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:
CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims the benefit of U.S. Application No. 60/870,493, filed Dec. 18, 2006, the entirety of which is incorporated by reference herein. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    Various scientific and patent publications are referred to herein. Each is incorporated by reference in its entirety. 
         [0003]    Aptamers are molecules that assume an appropriate shape “to fit” another molecule as in a lock and key mechanism. Aptamers can be used as diagnostic tools and/or therapeutics. They were first described in connection with a selection process called SELEX in 1990 by Tuerk and Gold (1). SELEX and its derivatives are based on starting with a large pool of molecules and enriching the pool through a series of iterations until the best binders are discovered. While this process has aided in the discovery of a number of high affinity binders, it is also cumbersome from the need to perform successive rounds of selection and amplification, ending with sequencing and testing of the sequenced aptamers (2). It is further complicated in that aptamers discovered through SELEX may not possess the desired attributes. For example, in the recent description of Cooperative Probe Assays and Tentacle Probes, low affinity binders may be preferable over high affinity binders in order to achieve greater specificity (3). Also the aptamers may need to be in a form such that they can undergo conformational changes producing an increase in signal. Thus there is a need for aptamer selection methods which are faster and easier than conventional SELEX and that allow selection of aptamers based on other properties than high affinity. 
         [0004]    Aptamer selection on a chip has been suggested (4). This approach has particular appeal due to the fact that a library of aptamers can be screened for selection and counterselection in a matter of hours, yielding not only information about which sequences function as aptamers, but also information on affinities and/or thermodynamic properties. Unfortunately, chip screening is limited due to library size. Until now, incomplete screenings have been performed requiring multiple steps (5) or using aptamers that are unusually small (hexamers) (4). It is conceivable that mathematical models could screen the library if appropriate algorithms were available, reducing the library to a size that could be placed on a chip. But to date, no algorithms exist that can sufficiently enrich the pool. And given that a 100 mer aptamer contains over 1e60 possible structures and even a library of 20 mers contains over 1e12 possibilities, present day computers could not perform all of the computations even if such an algorithm were to exist. The present invention has significant utility for chip based aptamer selection and enables the use of an enriched pool of nucleic acid sequences to define protein binding using both monovalent and multivalent constructs. 
       SUMMARY OF THE INVENTION 
       [0005]    The present invention relates, in part, to the selection of aptamers based on geometries to bypass SELEX. 
         [0006]    One aspect of the present invention provides for an algorithm for constructing a library of all possible aptamer geometries. Applications of the algorithm include, but are not limited to, design of tentacle probes, cooperative probe assays, drug constructs, cell targeting constructs, and synthetic antibodies. 
         [0007]    Another aspect of the present invention pertains to use of statistical data on current aptamers to further enrich the geometries to those which are most likely to bind. 
         [0008]    In certain embodiments, the present invention provides a process of geometric selection of aptamers on a chip. The process includes the steps of choosing an objective parameter for aptamer design, such as specificity, affinity, kinetics, inhibition, among others. In one aspect, an ideal size or range of sizes for a given aptamer is chosen. In another aspect, a pool is created based on desirable geometries. In some embodiments the desired geometries are all possible geometries, but in others a smaller subset of geometries may be used. In yet another aspect, the number of possible sequences possessing those geometries is reduced by further algorithims, such as GC content in stem and/or loop. In still another aspect, all or a part of the library is placed upon a chip with each sequence at a discrete location. In some embodiments, specific and nonspecific analyte are passed over the chip. In some embodiments, fluorescence is used to determine binding and binding characteristics. 
         [0009]    In certain embodiments, the aptamers described are composed of nucleic acids and/or nucleic acid analogues such as PNA&#39;s and LNA&#39;s. 
         [0010]    In further embodiments, the use of chip based selection can be applied to pairs or greater numbers of aptamers, where geometrically selected aptamers are placed in close proximity to each other either through attachment to a substrate or via linker. This provision allows screening for desirable aptamer qualities from cooperative or destructive interactions from pairs of aptamers. 
         [0011]    The present invention further relates to a kit for aptamer selection to a target analyte in a sample. The kit comprises one or more geometrically selected aptamers in the present invention. The kit can also comprise instructions on their use. When used in chip based selection, the kit may also contain a chip and reagents. 
         [0012]    Other aspects of the present invention are described throughout the specification. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0013]    The summary, as well as the following detailed description, is further understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings exemplary embodiments of the invention; however, the invention is not limited to the specific methods, compositions, and devices disclosed. In addition, the drawings are not necessarily drawn to scale. In the drawings: 
           [0014]      FIG. 1  depicts a probe that utilizes two relatively short or low affinity aptamers in order to achieve the desired specificity, where binding only occurs to a wild type analyte, and where the exemplary probe allows for changes in conformation in order to produce a change in fluorescent signal resulting in detection; 
           [0015]      FIG. 2  depicts a dopamine aptamer, additionally depicting a series of short aptamers linked together cooperatively via single stranded DNA, and where the stem content may be described as primarily GC content forming the most stable conformation of the given aptamer geometries; 
           [0016]      FIG. 3  depicts a design algorithm for a 20-mer aptamer, in which the minimum stem size is 2 bases as necessary to stabilize geometry, in which pocket sizes vary between 0 and 5 bases and are not necessarily symmetrical, in which the minimum loop size that will still allow for folding is 3 bases; 
           [0017]      FIG. 4  depicts an example of aptamer geometries where there are only a limited number of unique, stable aptamer geometries, which may be characterized by GC rich stems; 
           [0018]      FIG. 5  depicts a chip having an exemplary selection and counterselection of aptamer geometries; 
           [0019]      FIG. 6  depicts two specific aptamers combined cooperatively to achieve greater affinity; 
           [0020]      FIG. 7  illustrates an exemplary section of aptamer array following hybridization with 10 μM bovine serum albumin (BSA), in which aptamers having affinity for BSA appear as spots having a fluorescent intensity significantly above the background intensity; and 
           [0021]      FIG. 8  depicts exemplary aptamer geometries and sequences selected for HIV gp120 using the claimed geometric selection protocol—it was discovered that none of the aptamers binding to HIV gp120 bound to BSA. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0022]    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. 
         [0023]    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. 
         [0024]    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. 
         [0025]    To facilitate understanding of the invention set forth in the disclosure that follows, a number of terms are defined below. 
       Terms 
       [0026]    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. 
         [0027]    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. 
         [0028]    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. 
         [0029]    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. 
         [0030]    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. 
         [0031]    The terms “insertion” and “deletion” refer to extra or missing molecules in a complementary strand respectively. 
         [0032]    The term “label” refers to any atom or molecule that can be attached to a molecule for detection. 
         [0033]    The term “ligand” refers to any binder whether biological or non-biological of a target entity. 
         [0034]    The term “loop” refers to a single stranded segment of aptamer that is created by the aptamer folding back on itself. 
         [0035]    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. 
         [0036]    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. 
         [0037]    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”. 
         [0038]    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. 
         [0039]    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. 
         [0040]    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”. 
         [0041]    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. 
         [0042]    The term “stem” refers to a region of the aptamer which is folded on itself due to interactions between complementary strands. 
         [0043]    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. 
         [0044]    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. 
         [0045]    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. 
         [0046]    The term “variant” or “mutant” analyte refers to an analyte that is different than its wildtype counterpart. 
         [0047]    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 
         [0048]    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%. 
         [0049]    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. 
         [0050]    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. 
         [0051]    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. 
         [0052]    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. 
         [0053]    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. 
         [0054]    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. 
         [0055]    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. 
         [0056]    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. 
         [0057]    Additional discussion of the claimed invention follows. 
       Geometric Enrichment 
       [0058]    As discussed, the claimed invention includes a method for selecting aptamers using geometric enrichment. 
         [0059]    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. 
         [0060]    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. 
         [0061]    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): 
         [0000]    
       
         
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                   
                 Number 
                   
                   
                   
                   
                   
               
               
                   
                 Bases in 
                 of loops 
                   
                 Number 
               
               
                   
                 folded 
                 in folded 
                 Loop 
                 of 
                 Pocket 
                 Number 
                 Stem 
               
               
                   
                 region 
                 region 
                 Size 
                 pockets 
                 Size 
                 of Stems 
                 Size 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 Average 
                 24.26875 
                 1 
                 6.385938 
                 0.870313 
                 3.247378 
                 1.870313 
                 4.311406 
               
               
                 St Dev 
                 15.01247 
                 0 
                 5.209014 
                 1.192218 
                 1.874825 
                 1.192218 
                 1.191662 
               
               
                 Minimum 
                 9 
                 1 
                 3 
                 0 
                 1 
                 1 
                 2 
               
               
                 Maximum 
                 70 
                 1 
                 21 
                 5 
                 8 
                 6 
                 7.3 
               
               
                   
               
             
          
         
       
     
         [0062]    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. 
         [0063]    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  FIG. 3 . A table is made as set forth in Example I, in which all the possible combinations defined in the figure are mapped out. 
         [0064]    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. 
         [0065]    Suitable aptamer sizes for geometric enrichment are typically between 5 and 1000 bases, between 10 and 200 bases, between 10 and 100 bases. 
       Further Enrichment 
       [0066]    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): 
         [0000]    
       
         
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                   
                 Pocket 
                   
               
               
                   
                 Loop GC 
                 GC 
                 Stem GC 
               
               
                   
                 content 
                 content 
                 content 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Average 
                 0.387059 
                 0.500207 
                 0.689641 
               
               
                   
                 St Dev 
                 0.1804 
                 0.234697 
                 0.17954 
               
               
                   
                 Minimum 
                 0 
                 0 
                 0.25 
               
               
                   
                 Maximum 
                 0.89 
                 1 
                 1 
               
               
                   
                   
               
             
          
         
       
     
         [0067]    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. 
         [0068]    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. 
       Microarray Based Selection 
       [0069]    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. 
         [0070]    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. 
         [0071]    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. 
         [0072]    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. 
         [0073]    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. 
         [0074]    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. 
       Microarray Based Selection of Multivalent Aptamers 
       [0075]    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. 
         [0076]    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. 
         [0077]    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. 
         [0078]    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. 
         [0079]    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. 
         [0080]    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. 
         [0081]    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. 
       EXAMPLES AND ILLUSTRATIVE EMBODIMENTS 
     Example I 
     Example of Creating all Possible Geometries for an Aptamer 
       [0082]    In an exemplary embodiment of creating all possible geometries, the format shown in  FIG. 3  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  FIG. 3 . 
         [0000]    
       
         
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                 Seq # 
                 L 
                 S1 
                 S2 
                 S3 
                 P1 
                 P2 
                 P3 
                 P4 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 10 
                 10 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 2 
                 9 
                 6 
                 4 
                 0 
                 1 
                 0 
                 0 
                 0 
               
               
                 3 
                 9 
                 6 
                 4 
                 0 
                 0 
                 1 
                 0 
                 0 
               
               
                 4 
                 9 
                 4 
                 6 
                 0 
                 1 
                 0 
                 0 
                 0 
               
               
                 5 
                 9 
                 4 
                 6 
                 0 
                 0 
                 1 
                 0 
                 0 
               
               
                 6 
                 9 
                 4 
                 4 
                 0 
                 3 
                 0 
                 0 
                 0 
               
               
                 7 
                 9 
                 4 
                 4 
                 0 
                 0 
                 3 
                 0 
                 0 
               
               
                 8 
                 9 
                 4 
                 4 
                 0 
                 2 
                 1 
                 0 
                 0 
               
               
                 9 
                 9 
                 4 
                 4 
                 0 
                 1 
                 2 
                 0 
                 0 
               
               
                 10 
                 8 
                 12 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 11 
                 8 
                 6 
                 4 
                 0 
                 2 
                 0 
                 0 
                 0 
               
               
                 12 
                 8 
                 6 
                 4 
                 0 
                 0 
                 2 
                 0 
                 0 
               
               
                 13 
                 8 
                 6 
                 4 
                 0 
                 1 
                 1 
                 0 
                 0 
               
               
                 14 
                 8 
                 4 
                 6 
                 0 
                 2 
                 0 
                 0 
                 0 
               
               
                 15 
                 8 
                 4 
                 6 
                 0 
                 0 
                 2 
                 0 
                 0 
               
               
                 16 
                 8 
                 4 
                 6 
                 0 
                 1 
                 1 
                 0 
                 0 
               
               
                 17 
                 8 
                 4 
                 4 
                 0 
                 4 
                 0 
                 0 
                 0 
               
               
                 18 
                 8 
                 4 
                 4 
                 0 
                 0 
                 4 
                 0 
                 0 
               
               
                 19 
                 8 
                 4 
                 4 
                 0 
                 3 
                 1 
                 0 
                 0 
               
               
                 20 
                 8 
                 4 
                 4 
                 0 
                 1 
                 3 
                 0 
                 0 
               
               
                 21 
                 8 
                 4 
                 4 
                 0 
                 2 
                 2 
                 0 
                 0 
               
               
                 22 
                 7 
                 8 
                 4 
                 0 
                 1 
                 0 
                 0 
                 0 
               
               
                 23 
                 7 
                 8 
                 4 
                 0 
                 0 
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                 4 
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                 4 
                 4 
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                 2 
               
               
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                 4 
                 4 
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                 2 
               
               
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                 3 
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                 3 
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                 227 
                 3 
                 6 
                 4 
                 4 
                 2 
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                 1 
                 0 
               
               
                 228 
                 3 
                 6 
                 4 
                 4 
                 0 
                 2 
                 1 
                 0 
               
               
                 229 
                 3 
                 6 
                 4 
                 4 
                 2 
                 0 
                 0 
                 1 
               
               
                 230 
                 3 
                 6 
                 4 
                 4 
                 0 
                 2 
                 0 
                 1 
               
               
                 231 
                 3 
                 6 
                 4 
                 4 
                 1 
                 0 
                 2 
                 0 
               
               
                 232 
                 3 
                 6 
                 4 
                 4 
                 0 
                 1 
                 2 
                 0 
               
               
                 233 
                 3 
                 6 
                 4 
                 4 
                 1 
                 0 
                 0 
                 2 
               
               
                 234 
                 3 
                 6 
                 4 
                 4 
                 0 
                 1 
                 0 
                 2 
               
               
                 235 
                 3 
                 6 
                 4 
                 4 
                 1 
                 1 
                 1 
                 0 
               
               
                 236 
                 3 
                 6 
                 4 
                 4 
                 1 
                 1 
                 0 
                 1 
               
               
                 237 
                 3 
                 6 
                 4 
                 4 
                 1 
                 0 
                 1 
                 1 
               
               
                 238 
                 3 
                 6 
                 4 
                 4 
                 0 
                 1 
                 1 
                 1 
               
               
                 239 
                 3 
                 4 
                 12 
                 0 
                 1 
                 0 
                 0 
                 0 
               
               
                 240 
                 3 
                 4 
                 12 
                 0 
                 0 
                 1 
                 0 
                 0 
               
               
                 241 
                 3 
                 4 
                 10 
                 0 
                 3 
                 0 
                 0 
                 0 
               
               
                 242 
                 3 
                 4 
                 10 
                 0 
                 0 
                 3 
                 0 
                 0 
               
               
                 243 
                 3 
                 4 
                 10 
                 0 
                 2 
                 1 
                 0 
                 0 
               
               
                 244 
                 3 
                 4 
                 10 
                 0 
                 1 
                 2 
                 0 
                 0 
               
               
                 245 
                 3 
                 4 
                 8 
                 0 
                 5 
                 0 
                 0 
                 0 
               
               
                 246 
                 3 
                 4 
                 8 
                 0 
                 0 
                 5 
                 0 
                 0 
               
               
                 247 
                 3 
                 4 
                 8 
                 0 
                 4 
                 1 
                 0 
                 0 
               
               
                 248 
                 3 
                 4 
                 8 
                 0 
                 1 
                 4 
                 0 
                 0 
               
               
                 249 
                 3 
                 4 
                 8 
                 0 
                 3 
                 2 
                 0 
                 0 
               
               
                 250 
                 3 
                 4 
                 8 
                 0 
                 2 
                 3 
                 0 
                 0 
               
               
                 251 
                 3 
                 4 
                 6 
                 0 
                 5 
                 2 
                 0 
                 0 
               
               
                 252 
                 3 
                 4 
                 6 
                 0 
                 2 
                 5 
                 0 
                 0 
               
               
                 253 
                 3 
                 4 
                 6 
                 0 
                 4 
                 3 
                 0 
                 0 
               
               
                 254 
                 3 
                 4 
                 6 
                 0 
                 3 
                 4 
                 0 
                 0 
               
               
                 255 
                 3 
                 4 
                 6 
                 4 
                 2 
                 0 
                 1 
                 0 
               
               
                 256 
                 3 
                 4 
                 6 
                 4 
                 0 
                 2 
                 1 
                 0 
               
               
                 257 
                 3 
                 4 
                 6 
                 4 
                 2 
                 0 
                 0 
                 1 
               
               
                 258 
                 3 
                 4 
                 6 
                 4 
                 0 
                 2 
                 0 
                 1 
               
               
                 259 
                 3 
                 4 
                 6 
                 4 
                 1 
                 0 
                 2 
                 0 
               
               
                 260 
                 3 
                 4 
                 6 
                 4 
                 0 
                 1 
                 2 
                 0 
               
               
                 261 
                 3 
                 4 
                 6 
                 4 
                 1 
                 0 
                 0 
                 2 
               
               
                 262 
                 3 
                 4 
                 6 
                 4 
                 0 
                 1 
                 0 
                 2 
               
               
                 263 
                 3 
                 4 
                 6 
                 4 
                 1 
                 1 
                 1 
                 0 
               
               
                 264 
                 3 
                 4 
                 6 
                 4 
                 1 
                 1 
                 0 
                 1 
               
               
                 265 
                 3 
                 4 
                 6 
                 4 
                 1 
                 0 
                 1 
                 1 
               
               
                 266 
                 3 
                 4 
                 6 
                 4 
                 0 
                 1 
                 1 
                 1 
               
               
                 267 
                 3 
                 4 
                 4 
                 0 
                 5 
                 4 
                 0 
                 0 
               
               
                 268 
                 3 
                 4 
                 4 
                 0 
                 4 
                 5 
                 0 
                 0 
               
               
                 269 
                 3 
                 4 
                 4 
                 6 
                 2 
                 0 
                 1 
                 0 
               
               
                 270 
                 3 
                 4 
                 4 
                 6 
                 0 
                 2 
                 1 
                 0 
               
               
                 271 
                 3 
                 4 
                 4 
                 6 
                 2 
                 0 
                 0 
                 1 
               
               
                 272 
                 3 
                 4 
                 4 
                 6 
                 0 
                 2 
                 0 
                 1 
               
               
                 273 
                 3 
                 4 
                 4 
                 6 
                 1 
                 0 
                 2 
                 0 
               
               
                 274 
                 3 
                 4 
                 4 
                 6 
                 0 
                 1 
                 2 
                 0 
               
               
                 275 
                 3 
                 4 
                 4 
                 6 
                 1 
                 0 
                 0 
                 2 
               
               
                 276 
                 3 
                 4 
                 4 
                 6 
                 0 
                 1 
                 0 
                 2 
               
               
                 277 
                 3 
                 4 
                 4 
                 6 
                 1 
                 1 
                 1 
                 0 
               
               
                 278 
                 3 
                 4 
                 4 
                 6 
                 1 
                 1 
                 0 
                 1 
               
               
                 279 
                 3 
                 4 
                 4 
                 6 
                 1 
                 0 
                 1 
                 1 
               
               
                 280 
                 3 
                 4 
                 4 
                 6 
                 0 
                 1 
                 1 
                 1 
               
               
                 281 
                 3 
                 4 
                 4 
                 4 
                 4 
                 0 
                 1 
                 0 
               
               
                 282 
                 3 
                 4 
                 4 
                 4 
                 0 
                 4 
                 1 
                 0 
               
               
                 283 
                 3 
                 4 
                 4 
                 4 
                 4 
                 0 
                 0 
                 1 
               
               
                 284 
                 3 
                 4 
                 4 
                 4 
                 0 
                 4 
                 0 
                 1 
               
               
                 285 
                 3 
                 4 
                 4 
                 4 
                 1 
                 0 
                 4 
                 0 
               
               
                 286 
                 3 
                 4 
                 4 
                 4 
                 1 
                 0 
                 0 
                 4 
               
               
                 287 
                 3 
                 4 
                 4 
                 4 
                 0 
                 1 
                 4 
                 0 
               
               
                 288 
                 3 
                 4 
                 4 
                 4 
                 0 
                 1 
                 0 
                 4 
               
               
                 289 
                 3 
                 4 
                 4 
                 4 
                 3 
                 0 
                 2 
                 0 
               
               
                 290 
                 3 
                 4 
                 4 
                 4 
                 0 
                 3 
                 2 
                 0 
               
               
                 291 
                 3 
                 4 
                 4 
                 4 
                 3 
                 0 
                 0 
                 2 
               
               
                 292 
                 3 
                 4 
                 4 
                 4 
                 0 
                 3 
                 0 
                 2 
               
               
                 293 
                 3 
                 4 
                 4 
                 4 
                 2 
                 0 
                 3 
                 0 
               
               
                 294 
                 3 
                 4 
                 4 
                 4 
                 2 
                 0 
                 0 
                 3 
               
               
                 295 
                 3 
                 4 
                 4 
                 4 
                 0 
                 2 
                 3 
                 0 
               
               
                 296 
                 3 
                 4 
                 4 
                 4 
                 0 
                 2 
                 0 
                 3 
               
               
                 297 
                 3 
                 4 
                 4 
                 4 
                 3 
                 0 
                 1 
                 1 
               
               
                 298 
                 3 
                 4 
                 4 
                 4 
                 3 
                 1 
                 0 
                 1 
               
               
                 299 
                 3 
                 4 
                 4 
                 4 
                 3 
                 1 
                 1 
                 0 
               
               
                 300 
                 3 
                 4 
                 4 
                 4 
                 0 
                 3 
                 1 
                 1 
               
               
                 301 
                 3 
                 4 
                 4 
                 4 
                 1 
                 3 
                 0 
                 1 
               
               
                 302 
                 3 
                 4 
                 4 
                 4 
                 1 
                 3 
                 1 
                 0 
               
               
                 303 
                 3 
                 4 
                 4 
                 4 
                 1 
                 1 
                 3 
                 0 
               
               
                 304 
                 3 
                 4 
                 4 
                 4 
                 0 
                 1 
                 3 
                 1 
               
               
                 305 
                 3 
                 4 
                 4 
                 4 
                 1 
                 0 
                 3 
                 1 
               
               
                 306 
                 3 
                 4 
                 4 
                 4 
                 1 
                 1 
                 0 
                 3 
               
               
                 307 
                 3 
                 4 
                 4 
                 4 
                 1 
                 0 
                 1 
                 3 
               
               
                 308 
                 3 
                 4 
                 4 
                 4 
                 0 
                 1 
                 1 
                 3 
               
               
                 309 
                 3 
                 4 
                 4 
                 4 
                 2 
                 2 
                 1 
                 0 
               
               
                 310 
                 3 
                 4 
                 4 
                 4 
                 2 
                 2 
                 0 
                 1 
               
               
                 311 
                 3 
                 4 
                 4 
                 4 
                 1 
                 0 
                 2 
                 2 
               
               
                 312 
                 3 
                 4 
                 4 
                 4 
                 0 
                 1 
                 2 
                 2 
               
               
                 313 
                 3 
                 4 
                 4 
                 4 
                 2 
                 1 
                 2 
                 0 
               
               
                 314 
                 3 
                 4 
                 4 
                 4 
                 2 
                 1 
                 0 
                 2 
               
               
                 315 
                 3 
                 4 
                 4 
                 4 
                 2 
                 0 
                 2 
                 1 
               
               
                 316 
                 3 
                 4 
                 4 
                 4 
                 0 
                 2 
                 2 
                 1 
               
               
                 317 
                 3 
                 4 
                 4 
                 4 
                 1 
                 2 
                 2 
                 0 
               
               
                 318 
                 3 
                 4 
                 4 
                 4 
                 1 
                 2 
                 0 
                 2 
               
               
                 319 
                 3 
                 4 
                 4 
                 4 
                 2 
                 0 
                 1 
                 2 
               
               
                 320 
                 3 
                 4 
                 4 
                 4 
                 0 
                 2 
                 1 
                 2 
               
               
                 321 
                 3 
                 4 
                 4 
                 4 
                 2 
                 1 
                 1 
                 1 
               
               
                 322 
                 3 
                 4 
                 4 
                 4 
                 1 
                 2 
                 1 
                 1 
               
               
                 323 
                 3 
                 4 
                 4 
                 4 
                 1 
                 1 
                 2 
                 1 
               
               
                 324 
                 3 
                 4 
                 4 
                 4 
                 1 
                 1 
                 1 
                 2 
               
               
                   
               
             
          
         
       
     
       Example II 
     Example of Further Enrichment 
       [0083]    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  FIG. 4 . 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: 
         [0000]    
       
         
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                 Seq 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 # 
                 S1 
                 P1 
                 S2 
                 P3 
                 S3 
                 L 
                 S3 
                 P4 
                 S2 
                 P2 
                 S1 
               
               
                   
               
             
             
               
                   1 
                 GCCGCCG 
                   
                   
                   
                   
                 AAAAAAAAAA 
                   
                   
                   
                   
                 CGGCGGC 
                   
               
               
                   
               
               
                   2 
                 GCCGC 
                 A 
                 GC 
                   
                   
                 AAAAAAAAA 
                   
                   
                 GC 
                   
                 GCGGC 
               
               
                   
               
               
                   3 
                 GCCGC 
                   
                 GC 
                   
                   
                 AAAAAAAAA 
                   
                   
                 GC 
                 A 
                 GCGGC 
               
               
                   
               
               
                   4 
                 GCCG 
                 A 
                 GCC 
                   
                   
                 AAAAAAAAA 
                   
                   
                 GGC 
                   
                 CGGC 
               
               
                   
               
               
                   5 
                 GCCG 
                   
                 GCC 
                   
                   
                 AAAAAAAAA 
                   
                   
                 GGC 
                 A 
                 CGGC 
               
               
                   
               
               
                   6 
                 GCCG 
                 AAA 
                 GC 
                   
                   
                 AAAAAAAAA 
                   
                   
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                   7 
                 GCCG 
                   
                 GC 
                   
                   
                 AAAAAAAAA 
                   
                   
                 GC 
                 AAA 
                 CGGC 
               
               
                   
               
               
                   8 
                 GCCG 
                 AA 
                 GC 
                   
                   
                 AAAAAAAAA 
                   
                   
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                   9 
                 GCCG 
                 A 
                 GC 
                   
                   
                 AAAAAAAAA 
                   
                   
                 GC 
                 AA 
                 CGGC 
               
               
                   
               
               
                  10 
                 GCCGCCGC 
                   
                   
                   
                   
                 AAAAAAAA 
                   
                   
                   
                   
                 GCGGCGGC 
               
               
                   
               
               
                  11 
                 GCCGC 
                 AA 
                 GC 
                   
                   
                 AAAAAAAA 
                   
                   
                 GC 
                   
                 GCGGC 
               
               
                   
               
               
                  12 
                 GCCGC 
                   
                 GC 
                   
                   
                 AAAAAAAA 
                   
                   
                 GC 
                 AA 
                 GCGGC 
               
               
                   
               
               
                  13 
                 GCCGC 
                 A 
                 GC 
                   
                   
                 AAAAAAAA 
                   
                   
                 GC 
                 A 
                 GCGGC 
               
               
                   
               
               
                  14 
                 GCCG 
                 AA 
                 GCC 
                   
                   
                 AAAAAAAA 
                   
                   
                 GGC 
                   
                 CGGC 
               
               
                   
               
               
                  15 
                 GCCG 
                   
                 GCC 
                   
                   
                 AAAAAAAA 
                   
                   
                 GGC 
                 AA 
                 CGGC 
               
               
                   
               
               
                  16 
                 GCCG 
                 A 
                 GCC 
                   
                   
                 AAAAAAAA 
                   
                   
                 GGC 
                 A 
                 CGGC 
               
               
                   
               
               
                  17 
                 GCCG 
                 AAAA 
                 GC 
                   
                   
                 AAAAAAAA 
                   
                   
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                  18 
                 GCCG 
                   
                 GC 
                   
                   
                 AAAAAAAA 
                   
                   
                 GC 
                 AAAA 
                 CGGC 
               
               
                   
               
               
                  19 
                 GCCG 
                 AAA 
                 GC 
                   
                   
                 AAAAAAAA 
                   
                   
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                  20 
                 GCCG 
                 A 
                 GC 
                   
                   
                 AAAAAAAA 
                   
                   
                 GC 
                 AAA 
                 CGGC 
               
               
                   
               
               
                  21 
                 GCCG 
                 AA 
                 GC 
                   
                   
                 AAAAAAAA 
                   
                   
                 GC 
                 AA 
                 CGGC 
               
               
                   
               
               
                  22 
                 GCCGCC 
                 A 
                 GC 
                   
                   
                 AAAAAAA 
                   
                   
                 GC 
                   
                 GGCGGC 
               
               
                   
               
               
                  23 
                 GCCGCC 
                   
                 GC 
                   
                   
                 AAAAAAA 
                   
                   
                 GC 
                 A 
                 GGCGGC 
               
               
                   
               
               
                  24 
                 GCCGC 
                 A 
                 GCC 
                   
                   
                 AAAAAAA 
                   
                   
                 GGC 
                   
                 GCGGC 
               
               
                   
               
               
                  25 
                 GCCGC 
                   
                 GCC 
                   
                   
                 AAAAAAA 
                   
                   
                 GGC 
                 A 
                 GCGGC 
               
               
                   
               
               
                  26 
                 GCCGC 
                 AAA 
                 GC 
                   
                   
                 AAAAAAA 
                   
                   
                 GC 
                   
                 GCGGC 
               
               
                   
               
               
                  27 
                 GCCGC 
                   
                 GC 
                   
                   
                 AAAAAAA 
                   
                   
                 GC 
                 AAA 
                 GCGGC 
               
               
                   
               
               
                  28 
                 GCCGC 
                 AA 
                 GC 
                   
                   
                 AAAAAAA 
                   
                   
                 GC 
                 A 
                 GCGGC 
               
               
                   
               
               
                  29 
                 GCCGC 
                 A 
                 GC 
                   
                   
                 AAAAAAA 
                   
                   
                 GC 
                 AA 
                 GCGGC 
               
               
                   
               
               
                  30 
                 GCCG 
                 A 
                 GCCG 
                   
                   
                 AAAAAAA 
                   
                   
                 CGGC 
                   
                 CGGC 
               
               
                   
               
               
                  31 
                 GCCG 
                   
                 GCCG 
                   
                   
                 AAAAAAA 
                   
                   
                 CGGC 
                 A 
                 CGGC 
               
               
                   
               
               
                  32 
                 GCCG 
                 AAA 
                 GCC 
                   
                   
                 AAAAAAA 
                   
                   
                 GGC 
                   
                 CGGC 
               
               
                   
               
               
                  33 
                 GCCG 
                   
                 GCC 
                   
                   
                 AAAAAAA 
                   
                   
                 GGC 
                 AAA 
                 CGGC 
               
               
                   
               
               
                  34 
                 GCCG 
                 AA 
                 GCC 
                   
                   
                 AAAAAAA 
                   
                   
                 GGC 
                 A 
                 CGGC 
               
               
                   
               
               
                  35 
                 GCCG 
                 A 
                 GCC 
                   
                   
                 AAAAAAA 
                   
                   
                 GGC 
                 AA 
                 CGGC 
               
               
                   
               
               
                  36 
                 GCCG 
                 AAAAA 
                 GC 
                   
                   
                 AAAAAAA 
                   
                   
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                  37 
                 GCCG 
                   
                 GC 
                   
                   
                 AAAAAAA 
                   
                   
                 GC 
                 AAAAA 
                 CGGC 
               
               
                   
               
               
                  38 
                 GCCG 
                 AAAA 
                 GC 
                   
                   
                 AAAAAAA 
                   
                   
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                  39 
                 GCCG 
                 A 
                 GC 
                   
                   
                 AAAAAAA 
                   
                   
                 GC 
                 AAAA 
                 CGGC 
               
               
                   
               
               
                  40 
                 GCCG 
                 AAA 
                 GC 
                   
                   
                 AAAAAAA 
                   
                   
                 GC 
                 AA 
                 CGGC 
               
               
                   
               
               
                  41 
                 GCCG 
                 AA 
                 GC 
                   
                   
                 AAAAAAA 
                   
                   
                 GC 
                 AAA 
                 CGGC 
               
               
                   
               
               
                  42 
                 GCCGCCGCC 
                   
                   
                   
                   
                 AAAAAA 
                   
                   
                   
                   
                 GGCGGCGGC 
               
               
                   
               
               
                  43 
                 GCCGCC 
                 AA 
                 GC 
                   
                   
                 AAAAAA 
                   
                   
                 GC 
                   
                 GGCGGC 
               
               
                   
               
               
                  44 
                 GCCGCC 
                   
                 GC 
                   
                   
                 AAAAAA 
                   
                   
                 GC 
                 AA 
                 GGCGGC 
               
               
                   
               
               
                  45 
                 GCCGCC 
                 A 
                 GC 
                   
                   
                 AAAAAA 
                   
                   
                 GC 
                 A 
                 GGCGGC 
               
               
                   
               
               
                  46 
                 GCCGC 
                 AA 
                 GCC 
                   
                   
                 AAAAAA 
                   
                   
                 GGC 
                   
                 GCGGC 
               
               
                   
               
               
                  47 
                 GCCGC 
                   
                 GCC 
                   
                   
                 AAAAAA 
                   
                   
                 GGC 
                 AA 
                 GCGGC 
               
               
                   
               
               
                  48 
                 GCCGC 
                 A 
                 GCC 
                   
                   
                 AAAAAA 
                   
                   
                 GGC 
                 A 
                 GCGGC 
               
               
                   
               
               
                  49 
                 GCCGC 
                 AAAA 
                 GC 
                   
                   
                 AAAAAA 
                   
                   
                 GC 
                   
                 GCGGC 
               
               
                   
               
               
                  50 
                 GCCGC 
                   
                 GC 
                   
                   
                 AAAAAA 
                   
                   
                 GC 
                 AAAA 
                 GCGGC 
               
               
                   
               
               
                  51 
                 GCCGC 
                 AAA 
                 GC 
                   
                   
                 AAAAAA 
                   
                   
                 GC 
                 A 
                 GCGGC 
               
               
                   
               
               
                  52 
                 GCCGC 
                 A 
                 GC 
                   
                   
                 AAAAAA 
                   
                   
                 GC 
                 AAA 
                 GCGGC 
               
               
                   
               
               
                  53 
                 GCCGC 
                 AA 
                 GC 
                   
                   
                 AAAAAA 
                   
                   
                 GC 
                 AA 
                 GCGGC 
               
               
                   
               
               
                  54 
                 GCCG 
                 AA 
                 GCCG 
                   
                   
                 AAAAAA 
                   
                   
                 CGGC 
                   
                 CGGC 
               
               
                   
               
               
                  55 
                 GCCG 
                   
                 GCCG 
                   
                   
                 AAAAAA 
                   
                   
                 CGGC 
                 AA 
                 CGGC 
               
               
                   
               
               
                  56 
                 GCCG 
                 A 
                 GCCG 
                   
                   
                 AAAAAA 
                   
                   
                 CGGC 
                 A 
                 CGGC 
               
               
                   
               
               
                  57 
                 GCCG 
                 AAAA 
                 GCC 
                   
                   
                 AAAAAA 
                   
                   
                 GGC 
                   
                 CGGC 
               
               
                   
               
               
                  58 
                 GCCG 
                   
                 GCC 
                   
                   
                 AAAAAA 
                   
                   
                 GGC 
                 AAAA 
                 CGGC 
               
               
                   
               
               
                  59 
                 GCCG 
                 AAA 
                 GCC 
                   
                   
                 AAAAAA 
                   
                   
                 GGC 
                 A 
                 CGGC 
               
               
                   
               
               
                  60 
                 GCCG 
                 A 
                 GCC 
                   
                   
                 AAAAAA 
                   
                   
                 GGC 
                 AAA 
                 CGGC 
               
               
                   
               
               
                  61 
                 GCCG 
                 AA 
                 GCC 
                   
                   
                 AAAAAA 
                   
                   
                 GGC 
                 AA 
                 CGGC 
               
               
                   
               
               
                  62 
                 GCCG 
                 AAAAA 
                 GC 
                   
                   
                 AAAAAA 
                   
                   
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                  63 
                 GCCG 
                 A 
                 GC 
                   
                   
                 AAAAAA 
                   
                   
                 GC 
                 AAAAA 
                 CGGC 
               
               
                   
               
               
                  64 
                 GCCG 
                 AAAA 
                 GC 
                   
                   
                 AAAAAA 
                   
                   
                 GC 
                 AA 
                 CGGC 
               
               
                   
               
               
                  65 
                 GCCG 
                 AA 
                 GC 
                   
                   
                 AAAAAA 
                   
                   
                 GC 
                 AAAA 
                 CGGC 
               
               
                   
               
               
                  66 
                 GCCG 
                 AAA 
                 GC 
                   
                   
                 AAAAAA 
                   
                   
                 GC 
                 AAA 
                 CGGC 
               
               
                   
               
               
                  67 
                 GCCG 
                 A 
                 GC 
                 A 
                 GC 
                 AAAAAA 
                 GC 
                   
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                  68 
                 GCCG 
                   
                 GC 
                 A 
                 GC 
                 AAAAAA 
                 GC 
                   
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                  69 
                 GCCG 
                 A 
                 GC 
                   
                 GC 
                 AAAAAA 
                 GC 
                 A 
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                  70 
                 GCCG 
                   
                 GC 
                   
                 GC 
                 AAAAAA 
                 GC 
                 A 
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                  71 
                 GCCGCC 
                 A 
                 GCC 
                   
                   
                 AAAAA 
                   
                   
                 GGC 
                   
                 GGCGGC 
               
               
                   
               
               
                  72 
                 GCCGCC 
                   
                 GCC 
                   
                   
                 AAAAA 
                   
                   
                 GGC 
                 A 
                 GGCGGC 
               
               
                   
               
               
                  73 
                 GCCGCC 
                 AAA 
                 GC 
                   
                   
                 AAAAA 
                   
                   
                 GC 
                   
                 GGCGGC 
               
               
                   
               
               
                  74 
                 GCCGCC 
                   
                 GC 
                   
                   
                 AAAAA 
                   
                   
                 GC 
                 AAA 
                 GGCGGC 
               
               
                   
               
               
                  75 
                 GCCGCC 
                 AA 
                 GC 
                   
                   
                 AAAAA 
                   
                   
                 GC 
                 A 
                 GGCGGC 
               
               
                   
               
               
                  76 
                 GCCGCC 
                 A 
                 GC 
                   
                   
                 AAAAA 
                   
                   
                 GC 
                 AA 
                 GGCGGC 
               
               
                   
               
               
                  77 
                 GCCGC 
                 A 
                 GCCG 
                   
                   
                 AAAAA 
                   
                   
                 CGGC 
                   
                 GCGGC 
               
               
                   
               
               
                  78 
                 GCCGC 
                   
                 GCCG 
                   
                   
                 AAAAA 
                   
                   
                 CGGC 
                 A 
                 GCGGC 
               
               
                   
               
               
                  79 
                 GCCGC 
                 AAA 
                 GCC 
                   
                   
                 AAAAA 
                   
                   
                 GGC 
                   
                 GCGGC 
               
               
                   
               
               
                  80 
                 GCCGC 
                   
                 GCC 
                   
                   
                 AAAAA 
                   
                   
                 GGC 
                 AAA 
                 GCGGC 
               
               
                   
               
               
                  81 
                 GCCGC 
                 AA 
                 GCC 
                   
                   
                 AAAAA 
                   
                   
                 GGC 
                 A 
                 GCGGC 
               
               
                   
               
               
                  82 
                 GCCGC 
                 A 
                 GCC 
                   
                   
                 AAAAA 
                   
                   
                 GGC 
                 AA 
                 GCGGC 
               
               
                   
               
               
                  83 
                 GCCGC 
                 AAAAA 
                 GC 
                   
                   
                 AAAAA 
                   
                   
                 GC 
                   
                 GCGGC 
               
               
                   
               
               
                  84 
                 GCCGC 
                   
                 GC 
                   
                   
                 AAAAA 
                   
                   
                 GC 
                 AAAAA 
                 GCGGC 
               
               
                   
               
               
                  85 
                 GCCGC 
                 AAAA 
                 GC 
                   
                   
                 AAAAA 
                   
                   
                 GC 
                 A 
                 GCGGC 
               
               
                   
               
               
                  86 
                 GCCGC 
                 A 
                 GC 
                   
                   
                 AAAAA 
                   
                   
                 GC 
                 AAAA 
                 GCGGC 
               
               
                   
               
               
                  87 
                 GCCGC 
                 AAA 
                 GC 
                   
                   
                 AAAAA 
                   
                   
                 GC 
                 AA 
                 GCGGC 
               
               
                   
               
               
                  88 
                 GCCGC 
                 AA 
                 GC 
                   
                   
                 AAAAA 
                   
                   
                 GC 
                 AAA 
                 GCGGC 
               
               
                   
               
               
                  89 
                 GCCG 
                 A 
                 GCCGC 
                   
                   
                 AAAAA 
                   
                   
                 GCGGC 
                   
                 CGGC 
               
               
                   
               
               
                  90 
                 GCCG 
                   
                 GCCGC 
                   
                   
                 AAAAA 
                   
                   
                 GCGGC 
                 A 
                 CGGC 
               
               
                   
               
               
                  91 
                 GCCG 
                 AAA 
                 GCCG 
                   
                   
                 AAAAA 
                   
                   
                 CGGC 
                   
                 CGGC 
               
               
                   
               
               
                  92 
                 GCCG 
                   
                 GCCG 
                   
                   
                 AAAAA 
                   
                   
                 CGGC 
                 AAA 
                 CGGC 
               
               
                   
               
               
                  93 
                 GCCG 
                 AA 
                 GCCG 
                   
                   
                 AAAAA 
                   
                   
                 CGGC 
                 A 
                 CGGC 
               
               
                   
               
               
                  94 
                 GCCG 
                 A 
                 GCCG 
                   
                   
                 AAAAA 
                   
                   
                 CGGC 
                 AA 
                 CGGC 
               
               
                   
               
               
                  95 
                 GCCG 
                 AAAAA 
                 GCC 
                   
                   
                 AAAAA 
                   
                   
                 GGC 
                   
                 CGGC 
               
               
                   
               
               
                  96 
                 GCCG 
                   
                 GCC 
                   
                   
                 AAAAA 
                   
                   
                 GGC 
                 AAAAA 
                 CGGC 
               
               
                   
               
               
                  97 
                 GCCG 
                 AAAA 
                 GCC 
                   
                   
                 AAAAA 
                   
                   
                 GGC 
                 A 
                 CGGC 
               
               
                   
               
               
                  98 
                 GCCG 
                 A 
                 GCC 
                   
                   
                 AAAAA 
                   
                   
                 GGC 
                 AAAA 
                 CGGC 
               
               
                   
               
               
                  99 
                 GCCG 
                 AAA 
                 GCC 
                   
                   
                 AAAAA 
                   
                   
                 GGC 
                 AA 
                 CGGC 
               
               
                   
               
               
                 100 
                 GCCG 
                 AA 
                 GCC 
                   
                   
                 AAAAA 
                   
                   
                 GGC 
                 AAA 
                 CGGC 
               
               
                   
               
               
                 101 
                 GCCG 
                 AA 
                 GC 
                 A 
                 GC 
                 AAAAA 
                 GC 
                   
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                 102 
                 GCCG 
                   
                 GC 
                 A 
                 GC 
                 AAAAA 
                 GC 
                   
                 GC 
                 AA 
                 CGGC 
               
               
                   
               
               
                 103 
                 GCCG 
                 AA 
                 GC 
                   
                 GC 
                 AAAAA 
                 GC 
                 A 
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                 104 
                 GCCG 
                   
                 GC 
                   
                 GC 
                 AAAAA 
                 GC 
                 A 
                 GC 
                 AA 
                 CGGC 
               
               
                   
               
               
                 105 
                 GCCG 
                 A 
                 GC 
                 AA 
                 GC 
                 AAAAA 
                 GC 
                   
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                 106 
                 GCCG 
                   
                 GC 
                 AA 
                 GC 
                 AAAAA 
                 GC 
                   
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                 107 
                 GCCG 
                 A 
                 GC 
                   
                 GC 
                 AAAAA 
                 GC 
                 AA 
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                 108 
                 GCCG 
                   
                 GC 
                   
                 GC 
                 AAAAA 
                 GC 
                 AA 
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                 109 
                 GCCG 
                 A 
                 GC 
                 A 
                 GC 
                 AAAAA 
                 GC 
                   
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                 110 
                 GCCG 
                 A 
                 GC 
                   
                 GC 
                 AAAAA 
                 GC 
                 A 
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                 111 
                 GCCG 
                 A 
                 GC 
                 A 
                 GC 
                 AAAAA 
                 GC 
                 A 
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                 112 
                 GCCG 
                   
                 GC 
                 A 
                 GC 
                 AAAAA 
                 GC 
                 A 
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                 113 
                 GCCGCCGCCG 
                   
                   
                   
                   
                 AAAA 
                   
                   
                   
                   
                 CGGCGGCGGC 
               
               
                   
               
               
                 114 
                 GCCGCCG 
                 AA 
                 GC 
                   
                   
                 AAAA 
                   
                   
                 GC 
                   
                 CGGCGGC 
               
               
                   
               
               
                 115 
                 GCCGCCG 
                   
                 GC 
                   
                   
                 AAAA 
                   
                   
                 GC 
                 AA 
                 CGGCGGC 
               
               
                   
               
               
                 116 
                 GCCGCCG 
                 A 
                 GC 
                   
                   
                 AAAA 
                   
                   
                 GC 
                 A 
                 CGGCGGC 
               
               
                   
               
               
                 117 
                 GCCGCC 
                 AA 
                 GCC 
                   
                   
                 AAAA 
                   
                   
                 GGC 
                   
                 GGCGGC 
               
               
                   
               
               
                 118 
                 GCCGCC 
                   
                 GCC 
                   
                   
                 AAAA 
                   
                   
                 GGC 
                 AA 
                 GGCGGC 
               
               
                   
               
               
                 119 
                 GCCGCC 
                 A 
                 GCC 
                   
                   
                 AAAA 
                   
                   
                 GGC 
                 A 
                 GGCGGC 
               
               
                   
               
               
                 120 
                 GCCGCC 
                 AAAA 
                 GC 
                   
                   
                 AAAA 
                   
                   
                 GC 
                   
                 GGCGGC 
               
               
                   
               
               
                 121 
                 GCCGCC 
                   
                 GC 
                   
                   
                 AAAA 
                   
                   
                 GC 
                 AAAA 
                 GGCGGC 
               
               
                   
               
               
                 122 
                 GCCGCC 
                 AAA 
                 GC 
                   
                   
                 AAAA 
                   
                   
                 GC 
                 A 
                 GGCGGC 
               
               
                   
               
               
                 123 
                 GCCGCC 
                 A 
                 GC 
                   
                   
                 AAAA 
                   
                   
                 GC 
                 AAA 
                 GGCGGC 
               
               
                   
               
               
                 124 
                 GCCGCC 
                 AA 
                 GC 
                   
                   
                 AAAA 
                   
                   
                 GC 
                 AA 
                 GGCGGC 
               
               
                   
               
               
                 125 
                 GCCGC 
                 AA 
                 GCCG 
                   
                   
                 AAAA 
                   
                   
                 CGGC 
                   
                 GCGGC 
               
               
                   
               
               
                 126 
                 GCCGC 
                   
                 GCCG 
                   
                   
                 AAAA 
                   
                   
                 CGGC 
                 AA 
                 GCGGC 
               
               
                   
               
               
                 127 
                 GCCGC 
                 A 
                 GCCG 
                   
                   
                 AAAA 
                   
                   
                 CGGC 
                 A 
                 GCGGC 
               
               
                   
               
               
                 128 
                 GCCGC 
                 AAAA 
                 GCC 
                   
                   
                 AAAA 
                   
                   
                 GGC 
                   
                 GCGGC 
               
               
                   
               
               
                 129 
                 GCCGC 
                   
                 GCC 
                   
                   
                 AAAA 
                   
                   
                 GGC 
                 AAAA 
                 GCGGC 
               
               
                   
               
               
                 130 
                 GCCGC 
                 AAA 
                 GCC 
                   
                   
                 AAAA 
                   
                   
                 GGC 
                 A 
                 GCGGC 
               
               
                   
               
               
                 131 
                 GCCGC 
                 A 
                 GCC 
                   
                   
                 AAAA 
                   
                   
                 GGC 
                 AAA 
                 GCGGC 
               
               
                   
               
               
                 132 
                 GCCGC 
                 AA 
                 GCC 
                   
                   
                 AAAA 
                   
                   
                 GGC 
                 AA 
                 GCGGC 
               
               
                   
               
               
                 133 
                 GCCGC 
                 AAAAA 
                 GC 
                   
                   
                 AAAA 
                   
                   
                 GC 
                 A 
                 GCGGC 
               
               
                   
               
               
                 134 
                 GCCGC 
                 A 
                 GC 
                   
                   
                 AAAA 
                   
                   
                 GC 
                 AAAAA 
                 GCGGC 
               
               
                   
               
               
                 135 
                 GCCGC 
                 AAAA 
                 GC 
                   
                   
                 AAAA 
                   
                   
                 GC 
                 AA 
                 GCGGC 
               
               
                   
               
               
                 136 
                 GCCGC 
                 AA 
                 GC 
                   
                   
                 AAAA 
                   
                   
                 GC 
                 AAAA 
                 GCGGC 
               
               
                   
               
               
                 137 
                 GCCGC 
                 AAA 
                 GC 
                   
                   
                 AAAA 
                   
                   
                 GC 
                 AAA 
                 GCGGC 
               
               
                   
               
               
                 138 
                 GCCGC 
                 A 
                 GC 
                 A 
                 GC 
                 AAAA 
                 GC 
                   
                 GC 
                   
                 GCGGC 
               
               
                   
               
               
                 139 
                 GCCGC 
                   
                 GC 
                 A 
                 GC 
                 AAAA 
                 GC 
                   
                 GC 
                 A 
                 GCGGC 
               
               
                   
               
               
                 140 
                 GCCGC 
                 A 
                 GC 
                   
                 GC 
                 AAAA 
                 GC 
                 A 
                 GC 
                   
                 GCGGC 
               
               
                   
               
               
                 141 
                 GCCGC 
                   
                 GC 
                   
                 GC 
                 AAAA 
                 GC 
                 A 
                 GC 
                 A 
                 GCGGC 
               
               
                   
               
               
                 142 
                 GCCG 
                 AA 
                 GCCGC 
                   
                   
                 AAAA 
                   
                   
                 GCGGC 
                   
                 CGGC 
               
               
                   
               
               
                 143 
                 GCCG 
                   
                 GCCGC 
                   
                   
                 AAAA 
                   
                   
                 GCGGC 
                 AA 
                 CGGC 
               
               
                   
               
               
                 144 
                 GCCG 
                 A 
                 GCCGC 
                   
                   
                 AAAA 
                   
                   
                 GCGGC 
                 A 
                 CGGC 
               
               
                   
               
               
                 145 
                 GCCG 
                 AAAA 
                 GCCG 
                   
                   
                 AAAA 
                   
                   
                 CGGC 
                   
                 CGGC 
               
               
                   
               
               
                 146 
                 GCCG 
                   
                 GCCG 
                   
                   
                 AAAA 
                   
                   
                 CGGC 
                 AAAA 
                 CGGC 
               
               
                   
               
               
                 147 
                 GCCG 
                 AAA 
                 GCCG 
                   
                   
                 AAAA 
                   
                   
                 CGGC 
                 A 
                 CGGC 
               
               
                   
               
               
                 148 
                 GCCG 
                 A 
                 GCCG 
                   
                   
                 AAAA 
                   
                   
                 CGGC 
                 AAA 
                 CGGC 
               
               
                   
               
               
                 149 
                 GCCG 
                 AA 
                 GCCG 
                   
                   
                 AAAA 
                   
                   
                 CGGC 
                 AA 
                 CGGC 
               
               
                   
               
               
                 150 
                 GCCG 
                 AAAAA 
                 GCC 
                   
                   
                 AAAA 
                   
                   
                 GGC 
                 A 
                 CGGC 
               
               
                   
               
               
                 151 
                 GCCG 
                 A 
                 GCC 
                   
                   
                 AAAA 
                   
                   
                 GGC 
                 AAAAA 
                 CGGC 
               
               
                   
               
               
                 152 
                 GCCG 
                 AAAA 
                 GCC 
                   
                   
                 AAAA 
                   
                   
                 GGC 
                 AA 
                 CGGC 
               
               
                   
               
               
                 153 
                 GCCG 
                 AA 
                 GCC 
                   
                   
                 AAAA 
                   
                   
                 GGC 
                 AAAA 
                 CGGC 
               
               
                   
               
               
                 154 
                 GCCG 
                 AAA 
                 GCC 
                   
                   
                 AAAA 
                   
                   
                 GGC 
                 AAA 
                 CGGC 
               
               
                   
               
               
                 155 
                 GCCG 
                 A 
                 GCC 
                 A 
                 GC 
                 AAAA 
                 GC 
                   
                 GGC 
                   
                 CGGC 
               
               
                   
               
               
                 156 
                 GCCG 
                   
                 GCC 
                 A 
                 GC 
                 AAAA 
                 GC 
                   
                 GGC 
                 A 
                 CGGC 
               
               
                   
               
               
                 157 
                 GCCG 
                 A 
                 GCC 
                   
                 GC 
                 AAAA 
                 GC 
                 A 
                 GGC 
                   
                 CGGC 
               
               
                   
               
               
                 158 
                 GCCG 
                   
                 GCC 
                   
                 GC 
                 AAAA 
                 GC 
                 A 
                 GGC 
                 A 
                 CGGC 
               
               
                   
               
               
                 159 
                 GCCG 
                 AAAAA 
                 GC 
                   
                   
                 AAAA 
                   
                   
                 GC 
                 AAA 
                 CGGC 
               
               
                   
               
               
                 160 
                 GCCG 
                 AAA 
                 GC 
                   
                   
                 AAAA 
                   
                   
                 GC 
                 AAAAA 
                 CGGC 
               
               
                   
               
               
                 161 
                 GCCG 
                 AAAA 
                 GC 
                   
                   
                 AAAA 
                   
                   
                 GC 
                 AAAA 
                 CGGC 
               
               
                   
               
               
                 162 
                 GCCG 
                 A 
                 GC 
                 A 
                 GCC 
                 AAAA 
                 GGC 
                   
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                 163 
                 GCCG 
                   
                 GC 
                 A 
                 GCC 
                 AAAA 
                 GGC 
                   
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                 164 
                 GCCG 
                 A 
                 GC 
                   
                 GCC 
                 AAAA 
                 GGC 
                 A 
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                 165 
                 GCCG 
                   
                 GC 
                   
                 GCC 
                 AAAA 
                 GGC 
                 A 
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                 166 
                 GCCG 
                 AAA 
                 GC 
                 A 
                 GC 
                 AAAA 
                 GC 
                   
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                 167 
                 GCCG 
                   
                 GC 
                 A 
                 GC 
                 AAAA 
                 GC 
                   
                 GC 
                 AAA 
                 CGGC 
               
               
                   
               
               
                 168 
                 GCCG 
                 AAA 
                 GC 
                   
                 GC 
                 AAAA 
                 GC 
                 A 
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                 169 
                 GCCG 
                   
                 GC 
                   
                 GC 
                 AAAA 
                 GC 
                 A 
                 GC 
                 AAA 
                 CGGC 
               
               
                   
               
               
                 170 
                 GCCG 
                 A 
                 GC 
                 AAA 
                 GC 
                 AAAA 
                 GC 
                   
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                 171 
                 GCCG 
                 A 
                 GC 
                   
                 GC 
                 AAAA 
                 GC 
                 AAA 
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                 172 
                 GCCG 
                   
                 GC 
                 AAA 
                 GC 
                 AAAA 
                 GC 
                   
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                 173 
                 GCCG 
                   
                 GC 
                   
                 GC 
                 AAAA 
                 GC 
                 AAA 
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                 174 
                 GCCG 
                 AA 
                 GC 
                 AA 
                 GC 
                 AAAA 
                 GC 
                   
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                 175 
                 GCCG 
                   
                 GC 
                 AA 
                 GC 
                 AAAA 
                 GC 
                   
                 GC 
                 AA 
                 CGGC 
               
               
                   
               
               
                 176 
                 GCCG 
                 AA 
                 GC 
                   
                 GC 
                 AAAA 
                 GC 
                 AA 
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                 177 
                 GCCG 
                   
                 GC 
                   
                 GC 
                 AAAA 
                 GC 
                 AA 
                 GC 
                 AA 
                 CGGC 
               
               
                   
               
               
                 178 
                 GCCG 
                 AA 
                 GC 
                 A 
                 GC 
                 AAAA 
                 GC 
                   
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                 179 
                 GCCG 
                 AA 
                 GC 
                   
                 GC 
                 AAAA 
                 GC 
                 A 
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                 180 
                 GCCG 
                 AA 
                 GC 
                 A 
                 GC 
                 AAAA 
                 GC 
                 A 
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                 181 
                 GCCG 
                 A 
                 GC 
                 A 
                 GC 
                 AAAA 
                 GC 
                 GC 
                 AA 
                 CGGC 
               
               
                   
               
               
                 182 
                 GCCG 
                 A 
                 GC 
                   
                 GC 
                 AAAA 
                 GC 
                 A 
                 GC 
                 AA 
                 CGGC 
               
               
                   
               
               
                 183 
                 GCCG 
                   
                 GC 
                 A 
                 GC 
                 AAAA 
                 GC 
                 A 
                 GC 
                 AA 
                 CGGC 
               
               
                   
               
               
                 184 
                 GCCG 
                 A 
                 GC 
                 AA 
                 GC 
                 AAAA 
                 GC 
                   
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                 185 
                 GCCG 
                 A 
                 GC 
                 AA 
                 GC 
                 AAAA 
                 GC 
                 A 
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                 186 
                 GCCG 
                   
                 GC 
                 AA 
                 GC 
                 AAAA 
                 GC 
                 A 
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                 187 
                 GCCG 
                 A 
                 GC 
                   
                 GC 
                 AAAA 
                 GC 
                 AA 
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                 188 
                 GCCG 
                 A 
                 GC 
                 A 
                 GC 
                 AAAA 
                 GC 
                 AA 
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                 189 
                 GCCG 
                   
                 GC 
                 A 
                 GC 
                 AAAA 
                 GC 
                 AA 
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                 190 
                 GCCG 
                 A 
                 GC 
                 A 
                 GC 
                 AAAA 
                 GC 
                 A 
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                 191 
                 GCCGCCGC 
                 A 
                 GC 
                   
                   
                 AAA 
                   
                   
                 GC 
                   
                 GCGGCGGC 
               
               
                   
               
               
                 192 
                 GCCGCCGC 
                   
                 GC 
                   
                   
                 AAA 
                   
                   
                 GC 
                 A 
                 GCGGCGGC 
               
               
                   
               
               
                 193 
                 GCCGCCG 
                 A 
                 GCC 
                   
                   
                 AAA 
                   
                   
                 GGC 
                   
                 CGGCGGC 
               
               
                   
               
               
                 194 
                 GCCGCCG 
                   
                 GCC 
                   
                   
                 AAA 
                   
                   
                 GGC 
                 A 
                 CGGCGGC 
               
               
                   
               
               
                 195 
                 GCCGCCG 
                 AAA 
                 GC 
                   
                   
                 AAA 
                   
                   
                 GC 
                   
                 CGGCGGC 
               
               
                   
               
               
                 196 
                 GCCGCCG 
                   
                 GC 
                   
                   
                 AAA 
                   
                   
                 GC 
                 AAA 
                 CGGCGGC 
               
               
                   
               
               
                 197 
                 GCCGCCG 
                 AA 
                 GC 
                   
                   
                 AAA 
                   
                   
                 GC 
                 A 
                 CGGCGGC 
               
               
                   
               
               
                 198 
                 GCCGCCG 
                 A 
                 GC 
                   
                   
                 AAA 
                   
                   
                 GC 
                 AA 
                 CGGCGGC 
               
               
                   
               
               
                 199 
                 GCCGCC 
                 A 
                 GCCG 
                   
                   
                 AAA 
                   
                   
                 CGGC 
                   
                 GGCGGC 
               
               
                   
               
               
                 200 
                 GCCGCC 
                   
                 GCCG 
                   
                   
                 AAA 
                   
                   
                 CGGC 
                 A 
                 GGCGGC 
               
               
                   
               
               
                 201 
                 GCCGCC 
                 AAA 
                 GCC 
                   
                   
                 AAA 
                   
                   
                 GGC 
                   
                 GGCGGC 
               
               
                   
               
               
                 202 
                 GCCGCC 
                   
                 GCC 
                   
                   
                 AAA 
                   
                   
                 GGC 
                 AAA 
                 GGCGGC 
               
               
                   
               
               
                 203 
                 GCCGCC 
                 AA 
                 GCC 
                   
                   
                 AAA 
                   
                   
                 GGC 
                 A 
                 GGCGGC 
               
               
                   
               
               
                 204 
                 GCCGCC 
                 A 
                 GCC 
                   
                   
                 AAA 
                   
                   
                 GGC 
                 AA 
                 GGCGGC 
               
               
                   
               
               
                 205 
                 GCCGCC 
                 AAAAA 
                 GC 
                   
                   
                 AAA 
                   
                   
                 GC 
                   
                 GGCGGC 
               
               
                   
               
               
                 206 
                 GCCGCC 
                   
                 GC 
                   
                   
                 AAA 
                   
                   
                 GC 
                 AAAAA 
                 GGCGGC 
               
               
                   
               
               
                 207 
                 GCCGCC 
                 AAAA 
                 GC 
                   
                   
                 AAA 
                   
                   
                 GC 
                 A 
                 GGCGGC 
               
               
                   
               
               
                 208 
                 GCCGCC 
                 A 
                 GC 
                   
                   
                 AAA 
                   
                   
                 GC 
                 AAAA 
                 GGCGGC 
               
               
                   
               
               
                 209 
                 GCCGCC 
                 AAA 
                 GC 
                   
                   
                 AAA 
                   
                   
                 GC 
                 AA 
                 GGCGGC 
               
               
                   
               
               
                 210 
                 GCCGCC 
                 AA 
                 GC 
                   
                   
                 AAA 
                   
                   
                 GC 
                 AAA 
                 GGCGGC 
               
               
                   
               
               
                 211 
                 GCCGC 
                 A 
                 GCCGC 
                   
                   
                 AAA 
                   
                   
                 GCGGC 
                   
                 GCGGC 
               
               
                   
               
               
                 212 
                 GCCGC 
                   
                 GCCGC 
                   
                   
                 AAA 
                   
                   
                 GCGGC 
                 A 
                 GCGGC 
               
               
                   
               
               
                 213 
                 GCCGC 
                 AAA 
                 GCCG 
                   
                   
                 AAA 
                   
                   
                 CGGC 
                   
                 GCGGC 
               
               
                   
               
               
                 214 
                 GCCGC 
                   
                 GCCG 
                   
                   
                 AAA 
                   
                   
                 CGGC 
                 AAA 
                 GCGGC 
               
               
                   
               
               
                 215 
                 GCCGC 
                 AA 
                 GCCG 
                   
                   
                 AAA 
                   
                   
                 CGGC 
                 A 
                 GCGGC 
               
               
                   
               
               
                 216 
                 GCCGC 
                 A 
                 GCCG 
                   
                   
                 AAA 
                   
                   
                 CGGC 
                 AA 
                 GCGGC 
               
               
                   
               
               
                 217 
                 GCCGC 
                 AAAAA 
                 GCC 
                   
                   
                 AAA 
                   
                   
                 GGC 
                   
                 GCGGC 
               
               
                   
               
               
                 218 
                 GCCGC 
                   
                 GCC 
                   
                   
                 AAA 
                   
                   
                 GGC 
                 AAAAA 
                 GCGGC 
               
               
                   
               
               
                 219 
                 GCCGC 
                 AAAA 
                 GCC 
                   
                   
                 AAA 
                   
                   
                 GGC 
                 A 
                 GCGGC 
               
               
                   
               
               
                 220 
                 GCCGC 
                 A 
                 GCC 
                   
                   
                 AAA 
                   
                   
                 GGC 
                 AAAA 
                 GCGGC 
               
               
                   
               
               
                 221 
                 GCCGC 
                 AAA 
                 GCC 
                   
                   
                 AAA 
                   
                   
                 GGC 
                 AA 
                 GCGGC 
               
               
                   
               
               
                 222 
                 GCCGC 
                 AA 
                 GCC 
                   
                   
                 AAA 
                   
                   
                 GGC 
                 AAA 
                 GCGGC 
               
               
                   
               
               
                 223 
                 GCCGC 
                 AAAAA 
                 GC 
                   
                   
                 AAA 
                   
                   
                 GC 
                 AA 
                 GCGGC 
               
               
                   
               
               
                 224 
                 GCCGC 
                 AA 
                 GC 
                   
                   
                 AAA 
                   
                   
                 GC 
                 AAAAA 
                 GCGGC 
               
               
                   
               
               
                 225 
                 GCCGC 
                 AAAA 
                 GC 
                   
                   
                 AAA 
                   
                   
                 GC 
                 AAA 
                 GCGGC 
               
               
                   
               
               
                 226 
                 GCCGC 
                 AAA 
                 GC 
                   
                   
                 AAA 
                   
                   
                 GC 
                 AAAA 
                 GCGGC 
               
               
                   
               
               
                 227 
                 GCCGC 
                 AA 
                 GC 
                 A 
                 GC 
                 AAA 
                 GC 
                   
                 GC 
                   
                 GCGGC 
               
               
                   
               
               
                 228 
                 GCCGC 
                   
                 GC 
                 A 
                 GC 
                 AAA 
                 GC 
                   
                 GC 
                 AA 
                 GCGGC 
               
               
                   
               
               
                 229 
                 GCCGC 
                 AA 
                 GC 
                   
                 GC 
                 AAA 
                 GC 
                 A 
                 GC 
                   
                 GCGGC 
               
               
                   
               
               
                 230 
                 GCCGC 
                   
                 GC 
                   
                 GC 
                 AAA 
                 GC 
                 A 
                 GC 
                 AA 
                 GCGGC 
               
               
                   
               
               
                 231 
                 GCCGC 
                 A 
                 GC 
                 AA 
                 GC 
                 AAA 
                 GC 
                   
                 GC 
                   
                 GCGGC 
               
               
                   
               
               
                 232 
                 GCCGC 
                   
                 GC 
                 AA 
                 GC 
                 AAA 
                 GC 
                   
                 GC 
                 A 
                 GCGGC 
               
               
                   
               
               
                 233 
                 GCCGC 
                 A 
                 GC 
                   
                 GC 
                 AAA 
                 GC 
                 AA 
                 GC 
                   
                 GCGGC 
               
               
                   
               
               
                 234 
                 GCCGC 
                   
                 GC 
                   
                 GC 
                 AAA 
                 GC 
                 AA 
                 GC 
                 A 
                 GCGGC 
               
               
                   
               
               
                 235 
                 GCCGC 
                 A 
                 GC 
                 A 
                 GC 
                 AAA 
                 GC 
                   
                 GC 
                 A 
                 GCGGC 
               
               
                   
               
               
                 236 
                 GCCGC 
                 A 
                 GC 
                   
                 GC 
                 AAA 
                 GC 
                 A 
                 GC 
                 A 
                 GCGGC 
               
               
                   
               
               
                 237 
                 GCCGC 
                 A 
                 GC 
                 A 
                 GC 
                 AAA 
                 GC 
                 A 
                 GC 
                   
                 GCGGC 
               
               
                   
               
               
                 238 
                 GCCGC 
                   
                 GC 
                 A 
                 GC 
                 AAA 
                 GC 
                 A 
                 GC 
                 A 
                 GCGGC 
               
               
                   
               
               
                 239 
                 GCCG 
                 A 
                 GCCGCC 
                   
                   
                 AAA 
                   
                   
                 GGCGGC 
                   
                 CGGC 
               
               
                   
               
               
                 240 
                 GCCG 
                   
                 GCCGCC 
                   
                   
                 AAA 
                   
                   
                 GGCGGC 
                 A 
                 CGGC 
               
               
                   
               
               
                 241 
                 GCCG 
                 AAA 
                 GCCGC 
                   
                   
                 AAA 
                   
                   
                 GCGGC 
                   
                 CGGC 
               
               
                   
               
               
                 242 
                 GCCG 
                   
                 GCCGC 
                   
                   
                 AAA 
                   
                   
                 GCGGC 
                 AAA 
                 CGGC 
               
               
                   
               
               
                 243 
                 GCCG 
                 AA 
                 GCCGC 
                   
                   
                 AAA 
                   
                   
                 GCGGC 
                 A 
                 CGGC 
               
               
                   
               
               
                 244 
                 GCCG 
                 A 
                 GCCGC 
                   
                   
                 AAA 
                   
                   
                 GCGGC 
                 AA 
                 CGGC 
               
               
                   
               
               
                 245 
                 GCCG 
                 AAAAA 
                 GCCG 
                   
                   
                 AAA 
                   
                   
                 CGGC 
                   
                 CGGC 
               
               
                   
               
               
                 246 
                 GCCG 
                   
                 GCCG 
                   
                   
                 AAA 
                   
                   
                 CGGC 
                 AAAAA 
                 CGGC 
               
               
                   
               
               
                 247 
                 GCCG 
                 AAAA  
                 GCCG 
                   
                   
                 AAA 
                   
                   
                 CGGC 
                 A 
                 CGGC 
               
               
                   
               
               
                 248 
                 GCCG 
                 A 
                 GCCG 
                   
                   
                 AAA 
                   
                   
                 CGGC 
                 AAAA 
                 CGGC 
               
               
                   
               
               
                 249 
                 GCCG 
                 AAA  
                 GCCG 
                   
                   
                 AAA 
                   
                   
                 CGGC 
                 AA 
                 CGGC 
               
               
                   
               
               
                 250 
                 GCCG 
                 AA 
                 GCCG 
                   
                   
                 AAA 
                   
                   
                 CGGC 
                 AAA 
                 CGGC 
               
               
                   
               
               
                 251 
                 GCCG 
                 AAAAA 
                 GCC 
                   
                   
                 AAA 
                   
                   
                 GGC 
                 AA 
                 CGGC 
               
               
                   
               
               
                 252 
                 GCCG 
                 AA 
                 GCC 
                   
                   
                 AAA 
                   
                   
                 GGC 
                 AAAAA 
                 CGGC 
               
               
                   
               
               
                 253 
                 GCCG 
                 AAAA 
                 GCC 
                   
                   
                 AAA 
                   
                   
                 GGC 
                 AAA 
                 CGGC 
               
               
                   
               
               
                 254 
                 GCCG 
                 AAA 
                 GCC 
                   
                   
                 AAA 
                   
                   
                 GGC 
                 AAAA 
                 CGGC 
               
               
                   
               
               
                 255 
                 GCCG 
                 AA 
                 GCC 
                 A 
                 GC 
                 AAA 
                 GC 
                   
                 GGC 
                   
                 CGGC 
               
               
                   
               
               
                 256 
                 GCCG 
                   
                 GCC 
                 A 
                 GC 
                 AAA 
                 GC 
                   
                 GGC 
                 AA 
                 CGGC 
               
               
                   
               
               
                 257 
                 GCCG 
                 AA 
                 GCC 
                   
                 GC 
                 AAA 
                 GC 
                 A 
                 GGC 
                   
                 CGGC 
               
               
                   
               
               
                 258 
                 GCCG 
                   
                 GCC 
                   
                 GC 
                 AAA 
                 GC 
                 A 
                 GGC 
                 AA 
                 CGGC 
               
               
                   
               
               
                 259 
                 GCCG 
                 A 
                 GCC 
                 AA 
                 GC 
                 AAA 
                 GC 
                   
                 GGC 
                   
                 CGGC 
               
               
                   
               
               
                 260 
                 GCCG 
                   
                 GCC 
                 AA 
                 GC 
                 AAA 
                 GC 
                   
                 GGC 
                 A 
                 CGGC 
               
               
                   
               
               
                 261 
                 GCCG 
                 A 
                 GCC 
                   
                 GC 
                 AAA 
                 GC 
                 AA 
                 GGC 
                   
                 CGGC 
               
               
                   
               
               
                 262 
                 GCCG 
                   
                 GCC 
                   
                 GC 
                 AAA 
                 GC 
                 AA 
                 GGC 
                 A 
                 CGGC 
               
               
                   
               
               
                 263 
                 GCCG 
                 A 
                 GCC 
                 A 
                 GC 
                 AAA 
                 GC 
                   
                 GGC 
                 A 
                 CGGC 
               
               
                   
               
               
                 264 
                 GCCG 
                 A 
                 GCC 
                   
                 GC 
                 AAA 
                 GC 
                 A 
                 GGC 
                 A 
                 CGGC 
               
               
                   
               
               
                 265 
                 GCCG 
                 A 
                 GCC 
                 A 
                 GC 
                 AAA 
                 GC 
                 A 
                 GGC 
                   
                 CGGC 
               
               
                   
               
               
                 266 
                 GCCG 
                   
                 GCC 
                 A 
                 GC 
                 AAA 
                 GC 
                 A 
                 GGC 
                 A 
                 CGGC 
               
               
                   
               
               
                 267 
                 GCCG 
                 AAAAA 
                 GC 
                   
                   
                 AAA 
                   
                   
                 GC 
                 AAAA 
                 CGGC 
               
               
                   
               
               
                 268 
                 GCCG 
                 AAAA 
                 GC 
                   
                   
                 AAA 
                   
                   
                 GC 
                 AAAAA 
                 CGGC 
               
               
                   
               
               
                 269 
                 GCCG 
                 AA 
                 GC 
                 A 
                 GCC 
                 AAA 
                 GGC 
                   
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                 270 
                 GCCG 
                   
                 GC 
                 A 
                 GCC 
                 AAA 
                 GGC 
                   
                 GC 
                 AA 
                 CGGC 
               
               
                   
               
               
                 271 
                 GCCG 
                 AA 
                 GC 
                   
                 GCC 
                 AAA 
                 GGC 
                 A 
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                 272 
                 GCCG 
                   
                 GC 
                   
                 GCC 
                 AAA 
                 GGC 
                 A 
                 GC 
                 AA 
                 CGGC 
               
               
                   
               
               
                 273 
                 GCCG 
                 A 
                 GC 
                 AA 
                 GCC 
                 AAA 
                 GGC 
                   
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                 274 
                 GCCG 
                   
                 GC 
                 AA 
                 GCC 
                 AAA 
                 GGC 
                   
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                 275 
                 GCCG 
                 A 
                 GC 
                   
                 GCC 
                 AAA 
                 GGC 
                 AA 
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                 276 
                 GCCG 
                   
                 GC 
                   
                 GCC 
                 AAA 
                 GGC 
                 AA 
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                 277 
                 GCCG 
                 A 
                 GC 
                 A 
                 GCC 
                 AAA 
                 GGC 
                   
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                 278 
                 GCCG 
                 A 
                 GC 
                   
                 GCC 
                 AAA 
                 GGC 
                 A 
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                 279 
                 GCCG 
                 A 
                 GC 
                 A 
                 GCC 
                 AAA 
                 GGC 
                 A 
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                 280 
                 GCCG 
                   
                 GC 
                 A 
                 GCC 
                 AAA 
                 GGC 
                 A 
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                 281 
                 GCCG 
                 AAAA 
                 GC 
                 A 
                 GC 
                 AAA 
                 GC 
                   
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                 282 
                 GCCG 
                   
                 GC 
                 A 
                 GC 
                 AAA 
                 GC 
                   
                 GC 
                 AAAA 
                 CGGC 
               
               
                   
               
               
                 283 
                 GCCG 
                 AAAA 
                 GC 
                   
                 GC 
                 AAA 
                 GC 
                 A 
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                 284 
                 GCCG 
                   
                 GC 
                   
                 GC 
                 AAA 
                 GC 
                 A 
                 GC 
                 AAAA 
                 CGGC 
               
               
                   
               
               
                 285 
                 GCCG 
                 A 
                 GC 
                 AAAA 
                 GC 
                 AAA 
                 GC 
                   
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                 286 
                 GCCG 
                 A 
                 GC 
                   
                 GC 
                 AAA 
                 GC 
                 AAAA 
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                 287 
                 GCCG 
                   
                 GC 
                 AAAA 
                 GC 
                 AAA 
                 GC 
                   
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                 288 
                 GCCG 
                   
                 GC 
                   
                 GC 
                 AAA 
                 GC 
                 AAAA 
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                 289 
                 GCCG 
                 AAA 
                 GC 
                 AA 
                 GC 
                 AAA 
                 GC 
                   
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                 290 
                 GCCG 
                   
                 GC 
                 AA 
                 GC 
                 AAA 
                 GC 
                   
                 GC 
                 AAA 
                 CGGC 
               
               
                   
               
               
                 291 
                 GCCG 
                 AAA 
                 GC 
                   
                 GC 
                 AAA 
                 GC 
                 AA 
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                 292 
                 GCCG 
                   
                 GC 
                   
                 GC 
                 AAA 
                 GC 
                 AA 
                 GC 
                 AAA 
                 CGGC 
               
               
                   
               
               
                 293 
                 GCCG 
                 AA 
                 GC 
                 AAA 
                 GC 
                 AAA 
                 GC 
                   
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                 294 
                 GCCG 
                 AA 
                 GC 
                   
                 GC 
                 AAA 
                 GC 
                 AAA 
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                 295 
                 GCCG 
                   
                 GC 
                 AAA 
                 GC 
                 AAA 
                 GC 
                   
                 GC 
                 AA 
                 CGGC 
               
               
                   
               
               
                 296 
                 GCCG 
                   
                 GC 
                   
                 GC 
                 AAA 
                 GC 
                 AAA 
                 GC 
                 AA 
                 CGGC 
               
               
                   
               
               
                 297 
                 GCCG 
                 AAA 
                 GC 
                 A 
                 GC 
                 AAA 
                 GC 
                 A 
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                 298 
                 GCCG 
                 AAA 
                 GC 
                   
                 GC 
                 AAA 
                 GC 
                 A 
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                 299 
                 GCCG 
                 AAA 
                 GC 
                 A 
                 GC 
                 AAA 
                 GC 
                   
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                 300 
                 GCCG 
                   
                 GC 
                 A 
                 GC 
                 AAA 
                 GC 
                 A 
                 GC 
                 AAA 
                 CGGC 
               
               
                   
               
               
                 301 
                 GCCG 
                 A 
                 GC 
                   
                 GC 
                 AAA 
                 GC 
                 A 
                 GC 
                 AAA 
                 CGGC 
               
               
                   
               
               
                 302 
                 GCCG 
                 A 
                 GC 
                 A 
                 GC 
                 AAA 
                 GC 
                   
                 GC 
                 AAA 
                 CGGC 
               
               
                   
               
               
                 303 
                 GCCG 
                 A 
                 GC 
                 AAA 
                 GC 
                 AAA 
                 GC 
                   
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                 304 
                 GCCG 
                   
                 GC 
                 AAA 
                 GC 
                 AAA 
                 GC 
                 A 
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                 305 
                 GCCG 
                 A 
                 GC 
                 AAA 
                 GC 
                 AAA 
                 GC 
                 A 
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                 306 
                 GCCG 
                 A 
                 GC 
                   
                 GC 
                 AAA 
                 GC 
                 AAA 
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                 307 
                 GCCG 
                 A 
                 GC 
                 A 
                 GC 
                 AAA 
                 GC 
                 AAA 
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                 308 
                 GCCG 
                   
                 GC 
                 A 
                 GC 
                 AAA 
                 GC 
                 AAA 
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                 309 
                 GCCG 
                 AA 
                 GC 
                 A 
                 GC 
                 AAA 
                 GC 
                   
                 GC 
                 AA 
                 CGGC 
               
               
                   
               
               
                 310 
                 GCCG 
                 AA 
                 GC 
                   
                 GC 
                 AAA 
                 GC 
                 A 
                 GC 
                 AA 
                 CGGC 
               
               
                   
               
               
                 311 
                 GCCG 
                 A 
                 GC 
                 AA 
                 GC 
                 AAA 
                 GC 
                 AA 
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                 312 
                 GCCG 
                   
                 GC 
                 AA 
                 GC 
                 AAA 
                 GC 
                 AA 
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                 313 
                 GCCG 
                 AA 
                 GC 
                 AA 
                 GC 
                 AAA 
                 GC 
                   
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                 314 
                 GCCG 
                 AA 
                 GC 
                   
                 GC 
                 AAA 
                 GC 
                 AA 
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                 315 
                 GCCG 
                 AA 
                 GC 
                 AA 
                 GC 
                 AAA 
                 GC 
                 A 
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                 316 
                 GCCG 
                   
                 GC 
                 AA 
                 GC 
                 AAA 
                 GC 
                 A 
                 GC 
                 AA 
                 CGGC 
               
               
                   
               
               
                 317 
                 GCCG 
                 A 
                 GC 
                 AA 
                 GC 
                 AAA 
                 GC 
                   
                 GC 
                 AA 
                 CGGC 
               
               
                   
               
               
                 318 
                 GCCG 
                 A 
                 GC 
                   
                 GC 
                 AAA 
                 GC 
                 AA 
                 GC 
                 AA 
                 CGGC 
               
               
                   
               
               
                 319 
                 GCCG 
                 AA 
                 GC 
                 A 
                 GC 
                 AAA 
                 GC 
                 AA 
                 GC 
                   
                 CGGC 
               
               
                   
               
               
                 320 
                 GCCG 
                   
                 GC 
                 A 
                 GC 
                 AAA 
                 GC 
                 AA 
                 GC 
                 AA 
                 CGGC 
               
               
                   
               
               
                 321 
                 GCCG 
                 AA 
                 GC 
                 A 
                 GC 
                 AAA 
                 GC 
                 A 
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                 322 
                 GCCG 
                 A 
                 GC 
                 A 
                 GC 
                 AAA 
                 GC 
                 A 
                 GC 
                 AA 
                 CGGC 
               
               
                   
               
               
                 323 
                 GCCG 
                 A 
                 GC 
                 AA 
                 GC 
                 AAA 
                 GC 
                 A 
                 GC 
                 A 
                 CGGC 
               
               
                   
               
               
                 324 
                 GCCG 
                 A 
                 GC 
                 A 
                 GC 
                 AAA 
                 GC 
                 AA 
                 GC 
                 A 
                 CGGC 
               
               
                   
               
             
          
         
       
     
       Example III 
     Example of Chip Based Selection of Aptamers 
       [0084]    In an exemplary embodiment, following geometric enrichment and further enrichment, selection of aptamers is performed directly on a chip as shown in  FIG. 5 . 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. 
       Example IV 
     Example of Combining Two Aptamers for Enhanced Performance 
       [0085]    In an exemplary embodiment, individual aptamers which have been independently specific for the target are combined together as shown in  FIG. 6 . 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. 
       Example V 
     Example of Chip Based Selection of Aptamers for gp120 
       [0086]    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. 
         [0087]    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 ( FIG. 7 ). Ten aptamers were selected for HIV gp120 ( FIG. 8 ). The aptamers selected for HIV gp120 did not have the same geometries or content as the aptamers that were selected for BSA. 
         [0088]    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. (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase.  Science,  249, 505-510.   2. Gold, L. and Tuerk, C. (2004) U.S. Pat. No. 6,716,583.   3. West, J. A. A. and Satterfield, B. C. (2006) USA.   4. Mescalchin, A., Wunsche, W., Laufer, S. D., Grohmann, D., Restle, T. and Sczakiel, G. (2006) Specific binding of a hexanucleotide to HIV-1 reverse transcriptase: a novel class of bioactive molecules.  Nucleic Acids Res.      5. Asai, R., Nishimura, S. I., Aita, T. and Takahashi, K. (2004) In Vitro Selection of DNA Aptamers on Chips Using a Method for Generating Point Mutations.  Chemical and Biosensors,  37, 645-656.   6. Hirao, I., Spingola, M., Peabody, D. and Ellington, A. D. (1998) The limits of specificity: an experimental analysis with RNA aptamers to MS2 coat protein variants.  Mol Divers,  4, 75-89.   7. Cerchia, L., Duconge, F., Pestourie, C., Boulay, J., Aissouni, Y., Gombert, K., Tavitian, B., de Franciscis, V. and Libri, D. (2005) Neutralizing aptamers from whole-cell SELEX inhibit the RET receptor tyrosine kinase.  PLoS Biol,  3, e123.   8. Mannironi, C., Di Nardo, A., Fruscoloni, P. and Tocchini-Valentini, G. P. (1997) In vitro selection of dopamine RNA ligands.  Biochemistry,  36, 9726-9734.   9. Boiziau, C., Dausse, E., Yurchenko, L. and Toulme, J. J. (1999) DNA aptamers selected against the HIV-1 trans-activation-responsive RNA element form RNA-DNA kissing complexes.  J Biol Chem,  274, 12730-12737.   10. Rhodes, A., Deakin, A., Spaull, J., Coomber, B., Aitken, A., Life, P. and Rees, S. (2000) The generation and characterization of antagonist RNA aptamers to human oncostatin M.  J Biol Chem,  275, 28555-28561.   11. Ruckman, J., Green, L. S., Beeson, J., Waugh, S., Gillette, W. L., Henninger, D. D., Claesson-Welsh, L. and Janjic, N. 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