recently designed and synthesized high - performance modified - nucleic - acids ( hipernas ) such as 2-o - methyl rna ( 2-ome ) , 2-o - methoxyethyl rna ( moe ) , and 2,4-bridged nucleic acid / locked nucleic acid ( 2,4-bna / lna ) have improved performance compared to phosphorothioate antisense oligonucleotides ( aons ) . hipernas overcome the systemic antisense effects of these earlier antisense oligonucleotides and show promise as antisense therapeutics for the treatment of a variety of diseases [ 15 ] . however , more potent and less toxic aons are required , since several clinical trials of aon drugs carrying hipernas have been recently terminated due to the lack of efficacy or because of safety concerns . in addition , we previously described a unique modified nucleic acid , 2,4-bridged nucleic acid ( 2,4-bna ; also known as lna ) [ 9 , 10 ] . its high therapeutic efficacy is based on the extraordinarily high target binding of the original 2,4-bna / lna - based aon . 2,4-bna / lna - based aon is widely accepted as one of the most promising antisense drugs , so fine - tuning the structure of bna is the key for further improving the therapeutic potency and toxicological properties of aon . in this context , seth and coworkers developed bna analogues and elucidated their potency and safety in vivo . they reported bna or lna analogues with 2,4-bna / lna - like binding affinities and biological activities with increased nuclease resistance and reduced toxicity [ 1114 ] . we have also reported the synthesis and physicochemical properties of several novel bnas , including 2,4-bna and 2,4-bna [ 1517 ] , and recently demonstrated the biological activity of a 2,4-bna -based aon . . a wide range of functional groups can be easily introduced , and the introduction of nitrogen atoms would improve the stability of the duplex by reducing repulsions between phosphates in the backbone [ 18 , 19 ] ( figure 1 ) . 2,4-bna - modified oligonucleotides have very high rna affinity , similar to or even higher than their 2,4-bna / lna counterparts . moreover , 2,4-bna - modified oligonucleotides are more resistant to endonucleolytic cleavage by nucleases than their 2,4-bna / lna counterparts . although there is limited information regarding the biological activity or therapeutic potency of 2,4-bna - based aons , we have demonstrated the high systemic effects and safety of 2,4-bna / lna- and 2,4-bna - based 20-nucleotide - long ( 20-nt - long ) aons that target pcsk9 mrna . additionally , prakashet al . independently showed the high potency and the nontoxicity of 2,4-bna - based aons . straarup et al . recently shortened the length of 2,4-bna / lna - based phosphorothioate aons to eliminate the latent potency of 2,4-bna / lna drugs . these short phosphorothioate aons contain central 6- to 10-nt - long dna regions flanked by terminal 2- to 4-nt - long 2,4-bna / lna segments . these short ( 12- to 14-nt ) aons would be beneficial in terms of target specificity . the introduction of only small numbers of modifications into short 2,4-bna / lna - based aons can greatly increase target affinity . thus , short 2,4-bna / lna phosphorothioate aons can minimize length - dependent disadvantages such as phosphorothioate - related protein binding and rnase h inactivation [ 2123 ] while maintaining satisfactory affinity and specificity . additionally , short aons are easier to produce on a bulk scale and could exhibit more drug - like characteristics . based on the assumption that the strand - shortening strategy is also applicable to 2,4-bna - based aons , we shortened 2,4-bna - based phosphorothioate aons and directly compared their silencing activities against the corresponding 2,4-bna / lna - based apolipoprotein b ( apob)-targeting aons . a series of 2,4-bna / lna - based antisense 10- to 20-nucleotide - long phosphorothioate gapmers reported previously by straarup et al .   were prepared and used in this study . these aons were designed with complementary target sites for both cynomolgus monkey and human apob mrna sequences . the 10- to 16-nt - long aons can also target murine apob mrna ( genbank accession number nm_000384 and nm_009693 for human and mouse apob mrna , resp . ) . additionally , we prepared 2,4-bna - based counterparts in which all the 2,4-bna / lnas were substituted by 2,4-bna . the synthesis of 2,4-bna with pyrimidine bases was previously reported [ 16 , 17 ] ; the synthesis of 2,4-bna with purine bases is currently being optimized and will be reported elsewhere . ( ibaraki , osaka , japan ) using standard phosphoramidite procedures and purified using hplc . uv melting experiments were carried out using a shimazu uv-1650 spectrometer equipped with a tm analysis accessory . equimolar amounts of two single - stranded oligonucleotides were dissolved in 10  mm sodium phosphate buffer ( ph 7.2 ) containing 100  mm nacl to give a final strand concentration of 4.0  m . the mixture was annealed by heating at 90c followed by slow cooling to room temperature . the melting profile was recorded at 260  nm in the forward and reverse direction from 5 to 90c at a scan rate of 0.5c / min . for aon transfection experiments , huh-7 cells were seeded at 15  10 cells per well in 12-well plates . aons were transfected by using lipofectamine 2000 ( invitrogen , carlsbad , ca ) according to the manufacturer 's procedures . after a 4-hour transfection , cells were washed with pbs , fresh medium was added , and the cells were incubated for an additional 20 hours at 37c . after incubation , cells were collected and subjected to analyses . total rna was isolated from cultured cells using an rneasy mini kit ( qiagen ) according to the manufacturer 's procedure . gene expression was evaluated by a two - step quantitative reverse transcription - pcr method . reverse transcription of rna samples was performed by using a high capacity cdna reverse - transcription kit ( applied biosystems , foster city , ca ) , and quantitative pcr was performed using a fast taqman gene expression assay ( applied biosystems ) . the following primer sets were used for quantitative pcr . for human apob and gapdh , assay ids of hs01071209_m1 and hs02758991_g1 two days after transfection , the cultures were subjected to centrifugation at 4c , 10,000  rpm for 15  min . each supernatant was collected into an amicon ultra-4 centrifugal filter ultracel pl-10k ( millipore ) and centrifuged at 4c at 3,000  rpm for 1  h , and then each supernatant was added to individual vivaspin 500 units ( sartorius stedim biotech ) and centrifuged at 4c at 3,000  rpm for 0.5  h. each sample ( 9  l ) was added to 9  l of novex tris - glycine sds sample buffer ( 2x ) ( invitrogen ) and applied to a 38% nupage tris - acetate gel ( invitrogen ) . the separated proteins were transferred to a pvdf membrane ( millipore ) at 220  ma for 120  min . membranes were then incubated with 10  ml of blocking buffer ( blocking one ; nacalai tesque ) for 12  h at 4c . membranes were successively incubated with primary antibody of anti - human apob antibody ( r&d systems ) for 80  min at room temperature . then , each membrane was washed with pbs containing 0.1% tween ( pbst ) 4 times . membranes were incubated with goat anti - mouse igg - hrp antibody ( santa cruz biotechnology ) for 80  min at room temperature . chemiluminescent detection was performed using an ecl advance western blot detection kit ( amersham biosciences ) according to the manufacture 's procedure . application of linear regression techniques to plots of the expression of apob mrna levels versus the logarithm of transfection concentration allows estimation of the coefficient ( slope ) and intercept values using the following equation : apob mrna = coefficientlog ( concentration ) + intercept linear regression analysis was applied to 500 bootstrap sample sets obtained from three independent cellular assays . the coefficient and intercept of each regression line were compared using tukey 's test or one - sample t - test . to better understand the effect of strand shortening on 2,4-bna - based aons , a series of 2,4-bna - based antisense oligonucleotides of 10- to 20-nt - long phosphorothioate gapmers and 2,4-bna / lna - counterparts were synthesized ( table 1 ) . formation of a stable duplex with the target mrna is a minimum essential step of the onset of an antisense effect . we first evaluated the thermal stability of the duplex formed between each modified aon and the single - stranded 20-nt - long oligoribonucleotide complementary to apob - lna-20 and apob - nc-20 . as expected , the longer the strand or the greater the number of modifications , the higher the tm value . moreover , the tm values of 2,4-bna - based aons surpassed those of their 2,4-bna / lna - based counterparts for any given length ( table 1 ) , in good agreement with previous reports [ 11 , 17 ] . note that the exact tm values of lnas in table 1 are different from those given by straarup et al . , due to differences in composition of the measurement buffer solutions and in the length of the complementary rnas between the two studies . we next used in vitro mrna silencing assays to estimate the potency of 2,4-bna - based aons and to compare their potency directly to the corresponding 2,4-bna / lna - based aons . we used the huh-7 human hepatoma cell line , which expresses high levels of apob mrna in cells and secrets its protein into the medium . all the aons , except the 10-mers , apob - lna-10 , and apob - nc-10 , reduced apob mrna and protein expression ( and hence secreted protein ) levels in a dose - dependent manner in the cells and culture medium , respectively , ( figures 2(a ) and 2(b ) ) . apob - lna-10 did not reduce apob mrna levels even at concentrations above 64 nm . this may be because apob - lna-10 did not bind target mrna at 37c due to lack of affinity . apob - nc-10 also did not reduce apob mrna expression , despite its higher tm value compared to that of apob - lna-10 . application of linear regression techniques to plots of the expression of apob mrna levels versus the logarithm of transfection concentration allowed estimation of the coefficient ( slope ) and intercept values . statistical comparison of these parameters between arms with the identical length would provide useful guides for the efficacies of the aons . one - sample t - tests revealed that the coefficients ( slopes ) of the 10-mer aons had statistically insignificant downward slopes , suggesting that these 10-mers have little or no silencing effect ( figure 3 ) . a length - dependent decrease in potency in 14- to 20-nt - long 2,4-bna / lna and 2,4-bna - based aons was observed ( table 1 and figure 1 ) . in each series of aons , the apob - lna-14 and apob - nc-14 were the most potent , whereas the apob - lna-13 was the most potent aon reported by straarup et al . . ic50 values of the 15- , 14- , 13- , and 12-mer aons were so close to each other ( 0.5  nm ) that straarup et al . confirmed the order by conducting an in vivo silencing study and showed that 12- and 13-mer aons are the most potent . in contrast , we confirmed the order of in vitro silencing effects of all the entries in table 1 by using larger 12-well culture dishes . these differences in experimental conditions ( in vivo versus in vitro ) may explain the differences in the ic50 values of 2,4-bna / lna - based aons and their order of potency . nevertheless , we observed in both chemistries that shorter aons ( 16- , 14- , and 13-mer ) are statistically significantly more potent than 20-mer aons . because the 10-mers did not show any activity , activity is positively correlated with binding affinity and indicates the presence of a  threshold affinity . this suggests the presence of a  length penalty  [ 20 , 22 ] and the presence of an  optimal affinity ,  which might be a more appropriate description than threshold affinity . the variables that independently govern  length penalty  and  optimal affinity  remain largely unknown . however , plasma and intracellular proteins preferentially bind to the phosphorothioate internucleotide linkages , and these linkages are known to control rnase h activity [ 2326 ] . thus , the number of phosphorothioate linkages is partly related to the  length penalty . surprisingly , when compared to the identical length 2,4-bna / lna - based counterpart , the corresponding 2,4-bna - based aon showed stronger inhibitory activity . the differential in inhibitory activity is more pronounced in shorter ( 13- , 14- , and 16-mers ) aons than in the 20-mer ( table 1 and figure 4 ) . indeed , as shown in figure 3 , statistical comparison using the tukey test of the coefficients and intercepts of the regression lines , which indicate how efficiently and strongly aons reduce apob mrna , revealed that 2,4-bna - based 13- , 14- , and 16-mers exhibit significantly stronger inhibitory activity than their 2,4-bna / lna counterparts . these potency differences could not be explained simply by the higher tm of the 2,4-bna - based aons compared to their 2,4-bna / lna - based counterpart . 2,4-bna - based aons might exhibit less  length penalty  than their 2,4-bna / lna - based counterpart . a weaker affinity for protein binding or lower rnase h inhibitory activity of 2,4-bna - based aons could cause this potency difference . on the other hand , figure 4 shows a plot of ic50 versus tm , in which apob - nc-20 , which has the highest affinity , exhibited worse ic50 value than apob - lna-20 . these findings suggest that very high affinity possibly undermines antisense potency , although binding affinity to the target generally correlates positively with potency in the case of traditional small molecule drugs . the precise onset mechanism giving rise to this phenomenon remains unclear due to the lack of experimental data . however , the u- or v - shaped curves in figure 4 clearly indicate that the potency of aons is the result of a delicate balance of reward term and penalty term . thus , fine adjustment to the optimal affinity and elimination of the penalties such as excess protein binding and rnase h inhibitory activity could result in superior efficacy , represented as the lowest point on the u- or v - shaped curve .   in conclusion , we have shown that 2,4-bna - based aons targeting apob mrna have higher binding affinities to the target rna than do 2,4-bna / lna - based aons . additionally , in vitro transfection studies revealed the superior silencing effect of short 2,4-bna - based aons ( < 20-nt - long ) , indicating that 2,4-bna may have advantageous properties as short antisense drugs . we are currently investigating the potential and safety of 2,4-bna - based aons as therapeutic drugs .
the duplex stability with target mrna and the gene silencing potential of a novel bridged nucleic acid analogue are described . 
 the analogue , 2,4-bnanc antisense oligonucleotides ( aons ) ranging from 10- to 20-nt - long , targeted apolipoprotein b. 2,4-bnanc was directly compared to its conventional bridged ( or locked ) nucleic acid ( 2,4-bna / lna)-based counterparts . 
 melting temperatures of duplexes formed between 2,4-bnanc - based antisense oligonucleotides and the target mrna surpassed those of 2,4-bna / lna - based counterparts at all lengths . 
 an in vitro transfection study revealed that when compared to the identical length 2,4-bna / lna - based counterpart , the corresponding 2,4-bnanc - based antisense oligonucleotide showed significantly stronger inhibitory activity . 
 this inhibitory activity was more pronounced in shorter ( 13- , 14- , and 16-mer ) oligonucleotides . on the other hand , the 2,4-bnanc - based 20-mer aon exhibited the highest affinity but the worst ic50 value , indicating that very high affinity may undermine antisense potency . 
 these results suggest that the potency of aons requires a balance between reward term and penalty term . 
 balance of these two parameters would depend on affinity , length , and the specific chemistry of the aon , and fine - tuning of this balance could lead to improved potency . 
 we demonstrate that 2,4-bnanc may be a better alternative to conventional 2,4-bna / lna , even for  short  antisense oligonucleotides , which are attractive in terms of drug - likeness and cost - effective bulk production .