Abstract:
This invention is about the selection and development of aptamers that specifically bound HSA and GHSA. HSA and GHSA are associated with diabetes mellitus. The length of selected aptamers are around 46-106 bases, in which aptamers against HSA are consisting of 46-106 bases and aptamers against GHSA are consisting of 49-71 bases. All selected aptamers against HSA and GHSA can be potentially applied for detection and monitoring of diabetes mellitus in combination with blood glucose and HbAlC level. They also can applied in the drug development and drug delivery system in the diabetes mellitus and Alzheimer disease. In addition, chemical or fluorescence labeled these aptamers can be used for study function and location of HSA and GHSA.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention relates to aptamers bound human serum albumin and glycated human serum albumin 
       BACKGROUND OF THE INVENTION 
       [0002]    Human Serum Albumin (HSA) is 66.4 kDa abundant protein in human serum (50% of total protein) composing of 585 amino acids with the heart shape structure (Sugio,  Protein Eng,  Vol. 12, 1999, 439-446). Multifunctional HSA protein is associated with its structure that allowed to bind and transport a number of metabolizes such as fatty acids, metal ions, bilirubin and some drugs (Fanali,  Molecular Aspects of Medicine,  Vol. 33, 2012, 209-290). HSA concentration in serum is around 3.5-5 g/dL. Abnormal HSA level is resulting in abnormal function in human system and can be an indicator for some diseases. The high HSA level can be found in heart failure condition, Alzheimer and diabetes mellitus (Fanali,  Molecular Aspects of Medicine,  Vol. 33, 2012, 209-290). 
         [0003]    Glycated human serum albumin (GHSA) is glycation product of HSA protein, in which glucose sugar is non-enzymatically added on some amino acids (Lysine 199, 281, 439 etc.) of the HSA molecule (Fanali,  Molecular Aspects of Medicine,  Vol. 33 2012, 209-290). GHSA can be produced in condition with the high level of sugar concentration, which usually found in diabetes mellitus patients. Adding sugar on the GHSA molecule results in three-dimensional structure changes and interferes normal HSA protein functions, for examples lower binding affinity to bilirubin (up to 50%) and cis-parinaric acid (up to 20 times) (Shaklai,  Journal of Biological Chemistry,  Vol. 259, No. 6, 1984, 3812-3817). Therefore, GHSA level can be an indicator for diabetes mellitus complications and Alzheimer diseases (Shuvae,  Neurobiology of Aging,  Vol. 22, No. 3, 2001, 397-402). In addition, GHSA level is correlated with blood sugar and glycated hemoglobin (HbAlc) and its half-life is shorter than HbAlc, therefore GHSA level can be the better indicator for diabetes mellitus detection and monitoring (Wincour,  Clinical Biochemistry,  Vol. 22, 1989, 457-461, Worner,  International Journal of Pharmacology, Therapy, and Toxicology,  Vol. 31, No. 5, 1993, 218-222). 
         [0004]    In case of diabetic nephropathy, GHSA will interact with receptor in the mesangial cells, which are associated with the glomerular dysfunction (Cohen,  Clinical and Methodological Aspects. Diabetes Technology  &amp;  Therapeutics,  1999, Thomas,  Journal of  10  American Society of Nephrology,  Vol. 16, 2005, 2976-2984, Ziyadeh,  Kidney International,  Vol. 53, 1998, 631-638). In 1994 and 1995, Cohen and colleagues found that monoclonal antibody that specifically bound GHSA could retard the progression of diabetes nephropathy in mice and prevent the GHSA from causing further harm in the kidney (Cohen, U.S. Pat. No. 5,518, 720). Therefore this antibody have a potential for drug development in diabetes nephropathy complication. 
         [0005]    It has been found that GHSA is associated with the protein phosphorylation in retinal cell growth, resulting in diabetes retinopathy (Okumura,  Journal of Opthalmology,  Vol. 51, 2007, 231-243). In 2007, Higashimoto and his colleagues selected single stranded DNA (ssDNA) that specifically bound to GHSA in vitro and they also found that some selected aptamers could inhibit GHSA toxicity in retinal pericytes (Higashimoto,  Microvascular Research,  Vol. 74, 2007, 65-69, Inou, US patent number US/2009/0023672 A1), which can be developed for the anti-diabetes retinopathy drugs. 
       Human Serum Albumin Detection 
       [0000]    
       
         
           
             1. Dye-Binding Method: There are 2 types of GHSA detection by dye, Which are Bromcresol Green (BCG) and Bromcresol Purple (BCP).
           Bromcresol Green: In 1965, it has been proved that bromcresol green, which is negative charge molecule could bind HSA protein at pH 7-7.1. The absorbance of the binding complex could be detected by spectrophotometry at the absorption wavelength 615 nm. Increasing of HSA concentration is associated with decreasing of 615 nm absorption (Rodkev,  Clinical Chemistry,  Vol. 11, No 4, 1965). In 1976, BCG has been found to bind to other proteins (α- and β-globulin) in condition with the low HSA for examples kidney failure and dialysis patients (Gustafsson,  Clinical Chemistry,  Vol. 22, No. 616, 1976, Webster,  Clinica Chimica Acta,  Vol. 53, No. 109, 1974). Therefore, BCG can be only used for screening method.   Bromcresol Purple: Detection of HSA using BCP method was firstly used in 1970 by Louderback and his colleagues (Louderback,  Clinical Chemistry,  Vol. 14, 1970, 793-794) and future developed by Carter and his colleagues (Carter,  Microchem Journal,  Vol. 15, 1970, 531-539). In 1978, Andrew and colleagues invented automate system based on BCP method (Andrew  Clin Chem,  Vol. 24., No. 1, 1978, 80-86). The principle of BCP method is based on BCP charge, which is the higher positive charge dye than the BCG charge. The BCP can specifically bind to HSA, leading to more broaden absorption wavelength comparing with BCG method. In addition, BCP method can be used for detection of human serum albumin in the lower concentration. Therefore, BCP method is more popular method than BCG method. However, it has been reported that 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF), which can be found in kidney failure patients who have been done dialysis for a long period of time, interferes BCP method (Basil  Clinical Chemistry,  Vol. 55, No. 3, 2009, 583-584). Therefore, it is better to develop new method for HSA measurement that can be used in kidney failure patients and other abnormal conditions.   
         
             2. Immunochemical Assay: Immunochemical assay seems to be the most sensitive and specific method for HSA detection. Principle of the assay is depending on the affinity binding of HSA and antibody. The final products could be measured by detection of the turbidity, fluorescence intensity and UV absorption (Basil,  Clinica Chimica,  Vol. 258, 1997, 3-20). In order to get an accurate results, the assay requires several sample dilutions, leading to high cost and time consuming. However, this assay is suitable for detection of low HSA concentration in urine and other secretions. 
           
         
       
     
       Glycated Human Serum Albumin Detection 
       [0010]    Nowadays, glycated human serum albumin detection is based on the binding of boronic acid and cis-diol group of the glucose molecule on the GHSA protein. The most three popular methods are described below.
       1. Boronate Affinity Chromatography (BAC): Boronic acids, which are coated on the resin beads, will bind to glucose molecule on GHSA protein in the sample. Then the unbound molecules will be washed out and the remaining GHSA protein will be analyzed by measuring the absorption of tryptophan amino acids.   2. Enzyme Link Boronate Immunoassay (ELIBA): Antibodies against HSA protein will bind to both HSA and GHSA protein. After the binding of Horseradish Peroxidase (HPR) conjugated boronic molecule and cis-diol group on the GHSA protein, GHSA concentration can be analyzed using the similar method as ELISA (Ikeda,  Clinical Chemistry,  Vol. 44, No. 2, 1998, 256-263).   3. Enzymatic Assay: Amino acids with the glucose attachment will be digested by proteinase enzyme, resulting in single glycated amino acids. Then glycated amino acids will be oxidized by Ketonamine oxidase enzyme, leading to the formation of hydrogen peroxide. The amount of hydrogen peroxide molecule, which correlated with the concentration of GHSA, can be measured using peroxidase method. On the other hand, total HSA can be analyzed using BCP method as previously described and the percentage of glycation can be calculated (Kohzuma,  Journal of Diabetes Science and Technology,  Vol. 5, No. 6, 1455-1462).       
 
         [0014]    Previous HSA and GHSA detections are suitable for only screening method because they are lacking of specificity. The ideal method should be more specific, which is depending on the affinity binding of the specific binding molecules (antibody or aptamer) and HSA/GHSA. 
       Aptamers Against Human Serum Albumin and Glycated Human Serum Albumin 
       [0015]    Aptamer is a short ssDNA or RNA that specifically bind to target molecule using three-dimensional structure. Target molecules could be cells, proteins, metal ions, and toxin. The aptamer can be selected from the large aptamer library using the method called “Systematic Evolution of Ligands by Exponential Enrichment” or “SELEX” (Tuerk,  Science,  Vol. 249, 1990, 505-510, Ellington,  Nature,  Vol. 346, 1990, 818-822). The principle of the SELEX method is the repeating of aptamers selection against target molecule. The higher pressure condition will be added to each selection process to obtain higher specific binding aptamers. Then the selected aptamers will be amplified and the process will be repeated until the affinity binding of selected aptamer is constant. 
         [0016]    Aptamer is similar as antibody, in which they can bind specifically to target molecule. However, aptamer is more stable and easily to produce comparing with the antibody. It has been reported that aptamers could be developed and used as a drug, drug delivery and applied for diagnostic field (Kyung-Mi Song,  Sensors,  Vol. 12, 2012, 612-631). Aptamers against GHSA have been reported in 2007 by Higashimoto and colleagues. They also found that some selected aptamers could inhibit toxicity of GHSA in retinal pericyte (Higashimoto,  Microvascular Research,  2007, 65-69, US patent number US 2009/0023672 A1). 
         [0017]    The present invention is about aptamers against HSA and GHSA. Selected aptamers in this invention have higher binding affinity than that from the previous report and have a potential to be used in the diagnostic field and also drug development. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0018]      FIG. 1 : Transferring of ssDNA aptamer from polyacrylamide gel to nylon membrane. 
           [0019]      FIG. 2 : Predicted secondary structure of aptamer G8 (MFold program). 
           [0020]      FIG. 3 : The schematic showing indirect ELISA using aptamer. 
           [0021]      FIG. 4 : The schematic showing direct ELISA using antibody (left) and streptavidin (right). 
           [0022]      FIG. 5 : Graph shows the binding affinity of G8 aptamer and human serum albumin using direct ELISA and antibody dilutions 1:1000, 1:2000, 1:3000 and 1:4000. 
           [0023]      FIG. 6 : Graph shows the binding affinity of G8 aptamer and glycated human serum albumin using direct ELISA and streptavidin dilutions 1:1000, 1:2000, 1:3000 and 1:4000. 
           [0024]      FIG. 7 : Nucleotide sequences of G8 and clone 9 aptamer. 
           [0025]      FIG. 8 : Binding assay results of G8 aptamer and glycated human serum albumin in comparison with the results of clone 9 aptamer and glycated human serum albumin. 
           [0026]      FIG. 9 : Graph showing binding affinity of G8 and GHSA comparing with clone 9 and GHSA, which is calculated from  FIG. 8 . 
       
    
    
     SUMMARY OF THE INVENTION 
       [0027]    This invention is about the selection and development of aptamers that specifically bound HSA and GHSA. HSA and GHSA are associated with diabetes mellitus. The length of selected aptamers are around 46-106 bases, in which aptamers against HSA are consisting of 46-106 bases and aptamers against GHSA are consisting of 49-71 bases. All selected aptamers against HSA and GHSA have a potential to be applied for monitoring and drug development of diabetes mellitus and Alzheimer disease. In addition, chemical or fluorescence labeled these aptamers can be used for function and location study of HSA and GHSA. 
       DETAILED DESCRIPTION OF THE INVENTION 
       [0028]    This invention is about aptamer that specifically bound to proteins associated with diabetes mellitus, which are human serum albumin (HSA) and glycated human serum albumin (GHSA). Selected aptamers against HSA and GHSA in this invention are consisting of 46-106 bases and 49-71 bases, respectively. Nucleotide sequences of aptamers are shown in Table 1 and Table 2. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1  
               
             
             
               
                   
               
               
                 Nucleotide sequences of aptamers against human serum albumin 
               
             
          
           
               
                   
                 Nucleotide 
                   
                 SEQ ID 
               
               
                 Name 
                 number 
                 Nucleotide sequence 
                 No. 
               
               
                   
               
             
          
           
               
                 H1 
                 88 
                 AGATTGCACTTACTATCTCCAGGTCTCCCTGAC 
                 1 
               
               
                   
                   
                 CACAATAAAAGATAGCGTCCTGCTTGGAATGAA 
                   
               
               
                   
                   
                 GGGC AATTGAATAAGCTGGTAT 
                   
               
               
                   
               
               
                 H2 
                 88 
                 AGATTGCACTTACTATCTCCAACACACCCGACC 
                 2 
               
               
                   
                   
                 GGGCCCTTATTGCTGACCACCAAACTATGAACA 
                   
               
               
                   
                   
                 ACGG AATTG AATAAGCTGGTAT 
                   
               
               
                   
               
               
                 H3 
                 46 
                 AGATTGCACTTACTATCT CCACCCATATG 
                 3 
               
               
                   
                   
                 AATTGAATACCCTGGTTT 
                   
               
               
                   
               
               
                 H4 
                 106 
                 AGATTGCACTTACTATCTATCCCACCACAGAAC 
                 4 
               
               
                   
                   
                 CCCAGCCATGCAACCCCACAACAAGACCTCAA 
                   
               
               
                   
                   
                 CCACC AATTGAATAAGCTGGTAT 
                   
               
               
                   
                   
                 AATTGAATAAGCTGGTAT 
                   
               
               
                   
               
               
                 H8 
                 87 
                 ATACCAGCTTATTCAATTCCCCCGGCTTTGGTTT 
                 5 
               
               
                   
                   
                 AGAGGTAGTTGCTCATTACTTGTACGCTCCGGA 
                   
               
               
                   
                   
                 T GAGATAGTAAGTGCAATCT 
                   
               
               
                   
               
               
                 H10 
                 88 
                 ATACCAGCTTATTCAATTGTTAACCGGTATGTAT 
                 6 
               
               
                   
                   
                 AGGATTATGAAAATGCCGCCCATCGACCCTGTT 
                   
               
               
                   
                   
                 CC GAGATAGTAAGTGCAATCT 
                   
               
               
                   
               
               
                 H11 
                 87 
                 ATACCAGCTTATTCAATTCCCGTACTGAGGGGG 
                 7 
               
               
                   
                   
                 TCCTACCCCGTCTCGGCCCAGCATGTGGTTCGA 
                   
               
               
                   
                   
                 TG GAGATAGTAAGTGCAATCT 
                   
               
               
                   
               
               
                 H12 
                 106 
                 AGATTGCACTTACTATCTATCCCACCACAGAAC 
                 8 
               
               
                   
                   
                 CCCAGCCATGCAACCCCACAACAAGACCTCAA 
                   
               
               
                   
                   
                 CCACCAATTGAATAAGCTGGTAT AATTG 
                   
               
               
                   
                   
                 AATAAGCTGGTAT 
                   
               
               
                   
               
               
                 H13 
                 88 
                 AGATTGCACTTACTATCTTTGCGCTTGCAGAAC 
                 9 
               
               
                   
                   
                 TAGAAACAAACGCGCAACATTATTCGTACACCC 
                   
               
               
                   
                   
                 CCCC AATTGAATAAGCTGGTAT 
                   
               
               
                   
               
               
                 H14 
                 88 
                 ATACCAGCTTATTCAATTCGCGCACATATACAGG 
                 10 
               
               
                   
                   
                 GCTTTACCAGCGGGGAAGGTTAGCGACGCGAG 
                   
               
               
                   
                   
                 GGG GAGATAGTAAGTGCAATCT 
                   
               
               
                   
               
               
                 H16 
                 87 
                 ATACCAGCTTATTCAATTAAGATCCGGATAGCAA 
                 11 
               
               
                   
                   
                 TCTGCCGTAGTAGGTCAACGTGTCTGGGGGGTT 
                   
               
               
                   
                   
                 A TAGATAGTAAGTGCAATCT 
                   
               
               
                   
               
               
                 H17 
                 88 
                 AGATTGCACTTACTATCTCGCGAAGCCAACAAA 
                 12 
               
               
                   
                   
                 ATCAACCACCCCACTCTTTAATACATCCCGGGC 
                   
               
               
                   
                   
                 GCCC AATTGAATAAGCTGGTAT 
                   
               
               
                   
               
               
                 H18 
                 88 
                 AGATTGCACTTACTATCTCCAAACCACTACACC 
                 13 
               
               
                   
                   
                 CTTCTAACCCCCCTGTCTTCCTCGCTCTGACCA 
                   
               
               
                   
                   
                 CCTT AATTGAATAAGCTGGTAT 
                   
               
               
                   
               
               
                 H20 
                 88 
                 ATACCAGCTTATTCAATTGTCGTGTCTGGGCCAT 
                 14 
               
               
                   
                   
                 TGATGAGTCGTAGTGGGGTTTCGCTCTATCGGG 
                   
               
               
                   
                   
                 TG TAGATAGTAAGTGCAATCT 
                   
               
               
                   
               
               
                 H23 
                 106 
                 ATACCAGCTTATTCAATTATACCAGCTTATTCAAT 
                 15 
               
               
                   
                   
                 TGTAGAACAATACTCTGGTTAACACTCGTTACA 
                   
               
               
                   
                   
                 CGTTTATTCCCCTGACACT 
                   
               
               
                   
                   
                 GAGATAGTAAGTGCAATCT 
                   
               
               
                   
               
               
                 H24 
                 88 
                 AGATTGCACTTACTATCTATGCCAACATCCCCCC 
                 16 
               
               
                   
                   
                 CCTATTCACTAACCATCCTACTAACGTCCTCCGG 
                   
               
               
                   
                   
                 GT AATTGAATAAGCTGGTAT 
                   
               
               
                   
               
               
                 H25 
                 105 
                 ATACCAGCTTATTCAATTATACCAGCTTATTCAAT 
                 17 
               
               
                   
                   
                 TCGCACTTGTTTAATGCGCAAGTATCTTGGGTG 
                   
               
               
                   
                   
                 TAGTTGGTCGGTGTGATA 
                   
               
               
                   
                   
                 GAGATAGTAAGTGCAATCT 
                   
               
               
                   
               
               
                 H26 
                 89 
                 AGATTGCACTTACTATCTGCACACTACTAAACTA 
                 18 
               
               
                   
                   
                 CATATGTCCCCACTCCAACCTACTTGAATCGGG 
                   
               
               
                   
                   
                 TTC AATTGAATAAGCTGGTATA 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 2  
               
             
             
               
                   
               
               
                 Nucleotide sequences of aptamers against glycated human serum albumin 
               
             
          
           
               
                   
                 Nucleotide 
                   
                 SEQ ID 
               
               
                 Name 
                 number 
                 Nucleotide sequence 
                 No. 
               
               
                   
               
               
                 G1 
                 71 
                 TCTATCCCCCCAGCCTTCCCACTCCAACCCTGC 
                 19 
               
               
                   
                   
                 CGGGCCGCTGCATATAACTGAATTGAATAAGCT 
                   
               
               
                   
                   
                 GGTAT 
                   
               
               
                   
               
               
                 G2 
                 52 
                 TGGTACATCGACCATCACCGCACCTCACATATT 
                 20 
               
               
                   
                   
                 CCGAATTACTCCCGACGTA 
                   
               
               
                   
               
               
                 G3 
                 52 
                 TACATTGCTCCTGCGGAAAAATTGTCAAACCAT 
                 21 
               
               
                   
                   
                 CTACTGCGAAGCGTGTTTT 
                   
               
               
                   
               
               
                 G4 
                 49 
                 TAGGAGTAGGGGGTCGTAGACGGTTGGGGCGG 
                 22 
               
               
                   
                   
                 AACGGGCGTGGGGCATG 
                   
               
               
                   
               
               
                 G5 
                 53 
                 TGGTACATCGACCATCACCGCACCTCACATATT 
                 23 
               
               
                   
                   
                 CCGAATTACTCCCGACGTAT 
                   
               
               
                   
               
               
                 G7 
                 53 
                 TCGATGGTGGGCAGCCCCAGCACATTCCGTATG 
                 24 
               
               
                   
                   
                 TTAACCCCTGCGTTGCCATT 
                   
               
               
                   
               
               
                 G8 
                 49 
                 GGTGCGGTTCGTGCGGTTGTAGTACTCGTGGCC 
                 25 
               
               
                   
                   
                 GATAGAGGTAGTTTCG 
                   
               
               
                   
               
               
                 G10 
                 51 
                 TCATACTGGGTCATGTACTTAGCTGGTCGCAGC 
                 26 
               
               
                   
                   
                 GGGGACTGAGTTAGTGTT 
                   
               
               
                   
               
               
                 G11 
                 53 
                 TCCCACGCCCGCCCGTCGTTCACCCCTCCCCGC 
                 27 
               
               
                   
                   
                 TACCTCCCTATCCAACTGCG 
                   
               
               
                   
               
               
                 G12 
                 53 
                 TCCCCCCATCACACCCAAGCCGCAGCCACCGA 
                 28 
               
               
                   
                   
                 CATAGCAAGCATTGTCTTTCC 
                   
               
               
                   
               
               
                 G13 
                 52 
                 TCGGGGGGGCGTTGATTTTGTTGAAGGGAGGT 
                 29 
               
               
                   
                   
                 ATAGTGTCTGTCGGTCTGAT 
                   
               
               
                   
               
               
                 G14 
                 51 
                 TCCTGCCGAACTCCAAGATCTCCGCTCCGCTCA 
                 30 
               
               
                   
                   
                 CGCTGTGTATCCATGGGG 
                   
               
               
                   
               
               
                 G15 
                 53 
                 TAGTTCTAGGCCGCCCTCGTGATAACCCCCCTC 
                 31 
               
               
                   
                   
                 CATCTTCCCTACGATGTACT 
                   
               
               
                   
               
               
                 G17 
                 52 
                 TGGGTCATCGTCGTCTTAGGCGCGTGAAAGGG 
                 32 
               
               
                   
                   
                 GTAGGATGGCGGGTAGGATG 
                   
               
               
                   
               
               
                 G19 
                 52 
                 TGCAAGGTGGGCATTGGCATTGCGTAGCTAGGG 
                 33 
               
               
                   
                   
                 GGTGAAGGCGTGTGGTTTT 
                   
               
               
                   
               
               
                 G23 
                 71 
                 TCAGGCAAACACAATATACGCAATATCACGGTG 
                 34 
               
               
                   
                   
                 GAATTTCAAGGCCTTTCATCAATTGAATAAGCT 
                   
               
               
                   
                   
                 GGTAT 
                   
               
               
                   
               
               
                 G24 
                 53 
                 TCAAAAGCGCGCTAAGCCTAGTTCGACAACTT 
                 35 
               
               
                   
                   
                 CACCAACGACCCACTATTCGT 
                   
               
               
                   
               
               
                 G25 
                 51 
                 TCCCTAACCCGCTCTAACCAACCGCGCTCAGTC 
                 36 
               
               
                   
                   
                 CGACATCCGTAAACGGGC 
                   
               
               
                   
               
               
                 G26 
                 53 
                 TCCAACCCAGACCAACATTCCTCGCCTCCGCTA 
                 37 
               
               
                   
                   
                 TCTGCACCGCCACACATAAC 
               
               
                   
               
             
          
         
       
     
       EXAMPLE 1 
     Qualitative Binding Assay of Selected Aptamers and HSA/GHSA Using Electromobility Shift Assay (SMSA) 
     1. Small Scale Preparation of 5′Biotinylated Aptamers 
       [0000]    
       
         
           
             Step 1: Plasmid DNAs encoding selected aptamer sequences (Table 1 and Table 2) were diluted with sterile water to make 10 nM stock solution for PCR amplification. 
             Step 2: Stock solutions from step 1 were used as templates for 50 μL PCR reaction. The PCR reaction is described in Table 3 and 4. 
           
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 PCR reaction (50 μL total reaction) for  
               
               
                 5′ Biotinylated aptamer preparation 
               
             
          
           
               
                   
                 PCR composition 
                 Volume (μL) 
               
               
                   
                   
               
             
          
           
               
                   
                 Plasmid DNA encoding aptamer sequence 
                 1 
               
               
                   
                 *25 μM 5′ Biotinylated forward primer 
                 1 
               
               
                   
                 (5′. . .Biotin/ATACCAGCTTATTCAATT. . .3′) 
                   
               
               
                   
                 **25 μM 5′ Phosphorylated reward primer 
                 1 
               
               
                   
                 (5′ ...Phosphate/AGATTGCACTTACTATCT...3&#39;) 
                   
               
               
                   
                 10 mM dNTP 
                 1 
               
               
                   
                 10 × Thermo Pol Reaction buffer 
                 5 
               
               
                   
                 Steriled water 
                 40 
               
               
                   
                 5 U/μL Taq Polymerase 
                 1 
               
               
                   
                   
               
               
                   
                 Remark: 
               
               
                   
                 *5′ Biotinylated primer for EMSA analysis 
               
               
                   
                 **Phosphorylated primer for λ-Exonuclease digestion 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
             
               
               
             
               
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 PCR condition for 5′ Biotinylated  
               
               
                 aptamer preparation 
               
             
          
           
               
                 Step 
                 Process 
                 Temperature 
                 Time 
               
               
                   
               
             
          
           
               
                 1 
                 Heat 
                 94° C. 
                 5  
                 minutes 
               
               
                 2 
                 Denaturation 
                 94° C. 
                 1  
                 minutes 
               
               
                 3 
                 Annealing 
                 36° C. 
                 30  
                 Seconds 
               
               
                 4 
                 Extension 
                 72° C. 
                 30  
                 Seconds 
               
             
          
           
               
                 5 
                 Repeat step 2-4 for 34 rounds 
               
             
          
           
               
                 6 
                 Final extension 
                 72° C. 
                 10  
                 minutes 
               
             
          
           
               
                 7 
                 Cooling 
                  4° C. 
                 Until use 
               
               
                   
               
             
          
         
       
       
         
           
             Step 3: 1 μL of 20 U/μL Dpnl enzyme was added in the PCR product and incubated at 37° C. for 3 hours. DpnI enzyme will digested plasmid DNA template by cutting at all methyl groups of the plasmid DNA. 
             Step 4: Then 1 μL λ-Exonuclease enzyme was added in the PCR product and incubated at 37° C. for another 3 hours. Phosphorylated DNA strands will be digested by λ-Exonuclease enzyme. 
             Step 5: 5′ Biotinylated aptamers were purified using QIAquick PCR purification kit (QIAGEN). Then purified aptamers were diluted in 20 μL steriled water. The stock aptamers with concentration around 5-10 ng/μL were stored at −20° C. until use. 
           
         
       
     
       2. Binding Assay of Aptamers and Target Proteins (Human Serum Albumin and Glycated Human Serum Albumin) Using Gel Electrophoresis Followed by Southern Blot Analysis 
       [0000]    
       
         
           
             Step 1: 9 μL of 5′ Biotinylated aptamers from previous process with the concentration of 2-10 ng/μL were incubated at 65° C. for 5 minutes to denature secondary structure. After that the reaction was incubated at 4° C. for 1 minutes before use. 
             Step 2: 1 μL HSA was mixed with 9 μL of aptamer against HSA and 1 μL of 0.4 μg/μL GHSA was mixed with 9 μL of aptamer against GHSA. 
             Step 3: The reaction from step 2 was incubated at 25° C. for 1 hour and analyzed on 8% polyacrylamide gel at 100 V for 30 minutes. 
             Step 4: Aptamers on the polyacrylamide gel from step 3 were transferred to nylon membrane (Amersham Hybond-N+; GE Healthcare). Classical DNA/RNA transferring method was used. The transferring buffer was SSC (150 mM CaCl 2  and 15 mM Sodium citrate, pH 7.0) and incubation time was 12 h. The schematic of transferring set up is shown in  FIG. 1 . 
             Step 5: Aptamer analysis using Phototope®-Star Detection Kit (New England Biolabs)
           Nylon membrane was removed from the transferring set up and put in the clear plastic container containing solution A (5% Sodium Dodecyl Sulfate (SDS), 125 mM NaCl, 25 mM Sodium Phosphate, pH 7.5). The system was incubated at room temperature for 5 minutes with gentle shaking.   The solution was discarded and 10 μL of streptavidin in 20 mL of solution A was added in the reaction container and incubated at room temperature for 5 minutes with gentle shaking.   The solution was discarded. Then the membrane was washed 3 times by using solution B (1:10 of solution A in steriled water) with gentle shaking for 5 min. Then the washing buffer was discarded before the next round washing.   μL of Biotinylated alkaline phosphatase in 20 mL of solution A was added in the container. Then the system was incubated at room temperature for 5 minutes with gentle shaking. Then the solution is discarded.   The membrane was washed 3 times by solution C (10 mM Tris HCl, 10 mM NaCl, 1 mM Mg 2 Cl, pH 9.50) with gentle shaking and then solution is discarded.   CDP star was added on the membrane (until solution covered the membrane) and incubated at room temperature (dark) for 10 minutes with gentle shaking.   The nylon membrane was attached to the x-ray film in the film cassette for 1 minutes (dark room).   The x-ray film was removed from the cassette and dipped in developer solution until the aptamer band was appeared on the x-ray film.   The x-ray film was washed with clean water for 30 seconds, followed by soaking in fixer solution until the x-ray film was clear.   The x-ray film was washed in clean water for 30 seconds and air dried. Remark: Steps involved the x-ray film were perfoimed in the dark room.   Density of the aptamer band on the dried x-ray film was analyzed and the positive results were shown in Table 5 and Table 6.   
         
           
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 5  
               
             
             
               
                   
               
               
                 Aptamers bound human serum albumin with EMSA 
               
               
                 positive result 
               
             
          
           
               
                   
                 Nucleo- 
                   
                 SEQ 
               
               
                   
                 tide 
                   
                 ID 
               
               
                 Name 
                 number 
                 Nucleotide sequence 
                 No. 
               
               
                   
               
             
          
           
               
                 H8 
                 87 
                 ATACCAGCTTATTCAATTCCCCCGGCTTTGG 
                 5 
               
               
                   
                   
                 TTTAGAGGTAGTTGCTCATTACTTGTACGCT 
                   
               
               
                   
                   
                 CCGGAT GAGATAGTAAGTGCAATCT 
                   
               
               
                   
               
               
                 H14 
                 88 
                 ATACCAGCTTATTCAATTCGCGCACATATAC 
                 10 
               
               
                   
                   
                 AGGGCTTTACCAGCGGGGAAGGTTAGCGA 
                   
               
               
                   
                   
                 CGCGAGGGG GAGATAGTAAGTGCAATCT 
                   
               
               
                   
               
               
                 H17 
                 88 
                 AGATTGCACTTACTATCTCGCGAAGCCAAC 
                 12 
               
               
                   
                   
                 AAAATCAACCACCCCACTCTTTAATACATC 
                   
               
               
                   
                   
                 CCGGGCGCCC AATTGAATAAGCTGGTAT 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 6  
               
             
             
               
                   
               
               
                 Aptamers boundglycated human serum albumin with 
               
               
                 EMSA positive result 
               
             
          
           
               
                   
                 Nucleo- 
                   
                 SEQ 
               
               
                   
                 tide 
                   
                 ID 
               
               
                 Name 
                 number 
                 Nucleotide sequence 
                 No 
               
               
                   
               
               
                 G1 
                 71 
                 TCTATCCCCCCAGCCTTCCCACTCCAACCCT 
                 19 
               
               
                   
                   
                 GCCGGGCCGCTGCATATAACTGAATTGAATA 
                   
               
               
                   
                   
                 AGCTGGTAT 
                   
               
               
                   
               
               
                 G8 
                 49 
                 GGTGCGGTTCGTGCGGTTGTAGTACTCGTG 
                 25 
               
               
                   
                   
                 GCCGATAGAGGTAGTTTCG 
                   
               
               
                   
               
               
                 G10 
                 51 
                 TCATACTGGGTCATGTACTTAGCTGGTCGCA 
                 26 
               
               
                   
                   
                 GCGGGGACTGAGTTAGTGTT 
                   
               
               
                   
               
               
                 G12 
                 53 
                 TCCCCCCATCACACCCAAGCCGCAGCCACC 
                 28 
               
               
                   
                   
                 GACATAGCAAGCATTGTCTTTCC 
                   
               
               
                   
               
               
                 G15 
                 53 
                 TAGTTCTAGGCCGCCCTCGTGATAACCCCCC 
                 31 
               
               
                   
                   
                 TCCATCTTCCCTACGATGTACT 
               
               
                   
               
             
          
         
       
     
       EXAMPLE 2 
     Thermodynamic Properties of Selected Aptamers 
       [0050]    Aptamer usually binds to the target molecule using secondary structure folding, therefore thermodynamic properties of selected aptamers (Table 5 and Table 6) were characterized by using MFold program, which is free software and developed by Michael Zuker and Nick Markham from College of Arts and Sciences, State University of New York at Albany, USA (http://mfold.rna.albany.edu/?q=mfold/DNA-Folding-Form). Parameters used in this study were shown here and the result is shown in Table 7 and Table 8.
       Linear ssDNA   Temperature at 25° C.   0.1 M Mg 2+  concentration   5% Suboptimality number   Upper bound on the number of computed folding at 50       
 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Thermodynamic properties of aptamers against human serum albumin 
               
             
          
           
               
                   
                 Secondary 
                   
                   
                   
                   
               
               
                   
                 structure 
                 ΔG 
                 ΔH 
                 ΔS 
                 Tm 
               
               
                 Name 
                 number 
                 (kcal/mol) 
                 (kcal/mol) 
                 (cal/(K · mol)) 
                 (° C.) 
               
               
                   
               
             
          
           
               
                 H8 
                 1 
                 −10.47 
                 −166.60 
                 −523.6 
                 44.9 
               
               
                   
                 2 
                 −10.18 
                 −149.00 
                 −465.6 
                 46.8 
               
               
                   
                 3 
                 −9.97 
                 −138.10 
                 −429.7 
                 48.1 
               
               
                   
                 4 
                 −9.77 
                 −151.50 
                 −475.3 
                 45.5 
               
               
                 H14 
                 1 
                 −8.69 
                 −103.10 
                 −316.6 
                 52.4 
               
               
                   
                 2 
                 −8.10 
                 −91.00 
                 −278 
                 54.1 
               
               
                   
                 3 
                 −7.99 
                 −112.80 
                 −351.5 
                 47.7 
               
               
                   
                 4 
                 −7.84 
                 −116.70 
                 −365.1 
                 46.4 
               
               
                   
                 5 
                 −7.81 
                 −108.10 
                 −336.3 
                 48.2 
               
               
                 H17 
                 1 
                 −5.41 
                 −105.10 
                 −334.3 
                 41.1 
               
               
                   
                 2 
                 −4.98 
                 −111.40 
                 −356.9 
                 38.9 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 Thermodynamic properties of aptamer  
               
               
                 against glycated human serum albumin 
               
             
          
           
               
                   
                 Secondary 
                   
                   
                   
                   
               
               
                   
                 structure  
                 ΔG 
                 ΔH 
                 ΔS 
                 Tm 
               
               
                 Name 
                 number 
                 (kcal/mol) 
                 (kcal/mol) 
                 (cal/(K · mol)) 
                 (° C.) 
               
               
                   
               
             
          
           
               
                 G1 
                 1 
                 −5.59 
                 −90.10 
                 −283.4 
                 44.7 
               
               
                   
                 2 
                 −5.56 
                 −88.50 
                 −278.1 
                 44.9 
               
               
                   
                 3 
                 −5.36 
                 −82.60 
                 −259 
                 45.6 
               
               
                   
                 4 
                 −4.63 
                 −69.80 
                 −218.5 
                 46.1 
               
               
                 G8 
                 1 
                 −4.09 
                 −45.10 
                 −137.5 
                 54.7 
               
               
                   
                 2 
                 −3.86 
                 −52.30 
                 −162.4 
                 48.7 
               
               
                   
                 3 
                 −3.43 
                 −45.90 
                 −142.4 
                 49 
               
               
                   
                 4 
                 −3.28 
                 −53.30 
                 −167.7 
                 44.5 
               
               
                   
                 5 
                 −3.23 
                 −60.90 
                 −193.4 
                 41.6 
               
               
                   
                 6 
                 −3.17 
                 −61.20 
                 −194.6 
                 41.2 
               
               
                 G10 
                 1 
                 −8.16 
                 −81.00 
                 −244.3 
                 58.4 
               
               
                 G12 
                 1 
                 −3.68 
                 −55.20 
                 −172.7 
                 46.2 
               
               
                   
                 2 
                 −3.05 
                 −43.20 
                 −134.6 
                 47.6 
               
               
                 G15 
                 1 
                 −5.41 
                 −77.30 
                 −241.1 
                 47.4 
               
               
                   
               
             
          
         
       
     
         [0056]    The result shows that ΔG of selected aptamers were between −10.47 kcal/mol and −3.05 kcal/mol. The melting temperature (temperature at 50% aptamer structure is denatured) was 38.9-58.4° C. To maintain secondary structure formation, experiments involved these aptamers should be performed at lower temperature than 38.9° C. The secondary structure of G8 aptamer is shown in  FIG. 2 . 
       EXAMPLE 3 
     Semi-Quantitative Binding Assay of Selected Aptamers 
       [0057]    The binding assay of selected aptamers against HSA and aptamers against GHSA is deteimined by using Indirect Enzyme-Linked Immunosorbent Assay (Indirect ELISA) and Direct Enzyme-Linked Immunosorbent Assay (Direct ELISA), as described below. 
       3.1. Indirect Enzyme-Linked Immunosorbent Assay (Indirect ELISA) 
       [0058]    The principle of this experiment is based on two antibodies, which are antibodies against 5′ biotinylated aptamer and antibodies against the first antibody. The second antibody is conjugated with horseradish peroxidase enzyme (HRP), which can changes TMB color from blue to be yellow. The yellow color intensity is direct indicator for the target protein concentration. Schematic of the Indirect ELISA is shown in  FIG. 3 . Random selected aptamers in Table 5 and Table 6 were chosen for analysis using this method. 
       Indirect ELISA Protocol 
       [0000]    
       
         
           
             Step 1: 0.8 μg of BSA or HSA or GHSA was added in 50 μl of 0.05 M carbonate buffer in 96 well plate (50 μL/well) and incubated at 4° C. for 1 night. In this process, all proteins will be coated on the 96 well plate. 
             Step 2: The reaction from step 1 was washed 5 times with Phosphate buffer (PBST) (0.05% Tween) using ELISA washing machine (Fluido 2) and tapped for 3-5 times. 
             Step 3: 200 μL of blocking solution (PBST with 1%Tryptone) was added in the reaction and incubated at room temperature for 1 hour, then washed 5 times with PBST using Fluido 2 and tapped for 3-5 times. 
             Step 4: 1 μL of varied concentrations of 5′ Biotinylate aptamer (200, 20, 2 and 0.2 ng/μL) diluted in 50 μL PBST buffer were added in the reaction. After incubating at room temperature for 1 hour, the reaction was washed with PBST for 5 times using Fluido2 and tapped for 3-5 times. 
             Step 5: 50 μL of anti-biotin (1 st  antibody) with the dilution of 1:3840 in PBST was added in the reaction. After incubating at room temperature for 30 minutes, the reaction was washed with PBST for 5 times using Fluido2 and tapped for 3-5 times. 
             Step 6: 50 μL of anti-biotin antibody conjugated with HRP (2 nd  antibody) with the dilution of 1:10,000 was added in the reaction. After incubating at room temperature for 30 minutes, the reaction was washed with PBST for 5 times using Fluido2 and tapped for 3-5 times. 
             Step 7: 50 μL of TMB (HRP substrate) was added in the reaction. Then the reaction was incubated at room temperature (dark) for 30 minutes. 
             Step 8: 50 μL of 0.6 M H 2 SO 4  was added in the reaction and immediately measured the OD 450 nm  using spectrophotometer. 
           
         
       
     
         [0067]    The indirect ELISA result showed the positive results from G12 and H14 aptamer and more positive comparing with clone 9, which is the positive control aptamer from the previous study. These results indicated that selected aptamers from this invention bound GHSA tighter than that from the other study. The indirect ELISA result is shown in Table 9. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                 Binding assay of aptamers against human serum albumin  
               
               
                 and glycated human serum albumin using Indirect ELISA. 
               
             
          
           
               
                   
                 Aptamer 
                 Concentration 
                 Indirect ELISA  
                 Indirect ELISA 
               
               
                   
                 name 
                 (nM) 
                 with HSA 
                 with GHSA 
               
               
                   
                   
               
             
          
           
               
                   
                 Clone 9 
                 200 
                 + 
                 + 
               
               
                   
                   
                 20 
                 − 
                 + 
               
               
                   
                   
                 2 
                 + 
                 + 
               
               
                   
                   
                 0.2 
                 + 
                 − 
               
               
                   
                 G12 
                 200 
                 − 
                 − 
               
               
                   
                   
                 20 
                 − 
                 − 
               
               
                   
                   
                 2 
                 − 
                 + 
               
               
                   
                   
                 0.2 
                 − 
                 − 
               
               
                   
                 H14 
                 200 
                 + 
                 − 
               
               
                   
                   
                 20 
                 + 
                 − 
               
               
                   
                   
                 2 
                 + 
                 − 
               
               
                   
                   
                 0.2 
                 + 
                 − 
               
               
                   
                   
               
             
          
         
       
     
       3.2 Direct Enzyme-Linked Immunosorbent Assay (Direct ELISA) 
       [0068]    Direct ELISA was used for study the binding of the selected aptamer and HSA or GHSA. G8 aptamer was chosen to be a model for direct ELISA. The strategy is based on antibody or streptavidin conjugated HRP, which can change the TMB color from blue to be yellow. Color intensity is depending on concentration of aptamer bound human serum albumin. Short explanation of direct ELISA (based on antibody conjugated HRP and streptavidin conjugated HRP) is shown in  FIG. 4 . G8 aptamer was chosen to be a model for this study. 
       Direct ELISA Protocol 
       [0000]    
       
         
           
             Step 1: 1 μg proteins (Lysozyme, BSA, HSA or GHSA) in 50 μL of 0.05 M Carbonate Buffer were coated on 96-well plat and incubated at 4° C. overnight. 
             Step 2: The reaction was washed with PBST (0.5% Tween) for 5 times ELISA washing machine (Fluido 2) and tapped for 3-5 times. 
             Step 3: The reaction was incubated with 200 μL of 2% Tryptone in PBST at room temperature for 1 hour, then washed with PBST for 5 times and tapped for 3-5 times. 
             Step 4: 50 μL of 40 ng aptamer in PBST (1% Tryptone) was added in the reaction and incubated at room temperature for 1 hour then washing 5 times with PBST and tapped for 3-5 times. 
             Step 5:
           In case of direct ELISA using antibody, 50 μL of antibody in PBST (1%Tryptone) with dilution of 1:1000, 1:2000, 1:3000 and 1:4000 were added in the reaction.   
         
             In case of direct ELISA using streptavidin, 50 μL of streptavidin in PBST (1%Tryptone) with dilution of 1:1000, 1:2000, 1:3000 and 1:4000 were added in the reaction. 
           
         
       
     
         [0076]    Then the reaction was incubated at room temperature for 1 hour before washing 5 times with PBST and tapped for 3-5 times.
       Step 6: 50 μL of TMB was added in the reaction and incubated at room temperature for 30 minutes.   Step 7: The reaction was stopped by adding 50 μL of 0.6 M H 2 SO 4  and measured OD450 nm by using spectrophotometer.   Step 8: Results from direct ELISA using antibody and streptavidin were compared and the best dilution of antibody and streptavidin was chosen for future study.       
 
         [0080]    The result from direct ELISA using antibody showed similar OD450 from all proteins (Lysozyme, BSA, HSA and GHSA) indicating that either G8 aptamer or antibody was non-specific binding to proteins from all dilutions (1:1000, 1:2000, 1:3000 and 1:4000) as shown in  FIG. 5 . On the other hand, the result from direct ELISA using streptavidin showed that 
         [0081]    OD450 from GHSA is significant higher (5 times) than that from HSA (Streptavidin dilution 1:3000). The later result indicated that G8 aptamer specifically bound GHSA ( FIG. 6 ). 
       EXAMPLE 4 
     Quantitative Binding Study of G8 Aptamer and Glycated Human Serum Albumin 
       [0082]    The quantitative binding of G8 aptamer from this invention and clone 9 aptamer from the previous study were analyzed by electromobility shifted assay (EMSA) and results were compared. The EMSA protocol was described in the previous section. 4 ng of 5′ Biotinylated DNA aptamers (G8 and clone 9 sequences is shown in  FIG. 7 ) were incubated with varied amounts of glycated human serum albumin as shown below. 
         [0083]    (1) 0 ng 
         [0084]    (2) 0.0125 ng 
         [0085]    (3) 0.025 ng 
         [0086]    (4) 0.05 ng 
         [0087]    (5) 0.1 ng 
         [0088]    (6) 0.2 ng 
         [0089]    (7) 0.4 ng 
         [0090]    For the control experiment, selected aptamer was incubate with/without 0.4 μg human serum albumin at 25° C. for 1 hour, then samples were analyzed by electrophoresis following by southern blot analysis (similar as Example 1). The result is shown in  FIG. 8 . 
         [0091]    The density of shifted band, which is the binding of aptamer and GHSA, was analyzed using AlphaImager HP. Fraction of bound aptamer (Fa) and dissociation constant (Kd) were calculated using equations below. 
         [0000]        Fa=[T ]/( Ka+[T ]) and  Kd= 1/ Ka (At the optimal aptamer concentration,  Kd= 0.5 Fa )       Fa=aptamer concentration   [T]=GHSA concentration   Ka=Association constant, which is optimal GHSA concentration that bind to optimal aptamer concentration.   Kd=Dissociation Constant, which is an affinity binding of GHSA and aptamer (1/Ka)         
         [0096]    The result showed that G8 aptamer bound GHSA with the Kd of 0.08±0.1 μmole, which is higher affinity comparing with the binding of clone 9 and GHSA, as shown in  FIG. 9 . Therefore, selected aptamers against HSA and GHSA from this invention has a potential for development of HSA and GHSA analysis in other secretions and drug development in the diabetic retinopathy and also drug delivery. 
       BEST MODE FOR CARRYING OUT THE INVENTION 
       [0097]    Previously described in “DETAILED DESCRIPTION OF THE INVENTION” section. 
       INDUSTRIAL APPLICABILITY 
       [0000]    
       
         
           
             1. Human serum albumin (HSA) is normally found in human serum and urine. In case of abnormal liver functions, higher amount of HSA will be found from serum and urine. Therefore, aptamers specifically bound HSA can be potentially developed for analysis of HSA in both serum and urine. 
             2. Glycated human serum albumin (GHSA) can be highly produced within 2-3 weeks in diabetes mellitus patience. Therefore, aptamers specifically bound GHSA can be applied for an analysis of diabetes mellitus in combination with HbAl c level. 
             3. Selected aptamers in this invention could bind to HSA or GHSA. Therefore, these aptamers have a potential to be a drug for treatment of diabetes mellitus and abnormal liver functions. 
             4. Chemical or fluorescence labeled selected aptamers can be potentially used for study the binding position on the HSA or GHSA protein.