Patent Application: US-201515509800-A

Abstract:
the present invention relates to novel dna aptamers capable of binding gelsolin tightly and specifically . the invention further relates to the use of these aptamers to estimate the gelsolin levels in a given sample and purify bulk quantities of tagless gelsolin and its variants . the present invention thus eliminates the use of different animals / their tissues to produce gelsolin binding proteins , which are much more expensive and socially unacceptable methods as opposed to the synthesis of a dna molecule by in vitro pcr . using this strategy , bulk production of the gelsolin binding matrix can be carried out at much lower cost . also , the aptamers can be used to block binding of gelsolin to its binding partners for diagnostic and / or therapeutic applications .

Description:
reduced levels of plasma gelsolin have been reported in various diseases and repletion of exogenous gelsolin has shown improvement in animal models of burn and sepsis . it is anticipated that in future gelsolin levels will be examined in a number of medical cases . possibly , recombinant gelsolin and its functional variants will be used for therapeutic purposes . thus , it is imperative to provide novel entities which can bind to gelsolin as well as develop plasma gelsolin diagnostic kit ( s ) and ways to express and purify gelsolin and its versions . novel dna aptamers which can bind tightly and specifically to gelsolin will help in developing ways to quantify and purify gelsolin and its variants , and block functioning of gelsolin in undesirable pathways , thus projecting a potential therapeutic role of these aptamers . here , we demonstrate that these aptamers bind gelsolin and stop gelsolin mediated prion protein association . present invention describes novel dna aptamers and use thereof as gelsolin binding molecules . the present invention for the first time reports specific aptamers which are capable of binding to gelsolin . aptamers are short dna / rna / peptide molecules which can bind specifically to a target molecule ( pan & amp ; clawson , 2009 ). these are usually identified by systematic evolution of ligands by exponential enrichment ( selex ). selex method involves exposing a random sequence library to a specific target and amplifying the bound molecules which are then subjected to additional rounds of selection . after multiple rounds of selection , specific aptamers identified for binding to the target molecule are subjected to different modifications to improve their binding affinity and stability . gelsolin is a calcium dependent , multifunctional actin regulatory protein composed of six domains ( g1 - g6 ), which appear to have arisen from a triplication event followed by a duplication of an ancestral gene encoding a single domain ( kwiatkowski et al , 1986 ; osborn et al , 2008 ). it exists as three isoforms , two cytoplasmic and a secreted extracellular plasma isoform , all encoded by a single gene located on chromosome 9 in humans . this single gene is subjected to alternative transcriptional initiation and splice site selection in different tissues resulting in differences at the amino termini of the isoforms ( kwiatkowski et al , 1988 ; yin et al , 1984 ). mature plasma ( secreted ) gelsolin ( plasma gsn , formed after cleavage of 27 residue amino - terminal signal sequence ) differs from its cytoplasmic counterpart by an additional unique leader peptide ( 24 amino acids ) at its n - terminal ( kwiatkowski et al , 1986 ) and presence of a disulfide bond providing additional stability ( wen et al , 1996 ). the rest of the polypeptide is identical in sequence to the cytoplasmic form ( kwiatkowski et al , 1986 ). the third minor isoform called gelsolin - 3 is a non - secreted form containing an additional 11 amino acids at the n terminus as compared to the cytoplasmic counterpart . while cytoplasmic gelsolin gets expressed in a wide range of tissues , gelsolin - 3 expresses predominantly in oligodendrocytes in the brain , lungs and testis and , is involved in myelin remodeling during spiralization around the axon ( vouyiouklis & amp ; brophy , 1997 ). plasma gsn is mainly produced and secreted into blood by muscle cells ( kwiatkowski et al , 1988 ). clinical significance and the therapeutic importance of plasma gelsolin have been well illustrated in animal models as well as in patients with various diseases . a significant decrement in pgsn levels has been documented in a variety of illnesses ranging from minor / major trauma , infections to chronic inflammation ( peddada et al , 2012 ). patients with low pgsn levels were observed to have higher mortality rate , longer hospital stay and longer ventilation time in intensive care units as compared to healthy controls . pgsn levels were found to be increasing in patients recovering from diseases . furthermore , it has been confirmed that repletion with exogenous recombinant pgsn significantly increases the survival rate in animal models of different acute insults ( lee et al , 2007 ). however , without knowing the exact plasma gelsolin levels of healthy individuals as well as the patient , gelsolin replacement therapy cannot become a reality . till date , no “ diagnostic method ” exits in the literature , and kits / protocols based on these aptamers can be reliably used for the determination of plasma gelsolin levels of humans and other animals for clinical uses . the term “ diagnostic ” as used in above embodiment refers to the process / method of determining the value of a given entity which could help in finding out the reason / extent of a clinical condition and thus is of clinical use as well as for research purposes . the phrase “ given sample ” as used in above embodiment refers to any biological sample which includes but is not limited to blood , serum , plasma , urine , saliva obtained from human or other animals . the phrase “ plasma gelsolin ” as used in above embodiment refers to the gelsolin present in plasma sample of human or other animals . the phrase “ plasma gelsolin ” as used above refers to the gelsolin present in human / animal plasma which could be a mixture of plasma isoform and other isoforms of gelsolin released into blood due to cell necrosis . plasma isoform of gelsolin is the isoform of gelsolin ( protein ) which gets secreted into blood mainly from muscle cells . the phrase “ clinical use ” refers to the purpose of knowing the gelsolin levels in a given human / animal sample in order to determine the best treatment strategy for the human / animal from which the “ sample ” was obtained . the term “ treatment ” as used herein refers to an approach for obtaining beneficial or desired result including clinical results . beneficial or desired clinical results can include but are not limited to alleviation or amelioration of one or more symptoms or conditions , diminished extent of disease , stabilization of state of disease , slowing disease progression and also prolonging survival as compared to expected survival in the absence of treatment . the term “ functionalize ” as used herein refers to covalent binding of dna aptamers on silica / modified silica / agarose / sepharose beads by chemical reaction . the term “ affinity chromatography ” as used herein refers to binding and thus retention of gelsolin in mobile phase on the aptamers in the stationary phase of the chromatography protocol . accordingly the main embodiment of the present invention provides novel dna aptamers represented by seq id nos . 6 to 9 . another embodiment of the present invention provides dna aptamers as herein described capable of binding the protein gelsolin represented by seq id no . 1 or its variants represented by seq id no . 2 and 3 . another embodiment of the present invention provides dna aptamers as herein described useful for the quantification of gelsolin levels in a sample . another embodiment of the present invention provides a method for the quantification of gelsolin in a sample using the aptamers as herein described , wherein the steps comprise : [ a ] coating lug of streptavidin on a support using 100 mm nahco 3 buffer having ph 9 . 2 for 10 to 12 hours at a temperature of 4 degree c . ; [ b ] washing the coated support of step [ a ] with pbs and blocking with 3 % bsa in pbs for 2 hours followed by washing with pbs ; [ c ] immobilizing 2 um of the biotin labeled aptamer selected from seq id nos . 6 to 9 on the support of step [ b ] using te buffer having ph 8 supplemented with 2m nacl for 2 hours at room temperature ; [ d ] washing the coated support of step [ c ] 2 times with pbs containing 0 . 1 % tween - 20 ; [ e ] adding gelsolin in the range of 0 . 2 um - 5 nm or a sample diluted in selection buffer containing 0 . 1 % bsa to the support of step [ d ] and allowing to stand for 2 hours ; [ f ] washing the coated support of step [ e ] 4 times with pbs containing 0 . 1 % tween - 20 followed by adding anti - gelsolin antibodies and incubating for 10 to 12 hours at 4 degree c . ; [ g ] washing the support obtained in step [ f ] 4 times with pbs containing 0 . 1 % tween - 20 followed by adding secondary antibodies conjugated with horseradish peroxidase and incubating for 1 hour and then adding the substrate for horseradish peroxidase ; [ h ] terminating the reaction of step [ g ] with 2m h 2 so 4 after the development of blue colour and measuring the absorbance at 450 nm so as to determine the quantity of gelsolin present in the sample . yet another embodiment of the present invention provides a method for the quantification of gelsolin in a sample using the aptamers as herein described , wherein the steps comprise : [ a ] coating anti - gelsolin antibodies on a support using 100 mm nahco 3 buffer having ph 9 . 2 for 10 to 12 hours at a temperature of 4 degree c . ; [ b ] washing the coated support of step [ a ] with pbs and blocking with 3 % bsa in pbs for 2 hours followed by washing with pbs ; [ c ] adding gelsolin in the range of 0 . 2 um - 5 nm or a sample diluted in pbs containing 0 . 1 % bsa and 0 . 01 % tween - 20 to the support of step [ b ] and allowing to stand for 2 hours at room temperature ; [ d ] washing the coated support of step [ c ] 3 times with pbs containing 0 . 1 % tween - 20 ; [ e ] adding 1 um of the biotin labeled aptamer selected from seq id nos . 6 to 9 diluted in selection buffer containing 0 . 1 % bsa to the support of step [ d ] and allowing to stand for 2 hours ; [ f ] washing the coated support of step [ e ] 4 times with pbs containing 0 . 1 % tween - 20 followed by adding streptavidin conjugated with horseradish peroxidase and incubating for 1 hour and then adding the substrate for horseradish peroxidase ; [ g ] terminating the reaction of step [ f ] with 2m h 2 50 4 after the development of blue colour and measuring the absorbance at 450 nm so as to determine the quantity of gelsolin present in the sample . yet another embodiment of the present invention provides dna aptamers as herein described , useful for the purification of gelsolin from a mixture . a method for the purification of gelsolin from a mixture using the aptamers as herein described , wherein the steps comprise : [ a ] washing the cnbr activated sepharose beads with ice - cold double - distilled water and adding 10 mm potassium phosphate ( ph 8 ) and 5 ′ - phosphorylated oligos thereto to make a thick slurry of the activated beads ; [ b ] stirring the slurry obtained in step [ a ] at room temperature for 14 hours and washing with 1 m potassium phosphate ( ph 8 ) containing 1 m kcl ; [ c ] washing the beads obtained in step [ b ] with water , followed by resuspending in 10 mm tris - hcl ( ph 8 ) containing 300 mm nacl , 1 mm edta ; [ d ] pouring the slurry of beads obtained in step [ c ] in a pd - 10 column and washing with three column volumes of 40 mm tris buffer ph 8 containing 100 mm nacl and 2 mm egta ; [ e ] adding to the column obtained in step [ d ], the cell lysate containing gelsolin with ph adjusted to ph 8 and containing 2 mm egta ; [ f ] washing the column of step [ e ] obtained after initial loading with three column volumes of 40 mm tris buffer ph 8 containing 100 mm nacl and 3 mm cacl 2 ; [ g ] eluting the bound gelsolin from the column of step [ f ] by adding 40 mm tris buffer ph 8 containing 300 mm of nacl and 20 mm of cacl 2 . another embodiment of the present invention provides a kit for the detection of gelsolin using the aptamers as herein described , wherein the kit comprising : [ a ] a solid phase having immobilized thereon the aptamer selected from seq id nos . 6 to 9 ; [ b ] sample containing gelsolin ; [ c ] detection reagents containing anti - gelsolin antibodies and secondary antibodies conjugated with horseradish peroxidase which are capable of detecting the presence of gelsolin . the following examples are given by way of illustration and therefore should not be construed to limit the scope of the present invention . the amplified product of cdna clone obtained from nih mammalian gene collection - human gene clone id 4661084 was digested with xbai and xhoi , and subcloned into the vector backbone of pet303 / ct - his ( invitrogen , ny , usa ). the gelsolin variants were generated by site - directed or deletion mutagenesis as previously described ( peddada et al , 2013 ). the sequences of subcloned gelsolin dna were verified by automated dna sequencing ( applied biosystems , usa ). the plasmids thus created were used to transform e . coli bl21 ( de3 ) which was commercially procured from m / s invitrogen , usa vide catalogue number c6000 - 03 , for the heterologous protein expression . the transformed bacteria carrying pet303 / gelsolin and its mutants were deposited with mtcc , chandigarh , india an international depository authority recognized under the budapest treaty vide accession number mtcc5885 on 09 / 01 / 2014 . the bacteria mtcc 5885 were grown in lb broth ( merck , germany ) to a density of od 600 = 0 . 5 followed by induction of recombinant protein expression with 1 mm isopropyl - β - d - thiogalactoside ( iptg ) for 4 - 5 additional hours . the expressed proteins were purified by weak anion exchange chromatography ( de - 52 ). the target protein was immobilized onto cnbr activated sepharose beads as follows . proteins ( bsa and gelsolin ) were dialyzed against the 100 mm nahco 3 , ph 9 . 0 buffer and concentration was estimated by measuring the absorbance at 280 nm . cnbr activated resin was allowed to swell in 1 mm hcl for 15 min at room temperature and then equilibrated with coupling buffer ( 100 mm nahco 3 , ph 9 . 0 ). after equilibration , the resin was immediately transferred to protein solution and incubated with agitation overnight at 4 ° c . beads were collected by centrifugation at 2000 × g for 1 min and the protein concentration of the supernatant was determined ( it should be 10 - fold less than what was observed at step 1 ). the beads were washed 3 - 4 times with coupling buffer and then incubated with blocking buffer containing 1 m ethanolamine in coupling buffer for 2 hrs at room temperature . after incubation , beads were washed 4 times with a combination of low ph and high ph buffer and finally with 1 × pbs containing 0 . 01 % sodium azide . screening to identify the aptamers against the recombinant gelsolin and its variants : briefly , 2 nmol library of 76 b oligonucleotides including a central random nucleotide region of 30 - mer ( trilink biotechnologies , ca , usa ), representing 10 18 unique sequences was diluted in selection buffer ( 25 mm tris - hcl , ph 8 , 150 mm nacl , 2 mm cacl 2 , 5 mm mgcl 2 and 10 mm kcl and 0 . 01 % tween20 ) and incubated at 94 ° c . for 5 min and kept in ice for 10 min followed by an incubation of 20 min at room temperature . the dna was then allowed to bind to the target protein ( gsn ) conjugated sepharose beads for 1 hr at room temperature . resulting dna eluted after selection was purified by phenol - chloroform extraction , ethanol precipitated and resuspended in 10 ul te ( 10 mm tris - hcl , ph 8 , 1 mm edta ). the dna was pcr amplified in two 50 ul reaction mixtures containing 2 . 5 u of taq dna polymerase , 1 × taq buffer with ( nh4 ) 2 so 4 , 0 . 5 um of both selection primers ( seq id nos . 4 and 5 ), 2 . 5 mm mgcl 2 , 0 . 2 mm dntp and 2 . 5 ul of template . amplification conditions were 2 min at 94 ° c . ; 15 cycles of 10 s at 94 ° c ., 10 s at 62 ° c ., 10 s at 72 ° c . ; 2 min at 72 ° c . 90 ul of amplified dna was first subjected to negative selection using bsa conjugated sepharose beads and then incubated with gelsolin conjugated beads as earlier . after ten rounds of this selex method ( fig1 ), enriched aptamer library was subcloned using topo - ta cloning kit ( invitrogen , ny , usa ). the subcloned aptamers were subjected to automated dna sequencing and the individual aptamers were amplified by pcr as above for verification of the ability to bind gelsolin and variants by microtiter binding assays . the binding ability of the aptamers described herein ( l26f , 10 . 10r , l16f and l24f ) with recombinant gelsolin and its n - and c - terminal halves [ represented by seq id no . 2 and 3 , respectively ] was estimated using microtiter binding assay ( fig2 ). ( the sequences of gelsolin binding aptamers are represented by seq id nos . 6 - 9 .) briefly , different aptamers ( 100 nm ) or actin ( 100 nm ) extracted from chicken muscle ( peddada et al , 2013 ) were coated on to 96 well elisa plates in te ( 10 mm tris - hcl , ph 8 , 1 mm edta ) supplemented with 30 % ammonium sulfate and 100 mm nahco 3 buffer , ph 9 . 2 respectively overnight at 4 ° c . the wells were washed with pbs and blocked with 300 ul per well of 3 % bsa in phosphate buffered saline [ pbs ] for 2 hrs at room temperature . the wells were washed with pbs and were allowed to bind to 100 nm gelsolin diluted in selection buffer for 2 hrs at room temperature . the wells were then washed 4 times with pbs containing 0 . 1 % tween20 ( pbs - t ) and incubated with anti - gelsolin antibodies overnight at 4 ° c . the plates were washed 4 times with pbs - t and incubated with secondary antibodies conjugated with horseradish peroxidase at room temperature for 30 min followed by detection using 1 - step ultra tmb substrate ( pierce , rockford , usa ). the reactions were stopped with stop solution ( 2 m h 2 so 4 ) after the blue color had developed and absorbance was read at 450 nm using a microplate reader . to determine the affinity of different aptamers for gelsolin , 1 ug of streptavidin was coated on a support using 100 mm nahco 3 buffer having ph 9 . 2 for 10 to 12 hours at a temperature of 4 degree c . the wells were washed with pbs and blocked with 3 % bsa in pbs for 2 hours followed by washing with pbs . the aptamers , l26f ( seq id no : 6 ), 10 . 10r ( seq id no : 7 ), l16f ( seq id no : 8 ) and l24f ( seq id no : 9 ) were immobilized at a concentration range of 100 nm - 1 . 56 nm on to elisa plates using tris - edta , ph 8 supplemented 2m nacl for 2 hrs . the wells were washed with pbs containing 0 . 1 % tween 20 and were allowed to bind to 100 nm gelsolin diluted in selection buffer for 2 hrs at room temperature . after washing the bound gelsolin was detected using anti - gelsolin antibodies and secondary antibodies using standard procedures as above . using non linear curve fitting of the plots ( fig3 ), the dissociation constant , k d , values of l26f , 10 . 10r , l16f and l24f for binding to gelsolin were calculated to be 15 . 3 , 7 . 2 , 12 . 7 and 15 . 7 nm , respectively . similar results were obtained upon using hydrophobic and / or hydrophilic surface containing elisa plates , confirming equal efficacy of the immobilized aptamers to bind gelsolin and its variants from the solution . recombinant gelsolin was coated at a concentration of 1 . 56 nm on to elisa plate in 100 mm nahco 3 buffer , ph 9 . 2 at 4 ° c . overnight . the wells were washed with pbs and blocked with 300 ul per well of 3 % bsa in pbs for 2 hrs at room temperature . the wells were washed with pbs and were incubated with 50 nm biotin - labeled aptamers diluted in selection buffer for 2 hrs at room temperature . after washing , the bound aptamers were detected using streptavidin - horseradish peroxidase using standard procedures ( fig4 ). selected aptamers were bound to sepharose cl - 2b beads was done using cnbr activation and coupling protocols described earlier ( arndt - jovin et al , 1975 ; kadonaga & amp ; tjian , 1986 ). briefly , the sepharose beads were activated or derivatized using cnbr and then washed with ice - cold double - distilled water and 10 mm potassium phosphate ( ph 8 ) and 5 ′- phosphorylated oligos were added to a thick slurry of the activated beads . the mixture was stirred at room temperature for 14 hours and washed with 1 m potassium phosphate ( ph 8 ), 1 m kcl . then the beads were washed with water , followed by 10 mm tris - hcl ( ph 8 ) containing 300 mm nacl , 1 mm edta and 0 . 02 % sodium azide . the functionalized beads were stored at 10 ° c . in the latter buffer . for gelsolin purification experiments , the slurry of beads were poured in a pd - 10 column and washed with three column volumes of 40 mm tris buffer ph 8 containing 100 mm nacl and 2 mm egta . to this column , cell lysate with ph adjusted to ph 8 and containing 2 mm egta was added . after initial loading , the column was washed with 40 mm tris buffer ph 8 containing 100 mm of nacl and 3 mm of cacl 2 ( three column volumes ). finally , elution was done by adding 40 mm tris buffer ph 8 containing 300 mm nacl and 20 mm cacl 2 . each fraction was analyzed for the presence of gelsolin . results ( fig5 ) support that gelsolin binds to dna - immobilized column efficiently and elutes upon sensing higher levels of ca 2 + ions . final loading and elution conditions are still being worked upon to bind and purify much higher levels of gelsolin . after elution of gelsolin , the dna - sepharose was washed with 40 mm sodium acetate buffer ph 4 containing 300 mm nacl . column was regenerated by washing with 40 mm tris buffer ph 8 containing 100 mm nacl and 2 mm egta , and a second round of purification process was attempted . after washing the columns with 0 . 8 m nacl , and washing the columns with 10 × volume of 40 mm tris buffer ph 8 containing 100 mm nacl and 2 mm egta , five rounds of purification were done with little loss in efficiency in purifying tagless gelsolin . similar purification profiles were obtained upon functionalizing the claimed aptamers on other matrices as tested on agarose , superdex , sephadex , sephacryl , sephacryl fast flow . selected aptamers bind to gelsolin and does not allow it to bind prion causing prp protein gelsolin binds to prp protein and accelerates prion formation ( unpublished work ). using structural data analysis and western blots we have confirmed that gelsolin directly binds to prp . later , we observed that the aptamers bind to gelsolin ( fig6 ) and does not allow it to bind prp , thus blocking the gelsolin &# 39 ; s role in prion disease progression . in these experiments , elisa was performed to check whether binding of aptamer ( 10 . 10r ) to gelsolin can inhibit the interaction of gelsolin with prp . monoclonal antibody against gelsolin was first coated in the wells to immobilize gelsolin ( 2 μg / well ). after washings , prp and aptamer + prp ( mixture ) were added . also , in some wells , aptamer followed by prp was added , so that the aptamer and prp in solution and can competitively inhibit their interaction with gelsolin . bound prp was detected using anti - prp antibody ( rabbit ; 1 : 1000 dilutions ), which was detected by anti - rabbit igg antibody ( 1 : 3000 ). as mentioned earlier , absorbance values at 450 nm indicated the extent of bound prp and results showed that aptamer 10 . 10r can inhibit the interaction of gelsolin and prp . though the use of administration of recombinant gelsolin / its fragments has been reported earlier for therapeutic purposes in disease / health conditions accompanied by hypogelsolinemia , no reliable and mass producible gelsolin estimation or diagnostic method is available for therapeutic purposes . additionally , an efficient protocol for mass production of gelsolin ( particularly for constructs lacking any tags or groups for minimal step purification ) remains a challenge . the developed dna aptamers can replace the need for antibodies or actin — bringing in cost - effectiveness and better reproducibility in production and protocol steps without any loss of sensitivity and specificity . the use of immobilized aptamers on chromatographic material to purify gelsolin ( or its variants ), particularly after overproduction of gelsolin or its variants will have substantial advantage in purifying gelsolin lacking any affinity tags like his - tag , gst , mbp etc . currently , minimal step purification of tagless - gelsolin is achieved by columns formed of immobilized anti - gelsolin antibodies . another methodology involves ammonium sulphate based precipitation of gelsolin followed by its affinity binding on deae column , but this method requires 4 - 5 steps and suffers from significant loss of protein and time , during recovery from precipitated state . arndt - jovin d j , jovin t m , bahr w , frischauf a m , marquardt m ( 1975 ) covalent attachment of dna to agarose . improved synthesis and use in affinity chromatography . european journal of biochemistry / febs 54 : 411 - 418 ashish , paine m s , perryman p b , yang l , yin h l , krueger j k ( 2007 ) global structure changes associated with ca2 + activation of full - length human plasma gelsolin . j biol chem 282 : 25884 - 25892 bucki r , byfield f j , kulakowska a , mccormick m e , drozdowski w , namiot z , hartung t , janmey p a ( 2008 ) extracellular gelsolin binds lipoteichoic acid and modulates cellular response to proinflammatory bacterial wall components . journal of immunology 181 : 4936 - 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