Microsoft Technology Licensing, LLC

Jake Smith

Bichlien Hoang Nguyen

Karin Strauss

Yuan-Jyue Chen

Ranveer Chandra

Douglas M Carmean

Gagan Gupta

Richard P Rouse

Matthew David Turner

Robert Carlson

Recycling information is associated with objects through the use of molecular tags. The recycling information may describe the type of material that the object is made from as well as provide instructions for recycling. The molecular tags may be polynucleotides or other types of molecules including inorganic molecules. The molecular tags may be embedded within the object or attached to the surface of the object. At the end of the object's life, the molecular tags are read and the recycling information is used to appropriately recycle the object.

Molecular encoding of recycling information

De novo polynucleotide synthesis is performed with a substrate-bound polymerase. The polymerase is attached to a solid substrate such as a microelectrode array. The polymerase adds nucleotides to growing polynucleotides strands that are also attached to the solid substrate. Spatial control of polymerase activity is achieved by changing the rate of nucleotide polymerization at selected locations on the surface of the solid substrate. The rate of polymerization is changed by inhibiting or promoting activity of the polymerase. In some implementations, activation of electrodes in the microelectrode array changes the rate of nucleotide polymerization. Nucleotides are added to the growing polynucleotide strands at areas where the polymerase is active. By varying the locations where the substrate-bound polymerase is active and the species of nucleotide added, a population of polynucleotides with different, arbitrary sequences is synthesized on the surface of the solid substrate.

De novo polynucleotide synthesis with substrate-bound polymerase

Enzymatic polynucleotide synthesis with a template-independent polymerase is used to create multiple polynucleotides having different, arbitrary sequences on the surface of an array. The array provides a spatially-addressable substrate for solid-phase synthesis. Blocking groups are attached to the 3' ends of polynucleotides on the array. Prior to polynucleotide extension, the blocking groups are removed at a selected location on the array. In an implementation, the blocking groups are acyl groups removed with a negative voltage created at an electrode. The array is then incubated with the polymerase and a single species of nucleotide. Nucleotides are incorporated onto the 3′ ends of the polynucleotides without blocking groups. Washing removes the polymerase and free nucleotides. To create polynucleotides with different sequences at different locations on the array, the location where the blocking groups are removed and the species of nucleotide may be changed during repeated cycles of synthesis.

Spatial control of polynucleotide synthesis by strand capping

Multiple polynucleotides having different, arbitrary sequences are synthesized on the surface of an array by spatial control of polymerase activity. The polymerase is a template-independent polymerase such as terminal deoxynucleotidyl transferase (TdT). Spatial control of polymerase activity is implemented by localized changes in redox-pH conditions. A single species of nucleotide is added and incorporated on growing polynucleotide strands at locations on the array where the polymerase is active. A washing step removes the polymerase and free nucleotides. This process may be repeated multiple times changing both the location of polymerase activity and the species of nucleotide thereby synthesizing different polynucleotides in parallel on the surface of the array. Polymerase activity may be regulated by removing a blocking group attached to a His-tag sequence on the polymerase, a change in pH, or release of encapsulated inhibitors.

Spatially addressable control of polymerase activity

This disclosure provides electrochemically-cleavable linkers with cleavage potentials that are less than the redox potential of the solvent in which the linkers are used. In some applications, the solvent may be water or an aqueous buffer solution. The linkers may be used to link a nucleotide to a bound group. The linkers include a cleavable group which may be one of a methoxybenzyl alcohol, an ester, a propargyl thioether, or a trichloroethyl ether. The linkers may be cleaved in solvent by generating an electrode potential that is less than the redox potential of the solvent. In some implementations, an electrode array may be used to generate localized electrode potentials which selectively cleave linkers bound to the activated electrode. Uses for the linkers include attachment of blocking groups to nucleotides in enzymatic oligonucleotide synthesis.

Electrochemically-cleavable linkers

Electrode controlled hybridization is used to change local pH and selectively assemble oligonucleotide complexes on the surface of a microelectrode array. The oligonucleotide complexes have sticky ends that provide locations for subsequent oligonucleotide complexes to hybridize. The order in which specific oligonucleotide complexes are joined together encodes information. Controlled activation of individual electrodes in the microelectrode array creates negative voltages that reduces a buffer solution and raises the pH in proximity to the electrodes. At higher pH levels double-stranded oligonucleotides de-hybridize. Nicks between oligonucleotide complexes and oligonucleotides anchored to the microelectrode array are closed creating covalent attachments. De-hybridized single-stranded oligonucleotides are removed leaving only the oligonucleotides connected to microelectrode array. Thus, during a given round of synthesis, oligonucleotide complexes are added only to the locations on the microelectrode array where the electrodes are not activated.

Oligonucleotide assembly using pH based electrode controlled hybridization

Linker structures with minimal scar for enzymatic synthesis

Photon generating substrates for light-directed oligonucleotide synthesis are disclosed. Light is generated within a solid-state stack that supports growing oligonucleotides. The light may be generated by microLEDs, a pass-through liquid crystal panel, or an LCoS system. Light passes through a transmissive layer on which growing oligonucleotides are attached. Patterning of the light is controlled by selective activation of the microLEDs or by selective control of the transparency of a liquid crystal layer. Photolabile blocking groups are selectively removed by exposure to patterned light emitted from the photon generating substrate.

Photon generating substrates for oligonucleotide synthesis

Electrically controlled hybridization is used to selectively assemble oligonucleotides on the surface of a microelectrode array. Controlled activation of individual electrodes in the microelectrode array attracts oligonucleotides in solution to specific regions of the array where they hybridize to other oligonucleotides anchored on the array. The oligonucleotides that hybridize may provide locations for subsequent oligonucleotides to hybridize. The active electrodes and the oligonucleotides in solution may be varied during each round of synthesis. This allows for multiple oligonucleotides each with different and specific sequences to be created in parallel. This is accomplished without the use of phosphoramidite chemical synthesis or template-independent DNA polymerase enzymatic synthesis. Oligonucleotides created with these techniques may be used to encode digital data. Fully assembled oligonucleotides may be separated from the array and sequenced, stored, or otherwise processed.

Oligonucleotide assembly using electrically controlled hybridization

A switchable hydrophilic surface is created by attaching electrochemically switchable hydrophilicity polymers to the surface of a microelectrode array. Ferrocene polymers are one example of electrochemically switchable hydrophilicity polymers. Activation of electrodes in the microelectrode array changes the oxidation state of metal ions which switches the polymers between hydrophobic and hydrophilic conformations. Selective activation of electrodes can create patterns of wettability on the microelectrode array that may be varied in real time. The switchable hydrophilic surface may be used to control solid-phase synthesis of polymers. Growing polymers may be selectively extended at locations on the microelectrode array that are hydrophilic. The pattern of hydrophobic and hydrophilic regions can be changed during sequential rounds of synthesis to create a variety of different polymers at different locations on the surface of the microelectrode array.

Microelectrode array with a switchable hydrophilic surface

A universal template strand built with universal base analogs is used as a template for polynucleotide synthesis. The universal template strand can hybridize to any sequence of nucleotides. A new polynucleotide is synthesized by using a polymerase to extend a primer hybridized to the universal template strand. Unlike primer extension in polymerase chain reactions, base pairing with nucleotides in the template strand does not specify the sequence of the new polynucleotide. Instead, the sequence of the new polynucleotide is specified by the order of addition of protected nucleotides. After addition of a single species of protected nucleotide, the blocking group is removed and another protected nucleotide is added. The order of nucleotide addition can be varied to create any sequence. After synthesis, the polynucleotide can be dehybridized from the universal template strand. The universal template strand may then be reused to synthesize a different polynucleotide.

Growing community of inventors

Seattle, WA, United States of America

Jake Smith