Patent Application: US-95943507-A

Abstract:
disclosed are methods for performing aptamer preselection based on unique geometry and the content of stems or loops of the aptamer , which methods are capable of providing suitable binders and also permit selection of aptamers performed essentially entirely on a chip or other device . also disclosed are kits for aptamer selection .

Description:
the present invention may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures and examples , which form a part of this disclosure . it is to be understood that this invention is not limited to the specific devices , methods , applications , conditions or parameters described and / or shown herein , and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed invention . also , as used in the specification including the appended claims , the singular forms “ a ,” “ an ,” and “ the ” include the plural , and reference to a particular numerical value includes at least that particular value , unless the context clearly dictates otherwise . the term “ plurality ”, as used herein , means more than one . when a range of values is expressed , another embodiment includes from the one particular value and / or to the other particular value . similarly , when values are expressed as approximations , by use of the antecedent “ about ,” it will be understood that the particular value forms another embodiment . all ranges are inclusive and combinable . it is to be appreciated that certain features of the invention which are , for clarity , described herein in the context of separate embodiments , may also be provided in combination in a single embodiment . conversely , various features of the invention that are , for brevity , described in the context of a single embodiment , may also be provided separately or in any subcombination . further , reference to values stated in ranges include each and every value within that range . as used in this disclosure , the singular forms “ a ”, “ an ”, and “ the ” may refer to plural articles unless specifically stated otherwise . thus , for example , references to a method of manufacturing , derivatizing , or treating “ an analyte ” may include a mixture of one or more analytes . furthermore , the use of grammatical equivalents such as “ nucleic acids ”, “ polynucleotides ”, or “ oligonucleotides ” are not meant to imply differences among these terms unless specifically indicated . to facilitate understanding of the invention set forth in the disclosure that follows , a number of terms are defined below . the term “ aptamer ” refers to a molecule or series of molecules which assumes a shape that contributes toward binding of a target molecule or organism . the term “ complementary strand ” refers to a strand composed of opposite molecules in a pair as compared with the first strand . the pair will exhibit affinity for each other such as in electrostatic , hydrophobic , hydrophilic , magnetic or hydrogen bonding interactions . a common example of complementarity is in nucleic acid base pairing . the term “ cooperativity ” refers to the use of two or more aptamers in a set , where a binding event to one aptamer results in the presentation of bound analyte at an enhanced local concentration to a second aptamer , resulting in increases in kinetics , affinity , sensitivity and / or specificity of the reaction over what the second aptamer or set of aptamers would experience in a noncooperative setting such as in free solution . cooperativity can refer to enhanced characteristics contributing to the binding of an analyte or the inhibition of binding of an analyte . a cooperative aptamer is one that has two or more aptamers in close proximity that act cooperatively . the term “ geometric enrichment ” refers to the preselection of aptamers based on unique geometries . for a 20mer aptamer this corresponds to approximately 325 unique geometries in contrast with more than 1e12 randomers used in selex . geometric enrichment may refer to selection or consideration of all possible geometries or only a fraction of those geometries . the term “ characteristic ” refers to length , mass , volume , composition , geometry , or shape . as an example , a characteristic of an aptamer is the aptamer &# 39 ; s length . the terms “ insertion ” and “ deletion ” refer to extra or missing molecules in a complementary strand respectively . the term “ label ” refers to any atom or molecule that can be attached to a molecule for detection . the term “ ligand ” refers to any binder whether biological or non - biological of a target entity . the term “ loop ” refers to a single stranded segment of aptamer that is created by the aptamer folding back on itself . the term “ microarray ” refers to two or more unique aptamers or combinations of aptamers in a single screening in which target binding to one aptamer or combination of aptamers is distinguishable from binding to the others . the term “ mismatch ” in aptamer folding refers to a molecule in a complementary strand which does not allow for binding of the molecule opposite of it . in an aptamer - target complex , a mismatch indicates a variant target other than the wild type . the terms “ peptide ”, “ polypeptide ”, “ oligopeptide ”, or “ protein ” refers to two or more covalently linked , naturally occurring or synthetically manufactured amino acids . there is no intended distinction between the length of a “ peptide ”, “ polypeptide ”, “ oligopeptide ”, or “ protein ”. the term “ peptide nucleic acid ” or “ pna ” refers to an analogue of dna that has a backbone that comprises amino acids or derivatives or analogues thereof , rather than the sugar - phosphate backbone of nucleic acids ( dna and rna ). pna mimics the behavior of a natural nucleic acid and binds complementary nucleic acid strands . the term “ pocket ” refers to a single stranded segment of the aptamer that is created by mismatches , insertions or deletions in the complementary strand of the aptamer . the terms “ polynucleotide ”, “ oligonucleotide ” or “ nucleic acid ” refer to polydeoxyribonucleotides ( dna ), polyribonucleotides ( rna ), analogs and derivatives thereof . there is no intended distinction between the length of a “ polynucleotide ”, “ oligonucleotide ” or “ nucleic acid ”. a “ small organic molecule ” is a carbon - containing molecule which is typically less than about 2000 daltons . more typically , the small organic molecule is a carbon - containing molecule of less than about 1000 daltons . the small organic molecule may or may not be a biomolecule with known biological activity . the term “ stem ” refers to a region of the aptamer which is folded on itself due to interactions between complementary strands . the term “ substrate ” refers to a medium relatively large to the aptamer and can include the surface of a solid support , a nanotube , a cell , or a microorganism such as a bacterium , virus , or phage . suitable solid supports include , but are not limited to cyclo olefin polymers and copolymers , acrylamide , cellulose , nitrocellulose , glass , polystyrene , polyethylene vinyl acetate , polypropylene , polymethacrylate , polyethylene , polysilicates , polyethylene oxide , polycarbonates , teflon , fluorocarbons , nylon , silicon rubber , collagen , polyanhydrides , polyglycolic acid , polylactic acid , polyorthoesters , polypropylfumarate , glycosaminoglycans , and polyamino acids . a solid support or matrix can be in one of the many useful forms including thin films or membranes , plates such as various formats of microtiter plates , beads such as magnetic beads or latex beads , bottles , dishes , fibers , woven fibers , shaped polymers , particles , microarrays , microfluidic channels , microchips , microparticles such as microspheres , and nanoparticles . methods of attaching the capture and detection and capture probes to a surface are known in the art and include , without limitation , direct adhesion to the surface such as plastic , use of a capture agent , chemical coupling , and via a binding pair such as biotin - avidin . the detection and capture probes can independently have a tether to facilitate the attachments to the surface signals . the term “ target ” has reference to the molecule , compound or organism an aptamer is designed to bind . appropriate targets include both biological and non - biological entities . suitable biological targets include , but are not limited to , proteins , peptides , nucleic acid sequences , peptide nucleic acids , antibodies , antigens , receptors , molecules , biological cells , microorganisms , cellular organelles , cell membrane fragments , bacteriophage , bacteriophage fragments , whole viruses , viral fragments , and small molecules such as lipids , carbohydrates , amino acids , drug substances , and molecules for biological screening and testing . a target can also refer to a complex of two or more molecules , for example , a ribosome with both rna and protein elements or an enzyme with substrate attached . the term “ tentacle probe ” refers to a type of cooperative probe having a detection probe and a capture probe wherein the detection probe can change conformation and the change in conformation generates a change in detectable signal . in general , upon binding to a target analyte , the interactions between the detection probe and the target analyte shifts the equilibrium predominantly towards to an open conformation . the term “ variant ” or “ mutant ” analyte refers to an analyte that is different than its wildtype counterpart . the term “ wildtype ” as used herein refers to the typical form of an organism , strain , gene , or characteristic as it occurs in nature , as distinguished from mutant forms that can result from selective breeding in a first aspect , the present invention discloses methods for selecting one or more aptamers by geometric enrichment , comprising consideration of one or more characteristics of the one or more aptamers to so as to formulate one or more possible geometries of the one or more aptamers . additional detail regarding such methods is set forth in additional detail elsewhere herein . in some embodiments , the methods include consideration of more than 1 % of all possible geometries for a given aptamer length . in other embodiments , the methods may include more than 10 % of all possible geometries for a given aptamer length . in any of these embodiments , the gc content of a stem may be greater , on average , than 50 %. the claimed methods also include performing additional geometric enrichment on a substrate . suitable substrates include glass , polymers , and the like ; substrates suitable for forming microarrays are considered especially suitable . in some embodiments , attachment of one or more aptamers — preferably chosen or identified by geometric enrichment — are used to form a microarray . geometric enrichment may include monitoring binding to the microarray via fluorescence . techniques for such monitoring are known to those having ordinary skill in the art . in some configurations , the contrast between wild type and variant binding is used to select aptamers . binding over time of aptamers may also be used to determine the kinetics of individual aptamers . in some embodiments , binding over multiple concentrations of one or more aptamers is used to determine aptamer affinities . further , binding may also be used to determine inhibition of a process , which processes may be enzymatic in nature . binding may also be used to determine the acceleration of a process , including enzymatic processes . geometric enrichment may suitably be performed on a substrate , as described elsewhere herein . such substrates may comprise microarrays . enrichment may be performed by monitoring binding to the microarray via fluorescence , which may include using the contrast between wild type and variant binding is used to select aptamers . binding over time may also be used to observe kinetics of individual aptamers and , in some cases , to determine aptamer affinities , and or the inhibition or acceleration of processes , including enzymatic processes . two or more geometric enrichment - selected aptamers are linked directly or indirectly for further enrichment . one or more of such aptamers may be linked to a substrate so as to form a microarray , which microarray may be used to support enrichment , as described elsewhere herein . aptamers — including aptamers selected by geometric enrichment — may also be linked to one or more ligands . such ligands may be identified or isolated by a variety of methods known to those having ordinary skill in the art . aptamer - ligand combinations may be linked to a substrate to form a microarray , having application as described elsewhere herein . the claimed invention also provides kits , which kits include one or more geometrically enriched aptamers according to the claimed methods . such kits may be used to the method of claim 1 and instructions for using them to select the appropriate aptamer . kits suitably include instructions to enable to user to utilize the kits , although proper use of the kits will be apparent to those of ordinary skill in the art . kits may include one or more geometrically enrichment selected aptamers — which may also include ligands . as discussed , the claimed invention includes a method for selecting aptamers using geometric enrichment . in geometric enrichment , all the possible geometries are formulated for a given aptamer length or for a range of aptamer lengths . there are a number of methods in which this range of geometries can be produced . one example of a method to produce the available geometries involves making note of minimum requirements for aptamer geometry formation . for example , a stem cannot form without at least one base pair forming ; a pocket cannot exist without at least one base failing to base pair ; the loop on the end of an aptamer cannot be shorter than three base pairs and still fold on itself . in other embodiments , the geometries can be further refined by examining statistical trends among existing aptamers . for example , in a survey of 32 different aptamers with affinities toward 21 different targets , the following statistics were observed ( 6 - 26 ): by using the number of bases in each folded region ( aptamer ) one can ascertain the average number of loops , pockets and stems per base in the aptamer . one can also determine the standard deviations of these occurrences . accordingly , in some embodiments , these aptamer statistics can be used to further reduce the number of possible geometries . for example , it can be observed that there were no stem sizes below 2 bases in length . using this statistic , and the statistic on minimum loop size , it can be deduced that there can be no more than three pockets and one loop in a 20mer aptamer . one method of creating the list of all possible geometries is to use these rules to create a figure for a given aptamer size as shown in fig3 . a table is made as set forth in example i , in which all the possible combinations defined in the figure are mapped out . it should be noted that in some embodiments it is not necessary to use all the available geometries . in some embodiments , suspected geometries targeting a given epitope can be the focus of selection . suitable aptamer sizes for geometric enrichment are typically between 5 and 1000 bases , between 10 and 200 bases , between 10 and 100 bases . similar to geometric enrichment , in some embodiments statistical data can be used to further enrich the possible aptamer pool . for example , in a survey of 32 different aptamers with affinities toward 21 different targets , the following statistics were observed ( 6 - 26 ): by using this data , it is seen that stem gc content may be comparatively high . in fact , in some embodiments , it is preferred to use a gc rich stem greater than 50 %, greater than 60 %, greater than 75 % or even 100 % gc rich . such a stem stabilizes the aptamer geometry more than any other shape . by selecting stem with high gc content , the number of possible sequences conforming to a given geometry is greatly reduced , easily allowing chip selection of aptamers . this same methodology can be applied to loop content as well . in some embodiments , loop content may have less than 50 % gc content , less than 40 % gc content , less than 30 % gc content . in some embodiments , aptamer selection through geometric enrichment is greatly simplified by using an aptamer microarray . in some embodiments a library of similar geometries with identical or varying base content is placed on the microarray . in other embodiments , many different geometries with identical or varying base content are placed on the microarray . in some embodiments , the number of geometries represented on the microarray is greater than 1 %, greater than 10 %, or even greater than 50 % of the possible geometries . in some embodiments , target is allowed to hybridize with the aptamers on the microarray . in embodiments where target is labeled with a fluorescent substance , the excess target is washed away following hybridization . those geometries which exhibit the greatest fluorescence above background are chosen as candidate aptamers . in other embodiments , variant is allowed to hybridize with the aptamers on the microarray . in embodiments where variant is labeled with a fluorescent substance , the excess variant is washed away following hybridization . those geometries which exhibit fluorescence above background are eliminated as possible aptamers . in other embodiments it may be desirable to have aptamers that bind to both the wild type and variant . in this case , those geometries that exhibit binding to both the wild type and variant in microarray analysis are chosen as candidate aptamers . in still other embodiments , the microarray format is used to measure kinetic parameters of the aptamers before selection . in some embodiments , the microarray is monitored through label free detection means such as fluorescent polarization or surface plasmon resonance . binding over time is monitored to determine kinetic rates . those aptamers exhibiting the desired degree of binding and at the desired rate are selected as candidate aptamers . in yet other embodiments , thermodynamic parameters such as the affinity of binding are gleaned from the microarray . in some embodiments , the forward and reverse rate constants are determined as previously mentioned . the ratio of the forward to the reverse rate constant is used to find the affinity . in other embodiments , titrations of wild type target can be used to measure the fluorescence as a function of concentration . for an excess of target , the concentration at which binding is half maximal is equivalent to the dissociation constant . in some embodiments , it may be desirable to enhance the performance of individual aptamers by combining them with other aptamers . in some embodiments , geometrically enriched aptamers are placed in groups of two or more prior to selection . methods of placement together include but are not limited to indirect linkage to a substrate or direct linkage via polyethylene glycol , carbon chains , natural or modified nucleic acids , amino acids , or other linkers known to those skilled in the art . in some embodiments , the aptamers selected from an initial round of geometric enrichment may be placed together in a microarray . in some embodiments , geometrically enriched and selected aptamers are placed in groups of two or more prior to selection . methods of placement together include but are not limited to indirect linkage to a substrate or direct linkage via polyethylene glycol , carbon chains , natural or modified nucleic acids , amino acids , or other linkers known to those skilled in the art . in some embodiments , target is allowed to hybridize with the aptamers on the microarray . in embodiments where target is labeled with a fluorescent substance , the excess target is washed away following hybridization . those geometries which exhibit the greatest fluorescence above background are chosen as candidate aptamers . in other embodiments , variant is allowed to hybridize with the aptamers on the microarray . in embodiments where variant is labeled with a fluorescent substance , the excess variant is washed away following hybridization . those geometries which exhibit fluorescence above background are eliminated as possible aptamers . in other embodiments it may be desirable to have aptamers that bind to both the wild type and variant . in this case , those geometries that exhibit binding to both the wild type and variant in microarray analysis are chosen as candidate aptamers . in still other embodiments , the microarray format can be used to measure kinetic parameters of the aptamers before selection . in some embodiments , the microarray is monitored through label free detection means such as fluorescent polarization or surface plasmon resonance . binding over time is monitored to determine kinetic rates . those aptamers exhibiting the desired degree of binding and at the desired rate are selected as candidate aptamers . in yet other embodiments , thermodynamic parameters such as the affinity of binding are gleaned from the microarray . in some embodiments , the forward and reverse rate constants are determined as previously mentioned . the ratio of the forward to the reverse rate constant is used to find the affinity . in other embodiments , titrations of wild type target can be used to measure the fluorescence as a function of concentration . for an excess of target , the concentration at which binding is half maximal is equivalent to the dissociation constant . in an exemplary embodiment of creating all possible geometries , the format shown in fig3 was used to create all possible geometries for a 20mer . in the table below , seq #, l , s1 , s2 , s3 , p1 , p2 , p3 , p4 stand for sequence number , loop , stem 1 , stem 2 , stem 3 , pocket 1 , pocket 2 , pocket 3 , pocket 4 respectively . the numbers beside each sequence number represent the number of bases comprising each feature . each row adds up to a total of 20 bases in the aptamer . the location of each feature in the exemplary geometric aptamer is as shown in fig3 . in an exemplary embodiment of further enrichment , statistical measures governing existing aptamers are applied . high gc content is used in the stems to create the most stable aptamer geometries . in order to avoid alternate geometries to those intended , low gc content is used in the loops and pockets . in an exemplary embodiment , arbitrary sequences according to the above guidelines were chosen to form the stem , loop and pocket regions as follows : gccgccgccg ( for use in the stem ) and aaaaaaaaaaaaaaa ( for use in pockets and loops ). only the number of bases designated in the spreadsheet in example i were selected from the forgoing sequences . examples of these geometries are shown in fig4 . since all the unique geometries are represented and the stems are in their most stable form with high gc content , and the loop and pocket variability is limited to at rich sequences , the starting content has been greatly enriched . in an exemplary embodiment , four extra bases were added to the beginning of stem 1 in order to increase the number of geometries that assumed the predicted form . exemplary sequences are listed in the following table and are correlated with the table in example i for all possible geometries of a 20mer , where each column heading has the same meaning as defined in example i : in an exemplary embodiment , following geometric enrichment and further enrichment , selection of aptamers is performed directly on a chip as shown in fig5 . labeled analyte is passed across the microarray of aptamers for both wild type and variant . those aptamers which are specific to only the wildtype are selected for further examination and characterization . in an exemplary embodiment , individual aptamers which have been independently specific for the target are combined together as shown in fig6 . in an exemplary embodiment , the linker is polyethylene glycol . in an exemplary embodiment , each possible geometrically enriched aptamer is placed in a pair with another geometrically enriched aptamer and placed in a microarray . as in example iii , wild type and variant analyte is passed across the microarray , allowing for selection and counter selection of the appropriate aptamer pair . those aptamer pairs showing the highest affinity for the target and with no detectable affinity towards the variant are chosen . in an exemplary embodiment , following geometric enrichment and further enrichment , selection of aptamers was performed directly on a chip : aptamers hybridizations were performed on a tcan 4800 hs automated hybridization station according to the following protocol : 1 ) denature aptamers with 30 s wash at 85 ° c . with 0 . 1 % sds in di h 2 o , incubate at 85 ° c . for 30 s while shaking , wash at 85 ° c . for 30 s 2 ) repeat with di h 2 o 3 ) block with 0 . 1 % tween in pbs buffer and 5 mm mgcl 2 at 23 ° c . by washing for 30 s , incubating / shaking for 30 s , and washing for 30 more seconds 4 ) inject 100 μl of 1 to 10 μm protein ( bsa , gp120 ) and hybridize for 30 min while shaking 5 ) wash with 0 . 1 % tween in pbs buffer and 5 mm mgcl 2 at 23 ° c . for 10 s 6 ) wash with pbs buffer and 5 mm mgcl 2 at 23 ° c . for 20 s 7 ) dry and image on genepix 4000b scanner . alignment was performed using nimblescan v2 . 2 software and aptamers were selected by exceeding the average fluorescence plus three standard deviations of aptamers containing 100 % thymine in the variable loop regions . in order to be selected as an aptamer , 3 of the 4 replicates had to exceed this level of fluorescence ( fig7 ). ten aptamers were selected for hiv gp120 ( fig8 ). the aptamers selected for hiv gp120 did not have the same geometries or content as the aptamers that were selected for bsa . the examples set forth above are provided to give those of ordinary skill in the art with a complete disclosure and description of how to make and use the preferred embodiments of the present invention , and are not intended to limit the scope of what the inventors regard as their invention . modifications of the above - described modes for carrying out the invention that are obvious to persons of skill in the art are intended to be within the scope of the following claims . all publications , patents , and patent applications cited in this specification are incorporated herein by reference as if each such publication , patent or patent application were specifically and individually indicated to be incorporated herein by reference . 1 . tuerk , c . and gold , l . 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