BACKGROUND
huntington's disease  is an inherited disorder caused by expansions of cag repeats  at the n-terminus, within exon  <dig>  of the hd protein.
the extent of polyglutamine expansion is correlated with the severity of the symptoms and their onset  <cit>  while the pathology of the disease and neuronal cell death are thought to be associated with protein misfolding and protein aggregation.
these aggregates are usually seen in the nucleus but can also be found in the cytoplasm  <cit> .
protein aggregates develop via a complex biochemical process with intermediates being visible during the process.
polyq tracts within the pathogenic range induce a protein insolubility whereas htt with nonpathogenic length maintains a measured degree of solubility  <cit> .
consistent with the aggregate toxicity hypothesis, inhibition of aggregate formation has been shown to have beneficial effects on the progression of hd in the r6/ <dig> mouse model  <cit> .
the implication of the polyq aggregates in cytotoxicity validates them as targets for novel therapeutics.
despite the lack of details surrounding the molecular structure of the polyq aggregates, high throughput screening for compounds that inhibit their formation have produced some promising results.
several compounds, including congo red  <cit>  and clioquinol  <cit> , have been reported to inhibit the aggregation process in the r6/ <dig> mouse model but their neurotoxicity tempers enthusiasm.
thus, identifying molecules that show efficacy with minimal toxicity should be an important consideration in the search for hd therapeutics.
synthetic oligonucleotides  provide a model category of reagents that meet some of these requirements.
oligonucleotides are synthetic polymers that are produced in highly purified quantities in a cost-effective way and the technology surrounding odn synthesis has advanced dramatically in the last  <dig> years.
recently, parekh-olmedo et al.
showed that certain classes of odns can inhibit aggregation.
one of these groups is the g-rich oligonucleotide  class which have been used previously as aptamers to block protein function.
specifically, gros have been shown to bind directly to stat <dig> and interact with regions of the protein that enable dimerization  <cit>  and in another instance, gros have been shown to block the integration of the hiv into the host chromosome by interacting with the hiv integrase  <cit> .
in both cases, the gro forms a structure known as a g-quartet which arises from the association of four adjacent g-bases assembled into a cyclic conformation.
these structures are stabilized by von hoogstein hydrogen bonding  <cit>  and by base stacking interactions.
these molecules exhibit a very compact structure which allows them to interact productively with functionally important protein domains.
much of the focus on developing therapeutics that block aggregate formation comes from a wealth of data associating hd pathogenesis with the presence of cellular inclusion bodies.
but, recent evidence from in vitro  <cit>  and in vivo  <cit>  studies suggest that htt inclusions may not be toxic to the cell or lead to neuronal degeneration.
in fact, hayden and colleagues have created an exciting mouse model that shows no long term effect of htt inclusions on behavior or viability  <cit> .
it may be true that inclusion bodies are neuroprotective and eliminating them may actually increase the potential for neurotoxicity.
because of the known biological activity of gros in interacting with specific protein domains, we tested this type of oligonucleotide in assays that measure the aggregation activity of mutant htt fragment.
we report that g-rich oligonucleotides, which form g-quartets, inhibit aggregation of mutant htt fragment in biochemical and cell-based assays.
overall, our results suggest that g-rich oligonucleotides could be used to examine the relationship between cellular aggregates and toxicity in various model systems.
RESULTS
biochemical analyses of gros
the original observation that oligonucleotides bearing random sequences reduced htt aggregate formation  <cit>  prompted a closer examination of a potential role for odns in hd therapy.
we chose to utilize a biochemical/immunochemical assay system that enables rapid screening of compounds/molecules for the inhibition of aggregation  <cit> .
in this test, oligonucleotides were mixed with purified mutant htt fragment for  <dig> hours and then passed through a cellulose acetate membrane filter.
the percentage of aggregates remaining on the filter was detected by immunochemistry using a primary htt-antibody and a secondary anti-rabbit antibody conjugated to peroxidase.
signals from the sds insoluble aggregates were scanned and quantified.
a diagram of this assay, established by wang et al.
, is presented in figure  <dig>  in all preparations of the mutant protein, thrombin-directed cleavage of gst-q58htn was allowed to proceed for  <dig> minutes prior to the addition of the gro.
this cleavage generates an amino terminal polyglutamine fragment consisting of  <dig> amino acids of the human huntingtin with tract of  <dig> glutamine residues  <cit> .
the fragment is fused to gst to enable purification.
we will utilize the wang et al  <cit>  terminology, gst-q58-htn to designate the protein used in this assay.
the mixture was centrifuged to remove any aggregates that had already formed.
western blot analyses have shown that >95% of the gst-q58-htn is cleaved to completion by the thrombin .
this parameter is an important control for our study since a variety of agents are known to block the enzymatic cleavage reaction directed by thrombin  <cit> .
two known gros were tested for inhibitory activity in the biochemical assay described above.
odn t <dig> and odn t <dig> were designed by jing et al.
<cit>  and used as aptamers to inhibit the function of stat <dig> protein.
both of these molecules have been determined by circular dichroism  and nmr to have an intramolecular g-quartet structure, and similar cd spectra were seen by our lab .
the sequence of each is provided in figure 2a; t <dig> contains  <dig>   <dig> bases in length while t <dig> contains  <dig>   <dig> bases in length.
these two gros were tested in the biochemical screen for inhibitory activity of htt fragment aggregation.
gst-q58-htn was mixed with either t <dig> or t <dig> following thrombin cleavage and centrifugation  and then incubated for  <dig> hours.
aggregation was stopped by adding sds and 2-mercaptoethanol and heating at 99°c.
the aggregates, retained on the membrane after filtration, were quantitated following immunoblotting using an image analyses program .
three control reactions, designated 0-hour, 24-hour and congo red , were repeated for each experiment.
the 0-hour control displays the amount of aggregation at the start of the reaction, usually none.
the 24-hour control reflects the amount of gst-q58-htn aggregation when no inhibitor is added to the mixture.
the third control displays the degree of aggregation formed in the presence of congo red, a known inhibitor  <cit>  functioning as the positive control in the series.
as shown in figure 2b, both t <dig> and t <dig> are capable of inhibiting gst-q58-htn aggregation with a dose response visible in the samples with t <dig>  figure  <dig> represents the results of five experiments conducted in duplicate, followed by quantitation using imagequant analytical software.
a statistically significant difference is observed between each gro and the 24-hour control in each experiment.
the effect of gros on aggregation prompted an examination of the activity of a monotonic guanosine oligonucleotide  because this molecule can also form a g-quartet.
we chose  <dig> bases as a compromised length of t <dig>  and t <dig>  to establish the hdg series.
when a dose range of hdg was tested in the biochemical assay, inhibition of aggregation q58-htn fragment was readily observed .
a significant decrease was seen at  <dig> μm, a much lower final concentration than the inhibitory level found with either t <dig> or t <dig>  hdg was found to be unique in its inhibitory activity compared to other monotonic oligonucleotides.
huang et al.
<cit>  demonstrated that the flow-through fraction of reactions containing inhibitors of aggregation is comprised predominantly of monomeric htt fragments.
to verify that the flow-through in reactions bearing hdg  <dig> contains mutant htt fragments, we captured this fraction and placed it on blotting paper.
stacked membranes to capture monomers using the same antibody used to detect aggregates.
as seen in the inset for figure  <dig>  a positive reaction was observed indicating the presence of mutant htt fragment.
when 20-mers of a , t  or c  were tested at  <dig> μm and  <dig> μm, no inhibition of aggregation was observed .
quantitation after scanning revealed a large, statistically significant difference in the activity of hdg compared to any of the other monotonic odns .
taken together, our results suggest that hdg, a 20-mer containing all g residues, is a powerful inhibitor of aggregation of q58-htn fragment based on the results of the immunoblotting assay.
cd measures differences in the absorbance of right-handed and left-handed circularly polarized light and can be used to investigate dna helicity.
g-quadruplexes can exist as antiparallel monomers, dimers or tetramers or as parallel tetramers.
traditionally, antiparallel conformations are characterized by a positive ellipticity maximum at  <dig> nm and a negative minimum at  <dig> nm.
in contrast, the parallel conformation is characterized by a positive maximum at  <dig> nm and a negative minimum at  <dig> nm; however, recent results have shown some antiparallel structures to have some positive maximums at  <dig> nm and negative minimums at  <dig> nm  <cit> .
hdg was characterized by cd in order to gain a perspective view of its structure.
hdg was analyzed along with hda and t <dig> at  <dig> μm in  <dig> mm kcl and at 24°c.
cd spectropolarity was determined using an aviv model  <dig> spectrometer with an effective range of analysis from  <dig> nm to  <dig> nm .
hda has an unusual maximum absorbance at  <dig> nm with a smaller positive absorbance at  <dig> nm.
t <dig> and hdg, however, exhibit maximum positive absorbances at  <dig> nm and negative minimums at  <dig> nm, a distinct profile that matches closely with molecules known to adopt g-quartet structures.
hdg is a more effective inhibitor of gst-q58-htn aggregation than t <dig> which is known to adopt a dimer basket g-quartet conformation  suggesting that hdg's structure is a more active conformation in our assays.
further structural studies are ongoing.
inhibition of aggregate formation in hek <dig> cells
since hdg exhibited strong inhibitory activity of gst-q58-htn in a biochemical assay, we tested this molecule in a cell-based assay.
the human embryonic kidney cell line hek <dig> was transfected with plasmid pcdna <dig> -72httexon1-egfp , a construct that contains a fusion gene uniting the first exon of the hd gene containing a polyq repeat of  <dig> codons and the egfp gene .
this fragment of huntingtin differs from gst-q58-htn in both length of polyglutamine stretch and that it is fused to egfp rather than gst.
when transfected into hek <dig> cells, the gene is expressed and aggregates appear within  <dig> hours, reflected by the appearance of discrete green foci.
cells were photographed first under white light to verify that equal numbers of cells were present for each treatment and a representative sample is shown.
egfp foci were then imaged in the presence or absence of plasmid p72q.
in figure  <dig>  green fluorescent foci are evident when p72q is present but a significant reduction is seen in cells that have also received congo red.
importantly, inhibition of aggregate formation is only partially inhibited when a lower dosage of congo red is present, demonstrating a dose-dependent effect.
in figure  <dig>  a cell population in which hda was co-transfected with p72q is presented; hda appears to have had little effect on the number of aggregates formed in these cells.
as is the case in the biochemical assay , hda does not appear to inhibit aggregate formation in hek <dig> cells.
figure  <dig> illustrates the effect of hdg on aggregate formation.
the white light photograph again reveals that hdg has no detectable toxic effect on cells or cell growth at  <dig> nm , but a clear dose effect is seen on the number of aggregates when the level of hdg is increased .
these observations confirm results obtained in the biochemical assay using hdg as the inhibitor.
finally, in figure  <dig>  a panel of photographs reveals once again that hdg is an effective inhibitor of aggregate formation but that this positive activity can be reduced significantly when t residues are inserted at the 7th and 14th position of the hdg 20-mer , every third base of the hdg 20-mer  or every other base of the hdg 20-mer .
to validate these results and to further explore the relationship between the cell-based, and biochemical assays, we assayed hdg 20/ <dig>  hdg 20/ <dig> and hdg 20/ <dig> individually for activity in the immunoblot assay .
these results confirm the low level of activity observed for hdg 20/ <dig>  hdg 20/ <dig> and hdg 20/ <dig> respectively in the cell-based assay .
the correlation between the results obtained in the cell-based and immunoblot assay reveal a similar mode of action for the oligonucleotides.
we have preliminary evidence that the reduction in aggregates observed in the cell-based assay can be confirmed when aggregates isolated from transfected cells are passed through the immunoblot assay .
aggregate reduction in response to the addition of hdg can also be seen using facs analysis as the readout.
again, hek <dig> cells were transfected with p72q with or without hdg  and the reactions were allowed to proceed for  <dig> hours.
the cells were then processed for facs and measured for green fluorescence.
the y axis reflects the degree or intensity of fluorescence.
as seen in figure 13a, the background is gated at the far left of the graphic whereas expression of p72q produces a sharp peak of green fluorescence near the right edge of the profile .
this peak represents aggregated htt-egfp, scored by facs as cells containing high intensity egfp .
in figures 13c and 13d, the profile of cells treated with hda is represented and little detectable change is observed in the peak at the far right edge.
even as the level of hda is increased from  <dig> μm to  <dig>  μm, no significant reduction in aggregates is observed.
in contrast, cells treated with hdg exhibit a very different profile  as the peak representing aggregates is diminished in a dose-dependent fashion.
thus, taken together, the data suggest that hdg can inhibit aggregation formation in hek <dig> cells expressing the htt-egfp fusion protein from plasmid p72q.
finally, a derivative pc <dig> cell line, htt14a <dig>   <cit> , was used to measure the capacity of hdg to improve cellular viability.
this neuronal cell line is used as a standard in the field for studying the survival phenotype associated with aggregate formation.
in this assay, a truncated form of htt exon  <dig>  fused to enhanced green fluorescent protein  is induced to express by addition of muristerone to the culture.
after induction, cell viability decreases rapidly between  <dig> hours and  <dig> hours, respectively, as measured by a celltiter-glo luminescent cell viability assay, as shown in figure  <dig>  the addition of increasing doses of hdg  appears to arrest the drop in viability providing some level of neuroprotection.
the differences in these are statistically significant and a larger, survival study is underway to confirm and/or expand upon these results.
discussions and
CONCLUSIONS
intracellular aggregates of htt have long been considered phenotypic evidence of the neurodegenerative disorder huntington's disease.
it is, however, not clear how the appearance of such inclusion bodies relates to the pathogenesis of the disease.
a number of model systems have been designed to screen for therapeutic agents that can inhibit aggregation.
some of these assays measure the inhibition of fusion protein aggregation, proteins containing a fragment of htt  and a marker/reporter protein, often egfp.
the htt component of this fusion protein harbors an expanded polyq stretch.
we have examined the capacity of g-rich oligonucleotides that can adopt a g-quartet conformation to block aggregation.
a well-established biochemical assay was used to examine gros blockage of aggregation.
molecules t <dig> and t <dig> that are known to form intermolecular g-quartets were found to be effective inhibitors of aggregation.
both of these gros, which were used previously to inhibit stat <dig>  adopt conventional g-quartet structure with the g residues  in the center and a loop domain at the top and bottom  <cit> .
the gro hdg, which exhibits the highest level of activity in the aggregation assays, can also adopt a stable g-quartet structure and further studies to elucidate the details of the g-quartet structure adopted by hdg are currently being performed.
this molecule also may prevent or delay neurotoxicity in pc <dig> cells.
the gro, hdg, is unique among monotonic oligonucleotides containing  <dig> bases.
none of the related 20-mers, hda, hdc or hdt show reproducible inhibitory activity in the biochemical or cell-based assays.
furthermore, hdg displays a dose response with concentrations as low as  <dig> μm exhibiting substantial levels of aggregate reduction.
it is effective when added at the start of the q58-htn aggregation reaction but much less so when added after the process has begun .
thus, it is likely to block the nucleation phase of aggregation rather than the elongation phase, although our experiments were not designed to discriminate between these two phases  <cit> .
hdg is also quite active in blocking aggregation of the httexon1-egfp fusion protein aggregation in hek <dig> cells.
in this system, the fusion protein is produced from an expression plasmid and co-transfection with hdg prevents the appearance of green fluorescent foci in a dose-dependent inhibition.
importantly, the well-known aggregation inhibitor, congo red, was used as a positive control and displayed effects similar to hdg.
mtt viability assays reveal no cell toxicity or negative effects on cell growth as a function of oligonucleotide addition .
this result is not surprising since odns used as antisense or antigene therapy have been found to be practically inert in human cells with regard to cytotoxicity.
a number of clinical trials using odns have taken place and while the efficacy of such treatments may be questioned, significant adverse effects on cells or patients were not observed.
the lack of serious side effects from oligonucleotides is a virtue in the development of these molecules for use in hd patients.
for example, while the effective levels for gro actvitiy presented in this work are higher than those used for traditional pharmaceuticals, oligonucleotides are particularly well-tolerated in humans.
the level herein is not unusual and levels exceeding  <dig> mg/kg have been found to be both efficacious and nontoxic in various antisense therapies.
the higher amounts may be required because delivery to target cells or penetration into the cells may be less efficient than other drug treatments.
the potency of the g-quartet structure of hdg is demonstrated further in the activity of the modified hdgs.
using a type of reverse genetics strategy, we created several "mutant" hdgs; hdg 20/ <dig> wherein each 7th g was replaced with a t, hdg 20/ <dig> wherein each 3rd g was replaced with a t and hdg 20/ <dig> wherein every other g was substituted with a t residue.
none of these molecules were found to be effective inhibitors of aggregation.
the results presented in figure  <dig> most clearly illustrate the importance of the hdg g-quartet structure while support for this notion is also gained when t <dig> could not fully substitute for hdg in the hek <dig> assay.
the mechanism by which hdg exhibits its effect in these assays is not clear, but several paths can be suggested.
g-quartets created either by natural processes or by exogenous addition, are reactive structures in the cell.
they were first recognized in studies aimed at understanding the mechanism of cellular aging  <cit>  occurring naturally near the ends of chromosomes.
these telomeric structures are composed of repetitive blocks of the double-stranded dna sequence n with the guanines forming an overhang at the end of the telomere.
in humans, these sequence blocks can become extremely long, increasing the probability that they adopt secondary  structures.
such structures are known to inhibit telomerase activity and may explain why telomerase replication/activity in transformed cells is often absent or reduced  <cit> .
g-quartets formed within gros have also been shown to inhibit protein dimerization of such molecules as stat <dig>  <cit> .
they exert their activity by binding to specific domains within stat <dig> with a high degree of precision.
since mutant htt aggregation relies on a nucleation phase in which the mutant protein begin to assemble, hdg could block the transition between nucleation and elongation as aggregation  begins.
alternatively, hdg could block other enzymes involved in the development of the pathogenic phenotype, such as caspases which cleave the native protein perhaps producing a toxic fragment  <cit> .
bates and colleagues have shown that certain gros can bind to nucleolin in a variety of cancer cells with a high degree of specificity  <cit> .
in all of these cases, direct interactions with cellular proteins would be required and such a reaction is a well-documented characteristic of gros.
studies are now underway to determine which proteins are binding to hdg and if there is a difference between the composition of protein-gro complexes in mutant and wild-type cells.

 huntington's disease is an inherited disorder caused by expansions of cag repeats at the n-terminus, within exon of the hd protein. the extent of polyglutamine expansion is correlated with the severity of the symptoms and their onset while the pathology of the disease and neuronal cell death are thought to be associated with protein misfolding and protein aggregation. these aggregates are usually seen in the nucleus but can also be found in the cytoplasm. protein aggregates develop via a complex biochemical process with intermediates being visible during the process. polyq tracts within the pathogenic range induce a protein insolubility whereas htt with nonpathogenic length maintains a measured degree of solubility. consistent with the aggregate toxicity hypothesis, inhibition of aggregate formation has been shown to have beneficial effects on the progression of hd in the r6/ mouse model. the implication of the polyq aggregates in cytotoxicity validates them as targets for novel therapeutics. despite the lack of details surrounding the molecular structure of the polyq aggregates, high throughput screening for compounds that inhibit their formation have produced some promising results. several compounds, including congo red and clioquinol, have been reported to inhibit the aggregation process in the r6/ mouse model but their neurotoxicity tempers enthusiasm. thus, identifying molecules that show efficacy with minimal toxicity should be an important consideration in the search for hd therapeutics. synthetic oligonucleotides provide a model category of reagents that meet some of these requirements. oligonucleotides are synthetic polymers that are produced in highly purified quantities in a cost-effective way and the technology surrounding odn synthesis has advanced dramatically in the last years. recently, parekh-olmedo et al. showed that certain classes of odns can inhibit aggregation. one of these groups is the g-rich oligonucleotide class which have been used previously as aptamers to block protein function. specifically, gros have been shown to bind directly to stat and interact with regions of the protein that enable dimerization and in another instance, gros have been shown to block the integration of the hiv into the host chromosome by interacting with the hiv integrase. in both cases, the gro forms a structure known as a g-quartet which arises from the association of four adjacent g-bases assembled into a cyclic conformation. these structures are stabilized by von hoogstein hydrogen bonding and by base stacking interactions. these molecules exhibit a very compact structure which allows them to interact productively with functionally important protein domains. much of the focus on developing therapeutics that block aggregate formation comes from a wealth of data associating hd pathogenesis with the presence of cellular inclusion bodies. but, recent evidence from in vitro and in vivo studies suggest that htt inclusions may not be toxic to the cell or lead to neuronal degeneration. in fact, hayden and colleagues have created an exciting mouse model that shows no long term effect of htt inclusions on behavior or viability. it may be true that inclusion bodies are neuroprotective and eliminating them may actually increase the potential for neurotoxicity. because of the known biological activity of gros in interacting with specific protein domains, we tested this type of oligonucleotide in assays that measure the aggregation activity of mutant htt fragment. we report that g-rich oligonucleotides, which form g-quartets, inhibit aggregation of mutant htt fragment in biochemical and cell-based assays. overall, our results suggest that g-rich oligonucleotides could be used to examine the relationship between cellular aggregates and toxicity in various model systems.  biochemical analyses of gros the original observation that oligonucleotides bearing random sequences reduced htt aggregate formation prompted a closer examination of a potential role for odns in hd therapy. we chose to utilize a biochemical/immunochemical assay system that enables rapid screening of compounds/molecules for the inhibition of aggregation. in this test, oligonucleotides were mixed with purified mutant htt fragment for hours and then passed through a cellulose acetate membrane filter. the percentage of aggregates remaining on the filter was detected by immunochemistry using a primary htt-antibody and a secondary anti-rabbit antibody conjugated to peroxidase. signals from the sds insoluble aggregates were scanned and quantified. a diagram of this assay, established by wang et al., is presented in figure in all preparations of the mutant protein, thrombin-directed cleavage of gst-q58htn was allowed to proceed for minutes prior to the addition of the gro. this cleavage generates an amino terminal polyglutamine fragment consisting of amino acids of the human huntingtin with tract of glutamine residues. the fragment is fused to gst to enable purification. we will utilize the wang et al terminology, gst-q58-htn to designate the protein used in this assay. the mixture was centrifuged to remove any aggregates that had already formed. western blot analyses have shown that >95% of the gst-q58-htn is cleaved to completion by the thrombin. this parameter is an important control for our study since a variety of agents are known to block the enzymatic cleavage reaction directed by thrombin. two known gros were tested for inhibitory activity in the biochemical assay described above. odn t and odn t were designed by jing et al. and used as aptamers to inhibit the function of stat protein. both of these molecules have been determined by circular dichroism and nmr to have an intramolecular g-quartet structure, and similar cd spectra were seen by our lab. the sequence of each is provided in figure 2a; t contains bases in length while t contains bases in length. these two gros were tested in the biochemical screen for inhibitory activity of htt fragment aggregation. gst-q58-htn was mixed with either t or t following thrombin cleavage and centrifugation and then incubated for hours. aggregation was stopped by adding sds and 2-mercaptoethanol and heating at 99°c. the aggregates, retained on the membrane after filtration, were quantitated following immunoblotting using an image analyses program. three control reactions, designated 0-hour, 24-hour and congo red, were repeated for each experiment. the 0-hour control displays the amount of aggregation at the start of the reaction, usually none. the 24-hour control reflects the amount of gst-q58-htn aggregation when no inhibitor is added to the mixture. the third control displays the degree of aggregation formed in the presence of congo red, a known inhibitor functioning as the positive control in the series. as shown in figure 2b, both t and t are capable of inhibiting gst-q58-htn aggregation with a dose response visible in the samples with t figure represents the results of five experiments conducted in duplicate, followed by quantitation using imagequant analytical software. a statistically significant difference is observed between each gro and the 24-hour control in each experiment. the effect of gros on aggregation prompted an examination of the activity of a monotonic guanosine oligonucleotide because this molecule can also form a g-quartet. we chose bases as a compromised length of t and t to establish the hdg series. when a dose range of hdg was tested in the biochemical assay, inhibition of aggregation q58-htn fragment was readily observed. a significant decrease was seen at μm, a much lower final concentration than the inhibitory level found with either t or t hdg was found to be unique in its inhibitory activity compared to other monotonic oligonucleotides. huang et al. demonstrated that the flow-through fraction of reactions containing inhibitors of aggregation is comprised predominantly of monomeric htt fragments. to verify that the flow-through in reactions bearing hdg contains mutant htt fragments, we captured this fraction and placed it on blotting paper. stacked membranes to capture monomers using the same antibody used to detect aggregates. as seen in the inset for figure a positive reaction was observed indicating the presence of mutant htt fragment. when 20-mers of a, t or c were tested at μm and μm, no inhibition of aggregation was observed. quantitation after scanning revealed a large, statistically significant difference in the activity of hdg compared to any of the other monotonic odns. taken together, our results suggest that hdg, a 20-mer containing all g residues, is a powerful inhibitor of aggregation of q58-htn fragment based on the results of the immunoblotting assay. cd measures differences in the absorbance of right-handed and left-handed circularly polarized light and can be used to investigate dna helicity. g-quadruplexes can exist as antiparallel monomers, dimers or tetramers or as parallel tetramers. traditionally, antiparallel conformations are characterized by a positive ellipticity maximum at nm and a negative minimum at nm. in contrast, the parallel conformation is characterized by a positive maximum at nm and a negative minimum at nm; however, recent results have shown some antiparallel structures to have some positive maximums at nm and negative minimums at nm. hdg was characterized by cd in order to gain a perspective view of its structure. hdg was analyzed along with hda and t at μm in mm kcl and at 24°c. cd spectropolarity was determined using an aviv model spectrometer with an effective range of analysis from nm to nm. hda has an unusual maximum absorbance at nm with a smaller positive absorbance at nm. t and hdg, however, exhibit maximum positive absorbances at nm and negative minimums at nm, a distinct profile that matches closely with molecules known to adopt g-quartet structures. hdg is a more effective inhibitor of gst-q58-htn aggregation than t which is known to adopt a dimer basket g-quartet conformation suggesting that hdg's structure is a more active conformation in our assays. further structural studies are ongoing. inhibition of aggregate formation in hek cells since hdg exhibited strong inhibitory activity of gst-q58-htn in a biochemical assay, we tested this molecule in a cell-based assay. the human embryonic kidney cell line hek was transfected with plasmid pcdna -72httexon1-egfp, a construct that contains a fusion gene uniting the first exon of the hd gene containing a polyq repeat of codons and the egfp gene. this fragment of huntingtin differs from gst-q58-htn in both length of polyglutamine stretch and that it is fused to egfp rather than gst. when transfected into hek cells, the gene is expressed and aggregates appear within hours, reflected by the appearance of discrete green foci. cells were photographed first under white light to verify that equal numbers of cells were present for each treatment and a representative sample is shown. egfp foci were then imaged in the presence or absence of plasmid p72q. in figure green fluorescent foci are evident when p72q is present but a significant reduction is seen in cells that have also received congo red. importantly, inhibition of aggregate formation is only partially inhibited when a lower dosage of congo red is present, demonstrating a dose-dependent effect. in figure a cell population in which hda was co-transfected with p72q is presented; hda appears to have had little effect on the number of aggregates formed in these cells. as is the case in the biochemical assay, hda does not appear to inhibit aggregate formation in hek cells. figure illustrates the effect of hdg on aggregate formation. the white light photograph again reveals that hdg has no detectable toxic effect on cells or cell growth at nm, but a clear dose effect is seen on the number of aggregates when the level of hdg is increased. these observations confirm results obtained in the biochemical assay using hdg as the inhibitor. finally, in figure a panel of photographs reveals once again that hdg is an effective inhibitor of aggregate formation but that this positive activity can be reduced significantly when t residues are inserted at the 7th and 14th position of the hdg 20-mer, every third base of the hdg 20-mer or every other base of the hdg 20-mer. to validate these results and to further explore the relationship between the cell-based, and biochemical assays, we assayed hdg 20/ hdg 20/ and hdg 20/ individually for activity in the immunoblot assay. these results confirm the low level of activity observed for hdg 20/ hdg 20/ and hdg 20/ respectively in the cell-based assay. the correlation between the results obtained in the cell-based and immunoblot assay reveal a similar mode of action for the oligonucleotides. we have preliminary evidence that the reduction in aggregates observed in the cell-based assay can be confirmed when aggregates isolated from transfected cells are passed through the immunoblot assay. aggregate reduction in response to the addition of hdg can also be seen using facs analysis as the readout. again, hek cells were transfected with p72q with or without hdg and the reactions were allowed to proceed for hours. the cells were then processed for facs and measured for green fluorescence. the y axis reflects the degree or intensity of fluorescence. as seen in figure 13a, the background is gated at the far left of the graphic whereas expression of p72q produces a sharp peak of green fluorescence near the right edge of the profile. this peak represents aggregated htt-egfp, scored by facs as cells containing high intensity egfp. in figures 13c and 13d, the profile of cells treated with hda is represented and little detectable change is observed in the peak at the far right edge. even as the level of hda is increased from μm to μm, no significant reduction in aggregates is observed. in contrast, cells treated with hdg exhibit a very different profile as the peak representing aggregates is diminished in a dose-dependent fashion. thus, taken together, the data suggest that hdg can inhibit aggregation formation in hek cells expressing the htt-egfp fusion protein from plasmid p72q. finally, a derivative pc cell line, htt14a, was used to measure the capacity of hdg to improve cellular viability. this neuronal cell line is used as a standard in the field for studying the survival phenotype associated with aggregate formation. in this assay, a truncated form of htt exon fused to enhanced green fluorescent protein is induced to express by addition of muristerone to the culture. after induction, cell viability decreases rapidly between hours and hours, respectively, as measured by a celltiter-glo luminescent cell viability assay, as shown in figure the addition of increasing doses of hdg appears to arrest the drop in viability providing some level of neuroprotection. the differences in these are statistically significant and a larger, survival study is underway to confirm and/or expand upon these results. discussions and  intracellular aggregates of htt have long been considered phenotypic evidence of the neurodegenerative disorder huntington's disease. it is, however, not clear how the appearance of such inclusion bodies relates to the pathogenesis of the disease. a number of model systems have been designed to screen for therapeutic agents that can inhibit aggregation. some of these assays measure the inhibition of fusion protein aggregation, proteins containing a fragment of htt and a marker/reporter protein, often egfp. the htt component of this fusion protein harbors an expanded polyq stretch. we have examined the capacity of g-rich oligonucleotides that can adopt a g-quartet conformation to block aggregation. a well-established biochemical assay was used to examine gros blockage of aggregation. molecules t and t that are known to form intermolecular g-quartets were found to be effective inhibitors of aggregation. both of these gros, which were used previously to inhibit stat adopt conventional g-quartet structure with the g residues in the center and a loop domain at the top and bottom. the gro hdg, which exhibits the highest level of activity in the aggregation assays, can also adopt a stable g-quartet structure and further studies to elucidate the details of the g-quartet structure adopted by hdg are currently being performed. this molecule also may prevent or delay neurotoxicity in pc cells. the gro, hdg, is unique among monotonic oligonucleotides containing bases. none of the related 20-mers, hda, hdc or hdt show reproducible inhibitory activity in the biochemical or cell-based assays. furthermore, hdg displays a dose response with concentrations as low as μm exhibiting substantial levels of aggregate reduction. it is effective when added at the start of the q58-htn aggregation reaction but much less so when added after the process has begun. thus, it is likely to block the nucleation phase of aggregation rather than the elongation phase, although our experiments were not designed to discriminate between these two phases. hdg is also quite active in blocking aggregation of the httexon1-egfp fusion protein aggregation in hek cells. in this system, the fusion protein is produced from an expression plasmid and co-transfection with hdg prevents the appearance of green fluorescent foci in a dose-dependent inhibition. importantly, the well-known aggregation inhibitor, congo red, was used as a positive control and displayed effects similar to hdg. mtt viability assays reveal no cell toxicity or negative effects on cell growth as a function of oligonucleotide addition. this result is not surprising since odns used as antisense or antigene therapy have been found to be practically inert in human cells with regard to cytotoxicity. a number of clinical trials using odns have taken place and while the efficacy of such treatments may be questioned, significant adverse effects on cells or patients were not observed. the lack of serious side effects from oligonucleotides is a virtue in the development of these molecules for use in hd patients. for example, while the effective levels for gro actvitiy presented in this work are higher than those used for traditional pharmaceuticals, oligonucleotides are particularly well-tolerated in humans. the level herein is not unusual and levels exceeding mg/kg have been found to be both efficacious and nontoxic in various antisense therapies. the higher amounts may be required because delivery to target cells or penetration into the cells may be less efficient than other drug treatments. the potency of the g-quartet structure of hdg is demonstrated further in the activity of the modified hdgs. using a type of reverse genetics strategy, we created several "mutant" hdgs; hdg 20/ wherein each 7th g was replaced with a t, hdg 20/ wherein each 3rd g was replaced with a t and hdg 20/ wherein every other g was substituted with a t residue. none of these molecules were found to be effective inhibitors of aggregation. the results presented in figure most clearly illustrate the importance of the hdg g-quartet structure while support for this notion is also gained when t could not fully substitute for hdg in the hek assay. the mechanism by which hdg exhibits its effect in these assays is not clear, but several paths can be suggested. g-quartets created either by natural processes or by exogenous addition, are reactive structures in the cell. they were first recognized in studies aimed at understanding the mechanism of cellular aging occurring naturally near the ends of chromosomes. these telomeric structures are composed of repetitive blocks of the double-stranded dna sequence n with the guanines forming an overhang at the end of the telomere. in humans, these sequence blocks can become extremely long, increasing the probability that they adopt secondary structures. such structures are known to inhibit telomerase activity and may explain why telomerase replication/activity in transformed cells is often absent or reduced. g-quartets formed within gros have also been shown to inhibit protein dimerization of such molecules as stat. they exert their activity by binding to specific domains within stat with a high degree of precision. since mutant htt aggregation relies on a nucleation phase in which the mutant protein begin to assemble, hdg could block the transition between nucleation and elongation as aggregation begins. alternatively, hdg could block other enzymes involved in the development of the pathogenic phenotype, such as caspases which cleave the native protein perhaps producing a toxic fragment. bates and colleagues have shown that certain gros can bind to nucleolin in a variety of cancer cells with a high degree of specificity. in all of these cases, direct interactions with cellular proteins would be required and such a reaction is a well-documented characteristic of gros. studies are now underway to determine which proteins are binding to hdg and if there is a difference between the composition of protein-gro complexes in mutant and wild-type cells. 
here, we report that short guanosine monotonic oligonucleotides capable of adopting a g-quartet structure, are effective inhibitors of aggregation. taken together, our results suggest that a monotonic g-oligonucleotide, capable of adopting a g-quartet conformation is an effective inhibitor of aggregation. subsequent testing in a cell-based assay revealed that hdg was an effective inhibitor of aggregation of a fusion protein, comprised of a mutant htt fragment and green fluorescent protein. this oligonucleotide can also enable cell survival in pc cells overexpressing a mutant htt fragment fusion gene. single-stranded dna oligonucleotides capable of forming stable g-quartets can inhibit aggregation of the mutant htt fragment protein. the pathogenesis of the disease appears to center on the development of protein aggregates that arise initially from the misfolding of the mutant hd protein. our results reveal a new class of agents that could be developed as a therapeutic approach for huntington's disease.
 huntington's disease is an inherited autosomal dominant genetic disorder in which neuronal tissue degenerates. the pathogenesis of the disease appears to center on the development of protein aggregates that arise initially from the misfolding of the mutant hd protein. mutant huntingtin is produced by hd genes that contain an increased number of glutamine codons within the first exon and this expansion leads to the production of a protein that misfolds. recent studies suggest that mutant htt can nucleate protein aggregation and interfere with a multitude of normal cellular functions.  as such, efforts to find a therapy for hd have focused on agents that disrupt or block the mutant htt aggregation pathway. here, we report that short guanosine monotonic oligonucleotides capable of adopting a g-quartet structure, are effective inhibitors of aggregation. by utilizing a biochemical/immunoblotting assay as an initial screen, we identified a 20-mer, all g-oligonucleotide as an active molecule. subsequent testing in a cell-based assay revealed that hdg was an effective inhibitor of aggregation of a fusion protein, comprised of a mutant htt fragment and green fluorescent protein. taken together, our results suggest that a monotonic g-oligonucleotide, capable of adopting a g-quartet conformation is an effective inhibitor of aggregation. this oligonucleotide can also enable cell survival in pc cells overexpressing a mutant htt fragment fusion gene.  single-stranded dna oligonucleotides capable of forming stable g-quartets can inhibit aggregation of the mutant htt fragment protein. this activity maybe an important part of the pathogenecity of huntington's disease. our results reveal a new class of agents that could be developed as a therapeutic approach for huntington's disease. 