patient - derived fibroblast cell lines gm04281 , gm06151 , gm04719 , gm04869 , gm04717 , gm04795 , and gm09197 were obtained from the coriell institute ( camden , nj ) .
cells were maintained at 37 c and 5% co2 in minimal essential media eagle ( mem ) ( sigma , m4655 ) supplemented with 10% heat inactivated fetal bovine serum ( sigma ) and 0.5% mem nonessential amino acids ( sigma ) .
cells were plated in 6-well plates at 60,000 cells / well in supplemented mem two days prior to transfection .
stock solutions of pna - peptide conjugates were heated at 65 c for 5 min before use to dissolve any aggregates that may have formed .
pna - peptide conjugates were diluted to the appropriate concentration using optimem ( invitrogen , carlsbad , ca ) and then added to cells .
after 24 h , the media containing pna - peptides were removed and replaced by fresh supplemented mem .
sirnas or lnas were transfected to cells using rnaimax ( invitrogen ) according to the manufacturer 's instructions .
the appropriate amount of the lipid ( 3 l for 100 nm oligonucleotides ) were added to optimem containing oligonucleotides and the oligonucleotide - lipid mixture ( 250 l ) were incubated for 20 min .
optimem was added to the mixture to a final volume of 1.25 ml and then added to cells .
the protein concentration in each sample was quantified with bca assay ( thermo scientific , waltham , ma ) .
sds - page ( separating gel : 5 % acrylamide - bisacrylamide/34.7:1 , 450 mm tris - acetate ph 8.8 ; stacking gel : 4% acrylamide - bisacrylamide/34.7:1 , 150 mm tris - acetate ph 6.8 ) ( xt tricine running buffer , bio - rad , hercules , ca ) was used to separate wild - type and mutant htt proteins .
gels were run at 70v for 15 min followed by 100v for 4 h. for separation of htt variants containing shorter cag repeats , gels were run at 70 v for 15 min , then 110 v for 6 h. the electrophoresis apparatus was placed in ice - water bath to prevent overheating of the running buffer .
we monitored expression of actin protein to ensure even loading on protein in each lane .
in parallel with analysis for htt expression , portions of each protein lysate sample were analyzed for -actin expression by sds - page ( 7.5% acrylamide pre - cast gels ; bio - rad ) .
these gels were run at 70v for 15 min followed by 100v for 1 h. after gel electrophoresis , proteins were transferred to membrane ( hybond - c extra ; ge healthcare bio - sciences , piscataway , nj ) . ataxin-3 and
-actin were analyzed by sds - page ( 7.5 % acrylamide pre - cast gels ; bio - rad ) .
primary antibodies specific for each protein were obtained and used at the indicated dilution ratio : anti - htt antibody ( mab2166 .
1:10000 ; chemicon ) , anti - ataxin-3 antibody ( mab5360 ; 1:10000 ; chemicon ) , and anti--actin antibody ( 1:10,000 ; sigma ) .
hrp conjugate anti - mouse or anti - rabbit secondary antibody ( 1:10000 and 1:5000 , respectively ; jackson immunoresearch laboratories , west grove pa ) was used for visualizing proteins using supersignal west pico chemiluminescent substrate ( thermo scientific ) .
protein bands were quantified using imagej software ( rasband , w.s . , imagej , u. s. national institutes of health , bethesda , maryland , usa , http://rsb.info.nih.gov/ij/ , 19972007 ) .
yac128 mice ( fvbn / nj background strain ) were obtained from jackson labs ( stock number 004938 ) .
the male yac128 mice were crossed to wild - type ( wt ) female fvbn / nj mice and p1-p2 pups were collected and genotyped by pcr .
the primary cultures of striatal medium spiny neurons ( msn ) were established from yac128 and control wild type pups .
after dissociation , neurons were plated on poly - l - lysine ( sigma ) coated 12 mm round coverslips ( assistent ) in neurobasal - a medium supplemented with 2% b27 , 1 mm glutamine and penicillin - streptomycin ( all from invitrogen ) and kept at 37c in a 5% co2 environment .
the 13-div msn were exposed for 7 h to 250 m glutamate in neurobasal - a added to the culture medium . immediately after the treatment with glutamate
, neurons were fixed for 30 min in 4% paraformaldehyde plus 4% sucrose in pbs ( ph 7.4 ) , permeabilized for 5 min in 0.25% triton - x-100 , and stained by using the deadend fluorometric tunel system ( promega ) .
nuclei were counterstained with 5 m propidium iodine ( pi ) ( molecular probes ) .
coverslips were extensively washed with pbs and mounted in mowiol 488 ( polysciences ) . for quantification six to eight
randomly chosen microscopic fields containing 100300 msn each were cell - counted for yac128 and wild type cultures .
the number of tunel - positive neuronal nuclei was calculated as a fraction of pi - positive neuronal nuclei in each microscopic field .
the fractions of tunel - positive nuclei determined for each microscopic field were averaged and the results are presented as means  se ( n= number of fields counted ) .
msn cells were supported in culture by surrounding glial cells , but only msn cells were counted during the neuroprotection assay .
pna conjugate rep19 was added at the concentration of 0.25 m and 0.5 m , 4 days before the glutamate application .
msn were exposed to 250 m glutamate at 13-div for 7h , fixed , permeabilized and analyzed by tunel staining and pi counterstaining .
the data are presented as mean  se ( n = 68 microscopic fields , 100300 msn per field ) . for protein assays , after the addition of pna rep19 at 9-div , the 13-div msn were washed with ice - cold pbs and solubilized for 30 min at 4c in extraction buffer a ( 1% chaps , 137 mm nacl , 2.7 mm kcl , 4.3 mm na2hpo4 , 1.4 mm kh2po4 [ ph 7.2 ] , 5 mm edta , 5 mm egta , and protease inhibitors ) . extracts were clarified by centrifugation for 20 min at 10000  g at 4c and the upper solutions were collected for sds - page . for selectively harvesting msn ,
cells were incubated with cell dissociation solution ( sigma ) for 4 min at 37c and neuron basal - a medium with 10% fbs were added .
caspase-3 activity was measured by hydrolysis of acetyl - asp - glu - val - asp p - nitroanilide ( ac - devd - pna ) according to the manufacture instruction in a colorimetric assay kit ( sigma ) .
cell pellet was re - suspended in 15 l of 1x lysis buffer without protease inhibitor and incubated for 30 min on ice .
50 g of cell extract was mixed with 2 mm ac - devd - pna substrate to a total volume of 100 l of 1x assay buffer in a 96-well plate and incubated at 37 c overnight .
the number of cag repeats was estimated according to the published mrna sequence in genebank .
tata box binding protein ( tbp ) ( ~19 cag repeats , nm_003194 ) , aak1 ( 6 cag repeats , nm_014911 ) , atn1 ( 15 cag repeats , nm_001940 ) , foxp2 ( ~40 caacag repeats , nm_148899 ) , and pou3f2 ( ~6 cag repeats , nm_005604 ) .
protein lysates were analyzed by sds - page ( 7.5% acrylamide pre - cast gels ; bio - rad ) followed by western blotting with anti - tbp antibody ( 1:2000 ; sigma ) , anti - aak1 antibody ( 1:1000 ; abcam , cambridge ma ) , anti - atn1 antibody ( 1:300 , affinity bioreagents , golden co ) , anti - foxp2 antibody ( 1:1000 , abcam ) , or anti - pou3f2 antibody ( 1:1000 ; abnova , taipei , taiwan ) .
each data plot from dose response experiments for inhibition of htt or ataxin-3 was fit to the following model equation , y=100(1x/(n+x ) ) where y is % inhibition of protein and x is concentration of synthetic oligomers .
m and n are fitting parameters , where n is taken as the ic50 value .
patient - derived fibroblast cell lines gm04281 , gm06151 , gm04719 , gm04869 , gm04717 , gm04795 , and gm09197 were obtained from the coriell institute ( camden , nj ) .
cells were maintained at 37 c and 5% co2 in minimal essential media eagle ( mem ) ( sigma , m4655 ) supplemented with 10% heat inactivated fetal bovine serum ( sigma ) and 0.5% mem nonessential amino acids ( sigma ) .
cells were plated in 6-well plates at 60,000 cells / well in supplemented mem two days prior to transfection .
stock solutions of pna - peptide conjugates were heated at 65 c for 5 min before use to dissolve any aggregates that may have formed .
pna - peptide conjugates were diluted to the appropriate concentration using optimem ( invitrogen , carlsbad , ca ) and then added to cells .
after 24 h , the media containing pna - peptides were removed and replaced by fresh supplemented mem .
sirnas or lnas were transfected to cells using rnaimax ( invitrogen ) according to the manufacturer 's instructions .
the appropriate amount of the lipid ( 3 l for 100 nm oligonucleotides ) were added to optimem containing oligonucleotides and the oligonucleotide - lipid mixture ( 250 l ) were incubated for 20 min .
optimem was added to the mixture to a final volume of 1.25 ml and then added to cells .
cells were harvested with trypsin - edta solution ( invitrogen ) and lysed . the protein concentration in each sample
sds - page ( separating gel : 5 % acrylamide - bisacrylamide/34.7:1 , 450 mm tris - acetate ph 8.8 ; stacking gel : 4% acrylamide - bisacrylamide/34.7:1 , 150 mm tris - acetate ph 6.8 ) ( xt tricine running buffer , bio - rad , hercules , ca ) was used to separate wild - type and mutant htt proteins .
gels were run at 70v for 15 min followed by 100v for 4 h. for separation of htt variants containing shorter cag repeats , gels were run at 70 v for 15 min , then 110 v for 6 h. the electrophoresis apparatus was placed in ice - water bath to prevent overheating of the running buffer .
we monitored expression of actin protein to ensure even loading on protein in each lane . in parallel with analysis for htt expression , portions of each protein lysate sample were analyzed for -actin expression by sds - page ( 7.5% acrylamide pre - cast gels ; bio - rad ) .
these gels were run at 70v for 15 min followed by 100v for 1 h. after gel electrophoresis , proteins were transferred to membrane ( hybond - c extra ; ge healthcare bio - sciences , piscataway , nj ) . ataxin-3 and
-actin were analyzed by sds - page ( 7.5 % acrylamide pre - cast gels ; bio - rad ) .
primary antibodies specific for each protein were obtained and used at the indicated dilution ratio : anti - htt antibody ( mab2166 .
1:10000 ; chemicon ) , anti - ataxin-3 antibody ( mab5360 ; 1:10000 ; chemicon ) , and anti--actin antibody ( 1:10,000 ; sigma ) .
hrp conjugate anti - mouse or anti - rabbit secondary antibody ( 1:10000 and 1:5000 , respectively ; jackson immunoresearch laboratories , west grove pa ) was used for visualizing proteins using supersignal west pico chemiluminescent substrate ( thermo scientific ) .
protein bands were quantified using imagej software ( rasband , w.s . , imagej , u. s. national institutes of health , bethesda , maryland , usa , http://rsb.info.nih.gov/ij/ , 19972007 ) .
yac128 mice ( fvbn / nj background strain ) were obtained from jackson labs ( stock number 004938 ) . the male yac128 mice were crossed to wild - type ( wt ) female fvbn / nj mice and p1-p2 pups were collected and genotyped by pcr .
the primary cultures of striatal medium spiny neurons ( msn ) were established from yac128 and control wild type pups .
after dissociation , neurons were plated on poly - l - lysine ( sigma ) coated 12 mm round coverslips ( assistent ) in neurobasal - a medium supplemented with 2% b27 , 1 mm glutamine and penicillin - streptomycin ( all from invitrogen ) and kept at 37c in a 5% co2 environment .
the 13-div msn were exposed for 7 h to 250 m glutamate in neurobasal - a added to the culture medium . immediately after the treatment with glutamate , neurons were fixed for 30 min in 4% paraformaldehyde plus 4% sucrose in pbs ( ph 7.4 ) , permeabilized for 5 min in 0.25% triton - x-100 , and stained by using the deadend fluorometric tunel system ( promega ) .
nuclei were counterstained with 5 m propidium iodine ( pi ) ( molecular probes ) .
coverslips were extensively washed with pbs and mounted in mowiol 488 ( polysciences ) . for quantification six to eight
randomly chosen microscopic fields containing 100300 msn each were cell - counted for yac128 and wild type cultures .
the number of tunel - positive neuronal nuclei was calculated as a fraction of pi - positive neuronal nuclei in each microscopic field .
the fractions of tunel - positive nuclei determined for each microscopic field were averaged and the results are presented as means  se ( n= number of fields counted ) .
msn cells were supported in culture by surrounding glial cells , but only msn cells were counted during the neuroprotection assay .
pna conjugate rep19 was added at the concentration of 0.25 m and 0.5 m , 4 days before the glutamate application .
msn were exposed to 250 m glutamate at 13-div for 7h , fixed , permeabilized and analyzed by tunel staining and pi counterstaining .
the data are presented as mean  se ( n = 68 microscopic fields , 100300 msn per field ) . for protein assays , after the addition of pna rep19 at 9-div , the 13-div msn were washed with ice - cold pbs and solubilized for 30 min at 4c in extraction buffer a ( 1% chaps , 137 mm nacl , 2.7 mm kcl , 4.3 mm na2hpo4 , 1.4 mm kh2po4 [ ph 7.2 ] , 5 mm edta , 5 mm egta , and protease inhibitors ) . extracts were clarified by centrifugation for 20 min at 10000  g at 4c and the upper solutions were collected for sds - page . for selectively harvesting msn ,
cells were incubated with cell dissociation solution ( sigma ) for 4 min at 37c and neuron basal - a medium with 10% fbs were added .
caspase-3 activity was measured by hydrolysis of acetyl - asp - glu - val - asp p - nitroanilide ( ac - devd - pna ) according to the manufacture instruction in a colorimetric assay kit ( sigma ) .
cell pellet was re - suspended in 15 l of 1x lysis buffer without protease inhibitor and incubated for 30 min on ice .
50 g of cell extract was mixed with 2 mm ac - devd - pna substrate to a total volume of 100 l of 1x assay buffer in a 96-well plate and incubated at 37 c overnight .
the number of cag repeats was estimated according to the published mrna sequence in genebank .
tata box binding protein ( tbp ) ( ~19 cag repeats , nm_003194 ) , aak1 ( 6 cag repeats , nm_014911 ) , atn1 ( 15 cag repeats , nm_001940 ) , foxp2 ( ~40 caacag repeats , nm_148899 ) , and pou3f2 ( ~6 cag repeats , nm_005604 ) .
protein lysates were analyzed by sds - page ( 7.5% acrylamide pre - cast gels ; bio - rad ) followed by western blotting with anti - tbp antibody ( 1:2000 ; sigma ) , anti - aak1 antibody ( 1:1000 ; abcam , cambridge ma ) , anti - atn1 antibody ( 1:300 , affinity bioreagents , golden co ) , anti - foxp2 antibody ( 1:1000 , abcam ) , or anti - pou3f2 antibody ( 1:1000 ; abnova , taipei , taiwan ) .
each data plot from dose response experiments for inhibition of htt or ataxin-3 was fit to the following model equation , y=100(1x/(n+x ) ) where y is % inhibition of protein and x is concentration of synthetic oligomers .
m and n are fitting parameters , where n is taken as the ic50 value .
 many neurological disorders are caused by expanded trinucleotide repeats1 , including machado - joseph disease ( mjd)2 and huntington disease ( hd)3 . mjd and hd are caused by expanded cag repeats within the ataxin-3 ( atxn3 ) and huntingtin ( htt ) genes . inhibiting expression of atxn3 or htt are promising therapeutic strategies , but indiscriminant inhibition of wild - type and mutant alleles may lead to toxicity . 
 we hypothesized that expanded triplet repeat mrna might be preferentially recognized by complementary oligomers . 
 we observe selective inhibition of mutant ataxin-3 and htt protein expression by peptide nucleic acid ( pna ) and locked nucleic acid ( lna ) oligomers targeting cag repeats . 
 duplex rnas were less selective , suggesting an advantage for single - stranded oligomers . 
 inhibiting mutant htt expression protected cultured striatal neurons from an hd mouse model against glutamate - induced toxicity . 
 antisense oligomers that discriminate between wild - type and mutant genes on the basis of repeat length offer new options for treating mjd , hd , and other hereditary diseases . 
METHODS
Cell Culture and Transfection
Analysis of HTT and ataxin-3 expression
Neuronal cell glutamate susceptibility assay
Caspase-3 activity assay
Analysis of TBP, AAK1, ATN1, FOX2P, and POU3F2
Statistical analysis and curve fitting
Supplementary Material