matrix=BLOSUM62, Gap open=10, Gap extend=0.5. End gap penalty=false, End gap open=10 and End gap extend=0.5.
“Substitution” means that an amino acid residue is replaced by another amino acid residue. Preferably, the term “substitution” refers to the replacement of an amino acid residue by another selected from the naturally-occurring standard 20 amino acid residues, rare naturally occurring amino acid residues (e.g. hydroxyproline, hydroxylysine, allohydroxylysine, 6-N-methyllysine, N-ethylglycine, N-methylglycine, N-ethylasparagine, allo-isoleucine, N-methylisoleucine, N-methylvaline, pyroglutamate, aminobutyric acid, omithine, norleucine, norvaline), and non-naturally occurring amino acid residue, often made synthetically, (e.g. cyclohexyl-alanine). Preferably, the term “substitution” refers to the replacement of an amino acid residue by another selected from the naturally-occurring standard 20 amino acid residues. The sign “+” indicates a combination of substitutions. The amino acids are herein represented by their one-letter or three-letters code according to the following nomenclature: A: alanine (Ala); C: cysteine (Cys); D: aspartic acid (Asp); E: glutamic acid (Glu); F: phenylalanine (Phe); G: glycine (Gly); H: histidine (His); I: isoleucine (Tle); K: lysine (Lys); L: leucine (Leu); M: methionine (Met); N: asparagine (Asn); P: proline (Pro); Q: glutamine (Gin); R: arginine (Arg); S: serine (Ser); T: threonine (Thr); V: valine (Val); W: tryptophan (Trp) and Y: tyrosine (Tyr). In the present document, the following terminology is used to designate a substitution: L238A denotes that amino acid residue (Leucine, L) at position 238 of the parent sequence is changed to an Alanine (A). A132V/I/M denotes that amino acid residue (Alanine, A) at position 132 of the parent sequence is substituted by one of the following amino acids: Valine (V), Isoleucine (I), or Methionine (M). The substitution can be a conservative or non-conservative substitution. Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine, asparagine and threonine), hydrophobic amino acids (methionine, leucine, isoleucine, cysteine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine and serine).
| Number | Date | Country | Kind |
|---|---|---|---|
| 20185871.9 | Jul 2020 | EP | regional |
| 20306378.9 | Nov 2020 | EP | regional |
| 21305795.3 | Jun 2021 | EP | regional |
This application is a 371 of International Application Serial No. PCT/EP2021/069162, filed on Jul. 9, 2021, which application claims priority to EP21305795.3, filed on Jun. 10, 2021, EP20306378.9, filed on Nov. 13, 2020, and EP20185871.9, filed on Jul. 15, 2020, the disclosures of which are incorporated herein by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/069162 | 7/9/2021 | WO |
