Patent Application: US-22502302-A

Abstract:
the present invention concerns methods and reagents useful in modulating hiv gene expression in a variety of applications , including use in therapeutic , diagnostic , target validation , and genomic discovery applications . specifically , the invention relates to small interfering rna molecules capable of mediating rna interference against hiv polypeptide and polynucleotide targets .

Description:
[ 0135 ] fig1 shows a non - limiting example of a scheme for the synthesis of sirna molecules . the complimentary sirna sequence strands , strand 1 and strand 2 , are synthesized in tandem and are connected by a cleavable linkage , such as a nucleotide succinate or abasic succinate , which can be the same or different from the cleavable linker used for solid phase synthesis on a solid support . the synthesis can be either solid phase or solution phase , in the example shown , the synthesis is a solid phase synthesis . the synthesis is performed such that a protecting group , such as a dimethoxytrityl group , remains intact on the terminal nucleotide of the tandem oligonucleotide . upon cleavage and deprotection of the oligonucleotide , the two sirna strands spontaneously hybridize to form a sirna duplex , which allows the purification of the duplex by utilizing the properties of the terminal protecting group , for example by applying a trityl on purification method wherein only duplexes / oligonucleotides with the terminal protecting group are isolated . [ 0136 ] fig2 shows a maldi - tov mass spectrum of a purified sirna duplex synthesized by a method of the invention . the two peaks shown correspond to the predicted mass of the separate sirna sequence strands . this result demonstrates that the sirna duplex generated from tandem synthesis can be purified as a single entity using a simple trityl - on purification methodology . [ 0137 ] fig3 shows a non - limiting proposed mechanistic representation of target rna degradation involved in rnai . double stranded rna ( dsrna ), which is generated by rna dependent rna polymerase ( rdrp ) from foreign single stranded rna , for example viral , transposon , or other exogenous rna , activates the dicer enzyme which in turn generates sirna duplexes having terminal phosphate groups ( p ). an active sirna complex forms which recognizes a target rna , resulting in degradation of the target rna by the risc endonuclease complex or in the synthesis of additional rna by rna dependent rna polymerase ( rdrp ), which can activate dicer and result in additional sirna molecules , thereby amplifying the rnai response . [ 0138 ] fig4 shows non - limiting examples of chemically modified sirna constructs of the present invention . in the figure , n stands for any nucleotide ( adenosine , guanosine , cytosine , uridine , or optionally thymidine , for example thymidine can be substituted in the overhanging regions designated by parenthesis ( n n ). various modifications are shown for the sense and antisense strands of the sirna constructs . a the sense strand comprises 21 nucleotides having four phosphorothioate 5 ′ and 3 ′- terminal internucleotide linkages , wherein the two terminal 3 ′- nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2 ′- o - methyl modified nucleotides except for ( n n ) nucleotides , which can comprise naturally occurring ribonucleotides , deoxynucleotides , universal bases , or other chemical modifications described herein . the antisense strand comprises 21 nucleotides , wherein the two terminal 3 ′- nucleotides are optionally complimentary to the target rna sequence , and having one 3 ′- terminal phosphorothioate internucleotide linkage and four 5 ′- terminal phosphorothioate internucleotide linkages and wherein all pyrimidine nucleotides that may be present are 2 ′- deoxy - 2 ′- fluoro modified nucleotides except for ( n n ) nucleotides , which can comprise naturally occurring ribonucleotides , deoxynucleotides , universal bases , or other chemical modifications described herein . b the sense strand comprises 21 nucleotides wherein the two terminal 3 ′- nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2 ′- o - methyl modified nucleotides except for ( n n ) nucleotides , which can comprise naturally occurring ribonucleotides , deoxynucleotides , universal bases , or other chemical modifications described herein . the antisense strand comprises 21 nucleotides , wherein the two terminal 3 ′- nucleotides are optionally complimentary to the target rna sequence , and wherein all pyrimidine nucleotides that may be present are 2 ′- deoxy - 2 ′- fluoro modified nucleotides except for ( n n ) nucleotides , which can comprise naturally occurring ribonucleotides , deoxynucleotides , universal bases , or other chemical modifications described herein . c the sense strand comprises 21 nucleotides having 5 ′- and 3 ′- terminal cap moieties wherein the two terminal 3 ′- nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2 ′- o - methyl modified nucleotides except for ( n n ) nucleotides , which can comprise naturally occurring ribonucleotides , deoxynucleotides , universal bases , or other chemical modifications described herein . the antisense strand comprises 21 nucleotides , wherein the two terminal 3 ′- nucleotides are optionally complimentary to the target rna sequence , and wherein all pyrimidine nucleotides that may be present are 2 ′- deoxy - 2 ′- fluoro modified nucleotides except for ( n n ) nucleotides , which can comprise naturally occurring ribonucleotides , deoxynucleotides , universal bases , or other chemical modifications described herein . d the sense strand comprises 21 nucleotides having five phosphorothioate 5 ′ and 3 ′- terminal internucleotide linkages , wherein the two terminal 3 ′- nucleotides are optionally base paired and wherein all nucleotides are ribonucleotides except for ( n n ) nucleotides , which can comprise naturally occurring ribonucleotides , deoxynucleotides , universal bases , or other chemical modifications described herein . the antisense strand comprises 21 nucleotides , wherein the two terminal 3 ′- nucleotides are optionally complimentary to the target rna sequence , and having one 3 ′- terminal phosphorothioate internucleotide linkage and five 5 ′- terminal phosphorothioate internucleotide linkages and wherein all nucleotides are ribonucleotides except for ( n n ) nucleotides , which can comprise naturally occurring ribonucleotides , deoxynucleotides , universal bases , or other chemical modifications described herein . e the sense strand comprises 21 nucleotides wherein the two terminal 3 ′- nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2 ′- o - methyl nucleotides except for ( n n ) nucleotides , which can comprise naturally occurring ribonucleotides , deoxynucleotides , universal bases , or other chemical modifications described herein . the antisense strand comprises 21 nucleotides all having phosphorothioate internucleotide linkages , wherein the two terminal 3 ′- nucleotides are optionally complimentary to the target rna sequence , and wherein all nucleotides are ribonucleotides except for ( n n ) nucleotides , which can comprise naturally occurring ribonucleotides , deoxynucleotides , universal bases , or other chemical modifications described herein . f the sense strand comprises 21 nucleotides having 5 ′- and 3 ′- terminal cap moieties , wherein the two terminal 3 ′- nucleotides are optionally base paired and wherein all pyrimidine nucleotides that may be present are 2 ′- o - methyl nucleotides except for ( n n ) nucleotides , which can comprise naturally occurring ribonucleotides , deoxynucleotides , universal bases , or other chemical modifications described herein . the antisense strand comprises 21 nucleotides , wherein the two terminal 3 ′- nucleotides are optionally complimentary to the target rna sequence , and having one 3 ′- terminal phosphorothioate internucleotide linkage and wherein all pyrimidine nucleotides that may be present are 2 ′- deoxy - 2 ′- fluoro nucleotides except for ( n n ) nucleotides , which can comprise naturally occurring ribonucleotides , deoxynucleotides , universal bases , or other chemical modifications described herein . the antisense strand of constructs a - f comprise sequence complimentary to target rna sequence of the invention . [ 0139 ] fig5 shows non - limiting examples of specific chemically modified sirna sequences of the invention . a - f applies the chemical modifications described in fig4 a - f to a hiv sirna sequence . [ 0140 ] fig6 shows non - limiting examples of different sirna constructs of the invention . the examples shown ( constructs 1 , 2 , and 3 ) have 19 representative base pairs , however , different embodiments of the invention include any number of base pairs described herein . bracketed regions represent nucleotide overhangs , for example comprising between about 1 , 2 , 3 , or 4 nucleotides in length , preferably about 2 nucleotides . constructs 1 and 2 can be used independently for rnai activity . construct 2 can comprise a polynucleotide or non - nucleotide linker , which can optionally be designed as a biodegradable linker . in one embodiment , the loop structure shown in construct 2 can comprise a biodegradable linker that results in the formation of construct 1 in vivo and / or in vitro . in another example , construct 3 can be used to generate construct 2 under the same principle wherein a linker is used to generate the active sirna construct 2 in vivo and / or in vitro , which can optionally utilize another biodegradable linker to generate the active sirna construct 1 in vivo and / or in vitro . as such , the stability and / or activity of the sirna constructs can be modulated based on the design of the sirna construct for use in vivo or in vitro and / or in vitro . [ 0141 ] fig7 is a diagrammatic representation of a scheme utilized in generating an expression cassette to generate sirna hairpin constructs . ( a ) a dna oligomer is synthesized with a 5 ′- restriction site ( r1 ) sequence followed by a region having sequence identical ( sense region of sirna ) to a predetermined hiv target sequence , wherein the sense region comprises , for example , about 19 , 20 , 21 , or 22 nucleotides ( n ) in length , which is followed by a loop sequence of defined sequence ( x ), comprising , for example , between about 3 and 10 nucleotides . ( b ) the synthetic construct is then extended by dna polymerase to generate a hairpin structure having self complementary sequence that will result in a sirna transcript having specificity for an hiv target sequence and having self complementary sense and antisense regions . ( c ) the construct is heated ( for example to about 95 ° c .) to linearize the sequence , thus allowing extension of a complementary second dna strand using a primer to the 3 ′- restriction sequence of the first strand . the double stranded dna is then inserted into an appropriate vector for expression in cells . the construct can be designed such that a 3 ′- overhang results from the transcription , for example by engineering restriction sites and / or utilizing a poly - u termination region as described in paul et al ., 2002 , nature biotechnology , 29 , 505 - 508 . [ 0142 ] fig8 is a diagrammatic representation of a scheme utilized in generating an expression cassette to generate double stranded sirna constructs . ( a ) a dna oligomer is synthesized with a 5 ′- restriction ( r1 ) site sequence followed by a region having sequence identical ( sense region of sirna ) to a predetermined hiv target sequence , wherein the sense region comprises , for example , about 19 , 20 , 21 , or 22 nucleotides ( n ) in length , and which is followed by a 3 ′- restriction site ( r2 ) which is adjacent to a loop sequence of defined sequence ( x ). ( b ) the synthetic construct is then extended by dna polymerase to generate a hairpin structure having self complementary sequence . ( c ) the construct is processed by restriction enzymes specific to r1 and r2 to generate a double stranded dna which is then inserted into an appropriate vector for expression in cells . the transcription cassette is designed such that a u6 promoter region flanks each side of the dsdna which generates the separate sense and antisense strands of the sirna . poly t termination sequences can be added to the constructs to generate u overhangs in the resulting transcript . [ 0143 ] fig9 is a diagrammatic representation of a method used to determine target sites for sirna mediated rnai within a particular target nucleic acid sequence , such as messenger rna . ( a ) a pool of sirna oligonucleotides are synthesized wherein the antisense region of the sirna constructs has complementarity to target sites across the target nucleic acid sequence , and wherein the sense region comprises sequence complementary to the antisense region of the sirna . ( b ) the sequences are pooled and are inserted into vectors such that ( c ) transfection of a vector into cells results in the expression of the sirna . ( d ) cells are sorted based on phenotypic change that is associated with modulation of the target nucleic acid sequence . ( e ) the sirna is isolated from the sorted cells and is sequenced to identify efficacious target sites within the target nucleic acid sequence . rna interference refers to the process of sequence specific post transcriptional gene silencing in animals mediated by short interfering rnas ( sirna ) ( fire et al ., 1998 , nature , 391 , 806 ). the corresponding process in plants is commonly referred to as post transcriptional gene silencing or rna silencing and is also referred to as quelling in fungi . the process of post transcriptional gene silencing is thought to be an evolutionarily conserved cellular defense mechanism used to prevent the expression of foreign genes which is commonly shared by diverse flora and phyla ( fire et al ., 1999 , trends genet ., 15 , 358 ). such protection from foreign gene expression may have evolved in response to the production of double stranded rnas ( dsrna ) derived from viral infection or the random integration of transposon elements into a host genome via a cellular response that specifically destroys homologous single stranded rna or viral genomic rna . the presence of dsrna in cells triggers the rnai response though a mechanism that has yet to be fully characterized . this mechanism appears to be different from the interferon response that results from dsrna mediated activation of protein kinase pkr and 2 ′, 5 ′- oligoadenylate synthetase resulting in non - specific cleavage of mrna by ribonuclease l . the presence of long dsrnas in cells stimulates the activity of a ribonuclease iii enzyme referred to as dicer . dicer is involved in the processing of the dsrna into short pieces of dsrna known as short interfering rnas ( sirna ) ( berstein et al ., 2001 , nature , 409 , 363 ). short interfering rnas derived from dicer activity are typically about 21 to about 23 ( i . e ., about 21 , 22 or 23 ) nucleotides in length and comprise about 19 base pair duplexes . dicer has also been implicated in the excision of 21 and 22 nucleotide small temporal rnas ( strna ) from precursor rna of conserved structure that are implicated in translational control ( hutvagner et al ., 2001 , science , 293 , 834 ). the rnai response also features an endonuclease complex containing a sirna , commonly referred to as an rna - induced silencing complex ( risc ), which mediates cleavage of single stranded rna having sequence homologous to the sirna . cleavage of the target rna takes place in the middle of the region complementary to the guide sequence of the sirna duplex ( elbashir et al ., 2001 , genes dev ., 15 , 188 ). short interfering rna mediated rnai has been studied in a variety of systems . fire et al ., 1998 , nature , 391 , 806 , were the first to observe rnai in c . elegans . wianny and goetz , 1999 , nature cell biol ., 2 , 70 , describes rnai mediated by dsrna in mouse embryos . hammond et al ., 2000 , nature , 404 , 293 , describe rnai in drosophila cells transfected with dsrna . elbashir et al ., 2001 , nature , 411 , 494 , describe rnai induced by introduction of duplexes of synthetic 21 - nucleotide rnas in cultured mammalian cells including human embryonic kidney and hela cells . recent work in drosophila embryonic lysates has revealed certain requirements for sirna length , structure , chemical composition , and sequence that are essential to mediate efficient rnai activity . these studies have shown that 21 nucleotide sirna duplexes are most active when containing two nucleotide 3 ′- overhangs . furthermore , substitution of one or both sirna strands with 2 ′- deoxy or 2 ′- o - methyl nucleotides abolishes rnai activity , whereas substitution of 3 ′- terminal sirna nucleotides with deoxy nucleotides was shown to be tolerated . mismatch sequences in the center of the sirna duplex were also shown to abolish rnai activity . in addition , these studies also indicate that the position of the cleavage site in the target rna is defined by the 5 ′- end of the sirna guide sequence rather than the 3 ′- end ( elbashir et al ., 2001 , embo j ., 20 , 6877 ). other studies have indicated that a 5 ′- phosphate on the target - complementary strand of a sirna duplex is required for sirna activity and that atp is utilized to maintain the 5 ′- phosphate moiety on the sirna ( nykanen et al ., 2001 , cell , 107 , 309 ), however sirna molecules lacking a 5 ′- phosphate are active when introduced exogenously , suggesting that 5 ′- phosphorylation of sirna constructs may occur in vivo . synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods , and the therapeutic cost of such molecules is prohibitive . in this invention , small nucleic acid motifs (“ small ” refers to nucleic acid motifs no more than 100 nucleotides in length , preferably no more than 80 nucleotides in length , and most preferably no more than 50 nucleotides in length ; e . g ., individual sirna oligonucleotide sequences or sirna sequences synthesized in tandem ) are preferably used for exogenous delivery . the simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of protein and / or rna structure . exemplary molecules of the instant invention are chemically synthesized , and others can similarly be synthesized . oligonucleotides ( e . g ., certain modified oligonucleotides or portions of oligonucleotides lacking ribonucleotides ) are synthesized using protocols known in the art , for example as described in caruthers et al ., 1992 , methods in enzymology 211 , 3 - 19 , thompson et al ., international pct publication no . wo 99 / 54459 , wincott et al ., 1995 , nucleic acids res . 23 , 2677 - 2684 , wincott et al ., 1997 , methods mol . bio ., 74 , 59 , brennan et al ., 1998 , biotechnol bioeng ., 61 , 33 - 45 , and brennan , u . s . pat . no . 6 , 001 , 311 . all of these references are incorporated herein by reference . the synthesis of oligonucleotides makes use of common nucleic acid protecting and coupling groups , such as dimethoxytrityl at the 5 ′- end , and phosphoramidites at the 3 ′- end . in a non - limiting example , small scale syntheses are conducted on a 394 applied biosystems , inc . synthesizer using a 0 . 2 μmol scale protocol with a 2 . 5 min coupling step for 2 ′- o - methylated nucleotides and a 45 sec coupling step for 2 ′- deoxy nucleotides or 2 ′- deoxy - 2 ′- fluoro nucleotides . table ii outlines the amounts and the contact times of the reagents used in the synthesis cycle . alternatively , syntheses at the 0 . 2 μmol scale can be performed on a 96 - well plate synthesizer , such as the instrument produced by protogene ( palo alto , calif .) with minimal modification to the cycle . a 33 - fold excess ( 60 μl of 0 . 11 m = 6 . 6 μmol ) of 2 ′- o - methyl phosphoramidite and a 105 - fold excess of s - ethyl tetrazole ( 60 μl of 0 . 25 m = 15 μmol ) can be used in each coupling cycle of 2 ′- o - methyl residues relative to polymer - bound 5 ′- hydroxyl . a 22 - fold excess ( 40 μl of 0 . 11 m = 4 . 4 μmol ) of deoxy phosphoramidite and a 70 - fold excess of s - ethyl tetrazole ( 40 μl of 0 . 25 m = 10 μmol ) can be used in each coupling cycle of deoxy residues relative to polymer - bound 5 ′- hydroxyl . average coupling yields on the 394 applied biosystems , inc . synthesizer , determined by colorimetric quantitation of the trityl fractions , are typically 97 . 5 - 99 %. other oligonucleotide synthesis reagents for the 394 applied biosystems , inc . synthesizer include the following : detritylation solution is 3 % tca in methylene chloride ( abi ); capping is performed with 16 % n - methyl imidazole in thf ( abi ) and 10 % acetic anhydride / 10 % 2 , 6 - lutidine in thf ( abi ); and oxidation solution is 16 . 9 mm i 2 , 49 mm pyridine , 9 % water in thf ( perseptive ™). burdick & amp ; jackson synthesis grade acetonitrile is used directly from the reagent bottle . s - ethyltetrazole solution ( 0 . 25 m in acetonitrile ) is made up from the solid obtained from american international chemical , inc . alternately , for the introduction of phosphorothioate linkages , beaucage reagent ( 3h - 1 , 2 - benzodithiol - 3 - one 1 , 1 - dioxide , 0 . 05 m in acetonitrile ) is used . deprotection of the dna - based oligonucleotides is performed as follows : the polymer - bound trityl - on oligoribonucleotide is transferred to a 4 ml glass screw top vial and suspended in a solution of 40 % aq . methylamine ( 1 ml ) at 65 ° c . for 10 min . after cooling to − 20 ° c ., the supernatant is removed from the polymer support . the support is washed three times with 1 . 0 ml of etoh : mecn : h2o / 3 : 1 : 1 , vortexed and the supernatant is then added to the first supernatant . the combined supernatants , containing the oligoribonucleotide , are dried to a white powder . the method of synthesis used for rna including certain sirna molecules of the invention follows the procedure as described in usman et al ., 1987 , j . am . chem . soc ., 109 , 7845 ; scaringe et al ., 1990 , nucleic acids res ., 18 , 5433 ; and wincott et al ., 1995 , nucleic acids res . 23 , 2677 - 2684 wincott et al ., 1997 , methods mol . bio ., 74 , 59 , and makes use of common nucleic acid protecting and coupling groups , such as dimethoxytrityl at the 5 ′- end , and phosphoramidites at the 3 ′- end . in a non - limiting example , small scale syntheses are conducted on a 394 applied biosystems , inc . synthesizer using a 0 . 2 μmol scale protocol with a 7 . 5 min coupling step for alkylsilyl protected nucleotides and a 2 . 5 min coupling step for 2 ′- o - methylated nucleotides . table ii outlines the amounts and the contact times of the reagents used in the synthesis cycle . alternatively , syntheses at the 0 . 2 μmol scale can be done on a 96 - well plate synthesizer , such as the instrument produced by protogene ( palo alto , calif .) with minimal modification to the cycle . a 33 - fold excess ( 60 μl of 0 . 11 m = 6 . 6 μmol ) of 2 ′- o - methyl phosphoramidite and a 75 - fold excess of s - ethyl tetrazole ( 60 μl of 0 . 25 m = 15 μmol ) can be used in each coupling cycle of 2 ′- o - methyl residues relative to polymer - bound 5 ′- hydroxyl . a 66 - fold excess ( 120 μl of 0 . 11 m = 13 . 2 μmol ) of alkylsilyl ( ribo ) protected phosphoramidite and a 150 - fold excess of s - ethyl tetrazole ( 120 μl of 0 . 25 m = 30 μmol ) can be used in each coupling cycle of ribo residues relative to polymer - bound 5 ′- hydroxyl . average coupling yields on the 394 applied biosystems , inc . synthesizer , determined by colorimetric quantitation of the trityl fractions , are typically 97 . 5 - 99 %. other oligonucleotide synthesis reagents for the 394 applied biosystems , inc . synthesizer include the following : detritylation solution is 3 % tca in methylene chloride ( abi ); capping is performed with 16 % n - methyl imidazole in thf ( abi ) and 10 % acetic anhydride / 10 % 2 , 6 - lutidine in thf ( abi ); oxidation solution is 16 . 9 mm i 2 , 49 mm pyridine , 9 % water in thf ( perseptive ™). burdick & amp ; jackson synthesis grade acetonitrile is used directly from the reagent bottle . s - ethyltetrazole solution ( 0 . 25 m in acetonitrile ) is made up from the solid obtained from american international chemical , inc . alternately , for the introduction of phosphorothioate linkages , beaucage reagent ( 3h - 1 , 2 - benzodithiol - 3 - one 1 , 1 - dioxide 0 . 05 m in acetonitrile ) is used . deprotection of the rna is performed using either a two - pot or one - pot protocol . for the two - pot protocol , the polymer - bound trityl - on oligoribonucleotide is transferred to a 4 ml glass screw top vial and suspended in a solution of 40 % aq . methylamine ( 1 ml ) at 65 ° c . for 10 min . after cooling to − 20 ° c ., the supernatant is removed from the polymer support . the support is washed three times with 1 . 0 ml of etoh : mecn : h2o / 3 : 1 : 1 , vortexed and the supernatant is then added to the first supernatant . the combined supernatants , containing the oligoribonucleotide , are dried to a white powder . the base deprotected oligoribonucleotide is resuspended in anhydrous tea / hf / nmp solution ( 300 μl of a solution of 1 . 5 ml n - methylpyrrolidinone , 750 μl tea and 1 ml tea . 3hf to provide a 1 . 4 m hf concentration ) and heated to 65 ° c . after 1 . 5 h , the oligomer is quenched with 1 . 5 m nh 4 hco 3 . alternatively , for the one - pot protocol , the polymer - bound trityl - on oligoribonucleotide is transferred to a 4 ml glass screw top vial and suspended in a solution of 33 % ethanolic methylamine / dmso : 1 / 1 ( 0 . 8 ml ) at 65 ° c . for 15 min . the vial is brought to r . t . tea . 3hf ( 0 . 1 ml ) is added and the vial is heated at 65 ° c . for 15 min . the sample is cooled at − 20 ° c . and then quenched with 1 . 5 m nh 4 hco 3 . for purification of the trityl - on oligomers , the quenched nh 4 hco 3 solution is loaded onto a c - 18 containing cartridge that had been prewashed with acetonitrile followed by 50 mm teaa . after washing the loaded cartridge with water , the rna is detritylated with 0 . 5 % tfa for 13 min . the cartridge is then washed again with water , salt exchanged with 1 m nacl and washed with water again . the oligonucleotide is then eluted with 30 % acetonitrile . the average stepwise coupling yields are typically & gt ; 98 % ( wincott et al ., 1995 nucleic acids res . 23 , 2677 - 2684 ). those of ordinary skill in the art will recognize that the scale of synthesis can be adapted to be larger or smaller than the example described above including but not limited to 96 - well format , all that is important is the ratio of chemicals used in the reaction . alternatively , the nucleic acid molecules of the present invention can be synthesized separately and joined together post - synthetically , for example , by ligation ( moore et al ., 1992 , science 256 , 9923 ; draper et al ., international pct publication no . wo 93 / 23569 ; shabarova et al ., 1991 , nucleic acids research 19 , 4247 ; bellon et al ., 1997 , nucleosides & amp ; nucleotides , 16 , 951 ; bellon et al ., 1997 , bioconjugate chem . 8 , 204 ), or by hybridization following synthesis and / or deprotection . the sirna molecules of the invention can also be synthesized via a tandem synthesis methodology as described in example 1 herein , wherein both sirna strands are synthesized as a contiguous oligonucleotide sequence separated by a cleavable linker which is subsequently cleaved to provide separate sirna sequences that hybridize and permit purification of the sirna duplex . the tandem synthesis of sirna as described herein can be readily adapted to both multiwell / multiplate synthesis platforms such as 96 well or similarly larger multi - well platforms . the tandem synthesis of sirna as described herein can also be readily adapted to large scale synthesis platforms employing batch reactors , synthesis columns and the like . the nucleic acid molecules of the present invention can be modified extensively to enhance stability by modification with nuclease resistant groups , for example , 2 ′- amino , 2 ′- c - allyl , 2 ′- flouro , 2 ′- o - methyl , 2 ′- h ( for a review see usman and cedergren , 1992 , tibs 17 , 34 ; usman et al ., 1994 , nucleic acids symp . ser . 31 , 163 ). sirna constructs can be purified by gel electrophoresis using general methods or can be purified by high pressure liquid chromatography ( hplc ; see wincott et al ., supra , the totality of which is hereby incorporated herein by reference ) and re - suspended in water . in another aspect of the invention , sirna molecules of the invention are expressed from transcription units inserted into dna or rna vectors . the recombinant vectors can be dna plasmids or viral vectors . sirna expressing viral vectors can be constructed based on , but not limited to , adeno - associated virus , retrovirus , adenovirus , or alphavirus . the recombinant vectors capable of expressing the sirna molecules can be delivered as described herein , and persist in target cells . alternatively , viral vectors can be used that provide for transient expression of sirna molecules . chemically synthesizing nucleic acid molecules with modifications ( base , sugar and / or phosphate ) can prevent their degradation by serum ribonucleases , which can increase their potency ( see e . g ., eckstein et al ., international publication no . wo 92 / 07065 ; perrault et al ., 1990 nature 344 , 565 ; pieken et al ., 1991 , science 253 , 314 ; usman and cedergren , 1992 , trends in biochem . sci . 17 , 334 ; usman et al ., international publication no . wo 93 / 15187 ; and rossi et al ., international publication no . wo 91 / 03162 ; sproat , u . s . pat . no . 5 , 334 , 711 ; gold et al ., u . s . pat . no . 6 , 300 , 074 ; and burgin et al ., supra ; all of which are incorporated by reference herein ). all of the above references describe various chemical modifications that can be made to the base , phosphate and / or sugar moieties of the nucleic acid molecules described herein . modifications that enhance their efficacy in cells , and removal of bases from nucleic acid molecules to shorten oligonucleotide synthesis times and reduce chemical requirements are desired . there are several examples in the art describing sugar , base and phosphate modifications that can be introduced into nucleic acid molecules with significant enhancement in their nuclease stability and efficacy . for example , oligonucleotides are modified to enhance stability and / or enhance biological activity by modification with nuclease resistant groups , for example , 2 ′- amino , 2 ′- c - allyl , 2 ′- flouro , 2 ′- o - methyl , 2 ′- o - allyl , 2 ′- h , nucleotide base modifications ( for a review see usman and cedergren , 1992 , tibs . 17 , 34 ; usman et al ., 1994 , nucleic acids symp . ser . 31 , 163 ; burgin et al ., 1996 , biochemistry , 35 , 14090 ). sugar modification of nucleic acid molecules have been extensively described in the art ( see eckstein et al ., international publication pct no . wo 92 / 07065 ; perrault et al . nature , 1990 , 344 , 565 - 568 ; pieken et al . science , 1991 , 253 , 314 - 317 ; usman and cedergren , trends in biochem . sci ., 1992 , 17 , 334 - 339 ; usman et al . international publication pct no . wo 93 / 15187 ; sproat , u . s . pat . no . 5 , 334 , 711 and beigelman et al ., 1995 , j . biol . chem ., 270 , 25702 ; beigelman et al ., international pct publication no . wo 97 / 26270 ; beigelman et al ., u . s . pat . no . 5 , 716 , 824 ; usman et al ., u . s . pat . no . 5 , 627 , 053 ; woolf et al ., international pct publication no . wo 98 / 13526 ; thompson et al ., u . s . ser . no . 60 / 082 , 404 which was filed on apr . 20 , 1998 ; karpeisky et al ., 1998 , tetrahedron lett ., 39 , 1131 ; earnshaw and gait , 1998 , biopolymers ( nucleic acid sciences ), 48 , 39 - 55 ; verma and eckstein , 1998 , annu . rev . biochem ., 67 , 99 - 134 ; and burlina et al ., 1997 , bioorg . med . chem ., 5 , 1999 - 2010 ; all of the references are hereby incorporated in their totality by reference herein ). such publications describe general methods and strategies to determine the location of incorporation of sugar , base and / or phosphate modifications and the like into nucleic acid molecules without modulating catalysis , and are incorporated by reference herein . in view of such teachings , similar modifications can be used as described herein to modify the sirna nucleic acid molecules of the instant invention so long as the ability of sirna to promote rnai is cells is not significantly inhibited . while chemical modification of oligonucleotide internucleotide linkages with phosphorothioate , phosphorothioate , and / or 5 ′- methylphosphonate linkages improves stability , excessive modifications can cause some toxicity or decreased activity . therefore , when designing nucleic acid molecules , the amount of these internucleotide linkages should be minimized . the reduction in the concentration of these linkages should lower toxicity , resulting in increased efficacy and higher specificity of these molecules . small interfering rna ( sirna ) molecules having chemical modifications that maintain or enhance activity are provided . such a nucleic acid is also generally more resistant to nucleases than an unmodified nucleic acid . accordingly , the in vitro and / or in vivo activity should not be significantly lowered . in cases in which modulation is the goal , therapeutic nucleic acid molecules delivered exogenously should optimally be stable within cells until translation of the target rna has been modulated long enough to reduce the levels of the undesirable protein . this period of time varies between hours to days depending upon the disease state . improvements in the chemical synthesis of rna and dna ( wincott et al ., 1995 nucleic acids res . 23 , 2677 ; caruthers et al ., 1992 , methods in enzymology 211 , 3 - 19 ( incorporated by reference herein )) have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability , as described above . in one embodiment , nucleic acid molecules of the invention include one or more , for example , about 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 or more g - clamp nucleotides . a g - clamp nucleotide is a modified cytosine analog wherein the modifications confer the ability to hydrogen bond both watson - crick and hoogsteen faces of a complementary guanine within a duplex , see for example lin and matteucci , 1998 , j . am . chem . soc ., 120 , 8531 - 8532 . a single g - clamp analog substitution within an oligonucleotide can result in substantially enhanced helical thermal stability and mismatch discrimination when hybridized to complementary oligonucleotides . the inclusion of such nucleotides in nucleic acid molecules of the invention results in both enhanced affinity and specificity to nucleic acid targets , complimentary sequences , or template strands . in another embodiment , nucleic acid molecules of the invention include one or more , for example , about 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 or more lna “ locked nucleic acid ” nucleotides such as a 2 ′, 4 ′- c mythylene bicyclo nucleotide ( see for example wengel et al ., international pct publication no . wo 00 / 66604 and wo 99 / 14226 ). in another embodiment , the invention features conjugates and / or complexes of sirna molecules of the invention . such conjugates and / or complexes can be used to facilitate delivery of sirna molecules into a biological system , such as a cell . the conjugates and complexes provided by the instant invention can impart therapeutic activity by transferring therapeutic compounds across cellular membranes , altering the pharmacokinetics , and / or modulating the localization of nucleic acid molecules of the invention . the present invention encompasses the design and synthesis of novel conjugates and complexes for the delivery of molecules , including , but not limited to , small molecules , lipids , phospholipids , nucleosides , nucleotides , nucleic acids , antibodies , toxins , negatively charged polymers and other polymers , for example proteins , peptides , hormones , carbohydrates , polyethylene glycols , or polyamines , across cellular membranes . in general , the transporters described are designed to be used either individually or as part of a multi - component system , with or without degradable linkers . these compounds are expected to improve delivery and / or localization of nucleic acid molecules of the invention into a number of cell types originating from different tissues , in the presence or absence of serum ( see sullenger and cech , u . s . pat . no . 5 , 854 , 038 ). conjugates of the molecules described herein can be attached to biologically active molecules via linkers that are biodegradable , such as biodegradable nucleic acid linker molecules . the term “ biodegradable nucleic acid linker molecule ” as used herein , refers to a nucleic acid molecule that is designed as a biodegradable linker to connect one molecule to another molecule , for example , a biologically active molecule . the stability of the biodegradable nucleic acid linker molecule can be modulated by using various combinations of ribonucleotides , deoxyribonucleotides , and chemically modified nucleotides , for example , 2 ′- o - methyl , 2 ′- fluoro , 2 ′- amino , 2 ′- o - amino , 2 ′- c - allyl , 2 ′- o - allyl , and other 2 ′- modified or base modified nucleotides . the biodegradable nucleic acid linker molecule can be a dimer , trimer , tetramer or longer nucleic acid molecule , for example , an oligonucleotide of about 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , or 20 nucleotides in length , or can comprise a single nucleotide with a phosphorus - based linkage , for example , a phosphoramidate or phosphodiester linkage . the biodegradable nucleic acid linker molecule can also comprise nucleic acid backbone , nucleic acid sugar , or nucleic acid base modifications . the term “ biodegradable ” as used herein , refers to degradation in a biological system , for example enzymatic degradation or chemical degradation . the term “ biologically active molecule ” as used herein , refers to compounds or molecules that are capable of eliciting or modifying a biological response in a system . non - limiting examples of biologically active sirna molecules either alone or in combination with othe molecules contemplated by the instant invention include therapeutically active molecules such as antibodies , hormones , antivirals , peptides , proteins , chemotherapeutics , small molecules , vitamins , co - factors , nucleosides , nucleotides , oligonucleotides , enzymatic nucleic acids , antisense nucleic acids , triplex forming oligonucleotides , 2 , 5 - a chimeras , sirna , dsrna , allozymes , aptamers , decoys and analogs thereof . biologically active molecules of the invention also include molecules capable of modulating the pharmacokinetics and / or pharmacodynamics of other biologically active molecules , for example , lipids and polymers such as polyamines , polyamides , polyethylene glycol and other polyethers . the term “ phospholipid ” as used herein , refers to a hydrophobic molecule comprising at least one phosphorus group . for example , a phospholipid can comprise a phosphorus - containing group and saturated or unsaturated alkyl group , optionally substituted with oh , cooh , oxo , amine , or substituted or unsubstituted aryl groups . therapeutic nucleic acid molecules ( e . g ., sirna molecules ) delivered exogenously optimally are stable within cells until reverse trascription of the rna has been modulated long enough to reduce the levels of the rna transcript . the nucleic acid molecules are resistant to nucleases in order to function as effective intracellular therapeutic agents . improvements in the chemical synthesis of nucleic acid molecules described in the instant invention and in the art have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability as described above . in yet another embodiment , sirna molecules having chemical modifications that maintain or enhance enzymatic activity of proteins involved in rnai are provided . such nucleic acids are also generally more resistant to nucleases than unmodified nucleic acids . thus , in vitro and / or in vivo the activity should not be significantly lowered . use of the nucleic acid - based molecules of the invention will lead to better treatment of the disease progression by affording the possibility of combination therapies ( e . g ., multiple sirna molecules targeted to different genes ; nucleic acid molecules coupled with known small molecule modulators ; or intermittent treatment with combinations of molecules , including different motifs and / or other chemical or biological molecules ). the treatment of subjects with sirna molecules can also include combinations of different types of nucleic acid molecules , such as enzymatic nucleic acid molecules ( ribozymes ), allozymes , antisense , 2 , 5 - a oligoadenylate , decoys , aptamers etc . in another aspect a sirna molecule of the invention comprises one or more 5 ′ and / or a 3 ′- cap structure , for example on only the sense sirna strand , antisense sirna strand , or both sirna strands . by “ cap structure ” is meant chemical modifications , which have been incorporated at either terminus of the oligonucleotide ( see , for example , adamic et al ., u . s . pat . no . 5 , 998 , 203 , incorporated by reference herein ). these terminal modifications protect the nucleic acid molecule from exonuclease degradation , and can help in delivery and / or localization within a cell . the cap can be present at the 5 ′- terminus ( 5 ′- cap ) or at the 3 ′- terminal ( 3 ′- cap ) or can be present on both termini . in non - limiting examples : the 5 ′- cap is selected from the group comprising inverted abasic residue ( moiety ); 4 ′, 5 ′- methylene nucleotide ; 1 -( beta - d - erythrofuranosyl ) nucleotide , 4 ′- thio nucleotide ; carbocyclic nucleotide ; 1 , 5 - anhydrohexitol nucleotide ; l - nucleotides ; alpha - nucleotides ; modified base nucleotide ; phosphorodithioate linkage ; threo - pentofuranosyl nucleotide ; acyclic 3 ′, 4 ′- seco nucleotide ; acyclic 3 , 4 - dihydroxybutyl nucleotide ; acyclic 3 , 5 - dihydroxypentyl nucleotide , 3 ′- 3 ′- inverted nucleotide moiety ; 3 ′- 3 ′- inverted abasic moiety ; 3 ′- 2 ′- inverted nucleotide moiety ; 3 ′- 2 ′- inverted abasic moiety ; 1 , 4 - butanediol phosphate ; 3 ′- phosphoramidate ; hexylphosphate ; aminohexyl phosphate ; 3 ′- phosphate ; 3 ′- phosphorothioate ; phosphorodithioate ; or bridging or non - bridging methylphosphonate moiety . in yet another preferred embodiment , the 3 ′- cap is selected from a group comprising , 4 ′, 5 ′- methylene nucleotide ; 1 -( beta - d - erythrofuranosyl ) nucleotide ; 4 ′- thio nucleotide , carbocyclic nucleotide ; 5 ′- amino - alkyl phosphate ; 1 , 3 - diamino - 2 - propyl phosphate ; 3 - aminopropyl phosphate ; 6 - aminohexyl phosphate ; 1 , 2 - aminododecyl phosphate ; hydroxypropyl phosphate ; 1 , 5 - anhydrohexitol nucleotide ; l - nucleotide ; alpha - nucleotide ; modified base nucleotide ; phosphorodithioate ; threo - pentofuranosyl nucleotide ; acyclic 3 ′, 4 ′- seco nucleotide ; 3 , 4 - dihydroxybutyl nucleotide ; 3 , 5 - dihydroxypentyl nucleotide , 5 ′- 5 ′- inverted nucleotide moiety ; 5 ′- 5 ′- inverted abasic moiety ; 5 ′- phosphoramidate ; 5 ′- phosphorothioate ; 1 , 4 - butanediol phosphate ; 5 ′- amino ; bridging and / or non - bridging 5 ′- phosphoramidate , phosphorothioate and / or phosphorodithioate , bridging or non bridging methylphosphonate and 5 ′- mercapto moieties ( for more details see beaucage and iyer , 1993 , tetrahedron 49 , 1925 ; incorporated by reference herein ). by the term “ non - nucleotide ” is meant any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units , including either sugar and / or phosphate substitutions , and allows the remaining bases to exhibit their enzymatic activity . the group or compound is abasic in that it does not contain a commonly recognized nucleotide base , such as adenosine , guanine , cytosine , uracil or thymine and therefore lacks a base at the 1 ′- position . an “ alkyl ” group refers to a saturated aliphatic hydrocarbon , including straight - chain , branched - chain , and cyclic alkyl groups . preferably , the alkyl group has 1 to 12 carbons . more preferably , it is a lower alkyl of from 1 to 7 carbons , more preferably 1 to 4 carbons . the alkyl group can be substituted or unsubstituted . when substituted the substituted group ( s ) is preferably , hydroxyl , cyano , alkoxy , ═ o , ═ s , no 2 or n ( ch 3 ) 2 , amino , or sh . the term also includes alkenyl groups that are unsaturated hydrocarbon groups containing at least one carbon - carbon double bond , including straight - chain , branched - chain , and cyclic groups . preferably , the alkenyl group has 1 to 12 carbons . more preferably , it is a lower alkenyl of from 1 to 7 carbons , more preferably 1 to 4 carbons . the alkenyl group may be substituted or unsubstituted . when substituted the substituted group ( s ) is preferably , hydroxyl , cyano , alkoxy , ═ o , ═ s , no 2 , halogen , n ( ch 3 ) 2 , amino , or sh . the term “ alkyl ” also includes alkynyl groups that have an unsaturated hydrocarbon group containing at least one carbon - carbon triple bond , including straight - chain , branched - chain , and cyclic groups . preferably , the alkynyl group has 1 to 12 carbons . more preferably , it is a lower alkynyl of from 1 to 7 carbons , more preferably 1 to 4 carbons . the alkynyl group may be substituted or unsubstituted . when substituted the substituted group ( s ) is preferably , hydroxyl , cyano , alkoxy , ═ o , ═ s , no 2 or n ( ch 3 ) 2 , amino or sh . such alkyl groups may also include aryl , alkylaryl , carbocyclic aryl , heterocyclic aryl , amide and ester groups . an “ aryl ” group refers to an aromatic group that has at least one ring having a conjugated pi electron system and includes carbocyclic aryl , heterocyclic aryl and biaryl groups , all of which may be optionally substituted . the preferred substituent ( s ) of aryl groups are halogen , trihalomethyl , hydroxyl , sh , oh , cyano , alkoxy , alkyl , alkenyl , alkynyl , and amino groups . an “ alkylaryl ” group refers to an alkyl group ( as described above ) covalently joined to an aryl group ( as described above ). carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms . the carbon atoms are optionally substituted . heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms . suitable heteroatoms include oxygen , sulfur , and nitrogen , and include furanyl , thienyl , pyridyl , pyrrolyl , n - lower alkyl pyrrolo , pyrimidyl , pyrazinyl , imidazolyl and the like , all optionally substituted . an “ amide ” refers to an — c ( o )— nh — r , where r is either alkyl , aryl , alkylaryl or hydrogen . an “ ester ” refers to an — c ( o )— or ′, where r is either alkyl , aryl , alkylaryl or hydrogen . by “ nucleotide ” as used herein is as recognized in the art to include natural bases ( standard ), and modified bases well known in the art . such bases are generally located at the 1 ′ position of a nucleotide sugar moiety . nucleotides generally comprise a base , sugar and a phosphate group . the nucleotides can be unmodified or modified at the sugar , phosphate and / or base moiety , ( also referred to interchangeably as nucleotide analogs , modified nucleotides , non - natural nucleotides , non - standard nucleotides and other ; see , for example , usman and mcswiggen , supra ; eckstein et al ., international pct publication no . wo 92 / 07065 ; usman et al ., international pct publication no . wo 93 / 15187 ; uhlman & amp ; peyman , supra , all are hereby incorporated by reference herein ). there are several examples of modified nucleic acid bases known in the art as summarized by limbach et al ., 1994 , nucleic acids res . 22 , 2183 . some of the non - limiting examples of base modifications that can be introduced into nucleic acid molecules include , inosine , purine , pyridin - 4 - one , pyridin - 2 - one , phenyl , pseudouracil , 2 , 4 , 6 - trimethoxy benzene , 3 - methyl uracil , dihydrouridine , naphthyl , aminophenyl , 5 - alkylcytidines ( e . g ., 5 - methylcytidine ), 5 - alkyluridines ( e . g ., ribothymidine ), 5 - halouridine ( e . g ., 5 - bromouridine ) or 6 - azapyrimidines or 6 - alkylpyrimidines ( e . g . 6 - methyluridine ), propyne , and others ( burgin et al ., 1996 , biochemistry , 35 , 14090 ; uhlman & amp ; peyman , supra ). by “ modified bases ” in this aspect is meant nucleotide bases other than adenine , guanine , cytosine and uracil at 1 ′ position or their equivalents . in one embodiment , the invention features modified sirna molecules , with phosphate backbone modifications comprising one or more phosphorothioate , phosphorodithioate , methylphosphonate , phosphotriester , morpholino , amidate carbamate , carboxymethyl , acetamidate , polyamide , sulfonate , sulfonamide , sulfamate , formacetal , thioformacetal , and / or alkylsilyl , substitutions . for a review of oligonucleotide backbone modifications , see hunziker and leumann , 1995 , nucleic acid analogues : synthesis and properties , in modern synthetic methods , vch , 331 - 417 , and mesmaeker et al ., 1994 , novel backbone replacements for oligonucleotides , in carbohydrate modifications in antisense research , acs , 24 - 39 . by “ abasic ” is meant sugar moieties lacking a base or having other chemical groups in place of a base at the 1 ′ position , see for example adamic et al ., u . s . pat . no . 5 , 998 , 203 . by “ unmodified nucleoside ” is meant one of the bases adenine , cytosine , guanine , thymine , uracil joined to the 1 ′ carbon of β - d - ribo - furanose . by “ modified nucleoside ” is meant any nucleotide base which contains a modification in the chemical structure of an unmodified nucleotide base , sugar and / or phosphate . in connection with 2 ′- modified nucleotides as described for the present invention , by “ amino ” is meant 2 ′- nh 2 or 2 ′- o — nh 2 , which may be modified or unmodified . such modified groups are described , for example , in eckstein et al ., u . s . pat . no . 5 , 672 , 695 and matulic - adamic et al ., u . s . pat . no . 6 , 248 , 878 , which are both incorporated by reference in their entireties . various modifications to nucleic acid sirna structure can be made to enhance the utility of these molecules . such modifications will enhance shelf - life , half - life in vitro , stability , and ease of introduction of such oligonucleotides to the target site , e . g ., to enhance penetration of cellular membranes , and confer the ability to recognize and bind to targeted cells . a sirna molecule of the invention can be adapted for use to treat , for example conditions related to hiv infection and / or aids , alone or in combination with other therapies . for example , a sirna molecule can comprise a delivery vehicle , including liposomes , for administration to a subject , carriers and diluents and their salts , and / or can be present in pharmaceutically acceptable formulations . methods for the delivery of nucleic acid molecules are described in akhtar et al ., 1992 , trends cell bio ., 2 , 139 ; delivery strategies for antisense oligonucleotide therapeutics , ed . akhtar , 1995 , maurer et al ., 1999 , mol . membr . biol ., 16 , 129 - 140 ; hofland and huang , 1999 , handb . exp . pharmacol ., 137 , 165 - 192 ; and lee et al ., 2000 , acs symp . ser ., 752 , 184 - 192 , all of which are incorporated herein by reference . beigelman et al ., u . s . pat . no . 6 , 395 , 713 and sullivan et al ., pct wo 94 / 02595 , further describes the general methods for delivery of nucleic acid molecules . delivery of nucleic acid molecules of the invention to hematopoietic cells , such as t - cells , can be accomplished as is known in the art , see for example draper , u . s . pat . no . 6 , 622 , 854 ; phillips et al ., 1996 , nature medicine , 2 ( 10 ), 1154 - 1156 ; smith et al ., 1996 , antiviral research , 32 ( 2 ), 99 - 115 ; and rudoll et al ., 1996 , gene therapy , 3 ( 8 ), 695 - 705 . these protocols can be utilized for the delivery of virtually any nucleic acid molecule . nucleic acid molecules can be administered to cells by a variety of methods known to those of skill in the art , including , but not restricted to , encapsulation in liposomes , by iontophoresis , or by incorporation into other vehicles , such as hydrogels , cyclodextrins , biodegradable nanocapsules , and bioadhesive microspheres , or by proteinaceous vectors ( o &# 39 ; hare and normand , international pct publication no . wo 00 / 53722 ). alternatively , the nucleic acid / vehicle combination is locally delivered by direct injection or by use of an infusion pump . direct injection of the nucleic acid molecules of the invention , whether subcutaneous , intramuscular , or intradermal , can take place using standard needle and syringe methodologies , or by needle - free technologies such as those described in conry et al ., 1999 , clin . cancer res ., 5 , 2330 - 2337 and barry et al ., international pct publication no . wo 99 / 31262 . the molecules of the instant invention can be used as pharmaceutical agents . pharmaceutical agents prevent , modulate the occurrence , or treat ( alleviate a symptom to some extent , preferably all of the symptoms ) of a disease state in a subject . thus , the invention features a pharmaceutical composition comprising one or more nucleic acid ( s ) of the invention in an acceptable carrier , such as a stabilizer , buffer , and the like . the polynucleotides of the invention can be administered ( e . g ., rna , dna or protein ) and introduced into a subject by any standard means , with or without stabilizers , buffers , and the like , to form a pharmaceutical composition . when it is desired to use a liposome delivery mechanism , standard protocols for formation of liposomes can be followed . the compositions of the present invention can also be formulated and used as tablets , capsules or elixirs for oral administration , suppositories for rectal administration , sterile solutions , suspensions for injectable administration , and the other compositions known in the art . the present invention also includes pharmaceutically acceptable formulations of the compounds described . these formulations include salts of the above compounds , e . g ., acid addition salts , for example , salts of hydrochloric , hydrobromic , acetic acid , and benzene sulfonic acid . a pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration , e . g ., systemic administration , into a cell or subject , including for example a human . suitable forms , in part , depend upon the use or the route of entry , for example oral , transdermal , or by injection . such forms should not prevent the composition or formulation from reaching a target cell ( i . e ., a cell to which the negatively charged nucleic acid is desirable for delivery ). for example , pharmacological compositions injected into the blood stream should be soluble . other factors are known in the art , and include considerations such as toxicity and forms that prevent the composition or formulation from exerting its effect . by “ systemic administration ” is meant in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body . administration routes which lead to systemic absorption include , without limitation : intravenous , subcutaneous , intraperitoneal , inhalation , oral , intrapulmonary and intramuscular . each of these administration routes expose the sirna molecules of the invention to an accessible diseased tissue . the rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size . the use of a liposome or other drug carrier comprising the compounds of the instant invention can potentially localize the drug , for example , in certain tissue types , such as the tissues of the reticular endothelial system ( res ). a liposome formulation that can facilitate the association of drug with the surface of cells , such as , lymphocytes and macrophages is also useful . this approach can provide enhanced delivery of the drug to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells , such as cancer cells . by “ pharmaceutically acceptable formulation ” is meant , a composition or formulation that allows for the effective distribution of the nucleic acid molecules of the instant invention in the physical location most suitable for their desired activity . non - limiting examples of agents suitable for formulation with the nucleic acid molecules of the instant invention include : p - glycoprotein inhibitors ( such as pluronic p85 ), which can enhance entry of drugs into the cns ( jolliet - riant and tillement , 1999 , fundam . clin . pharmacol ., 13 , 16 - 26 ); biodegradable polymers , such as poly ( dl - lactide - coglycolide ) microspheres for sustained release delivery after intracerebral implantation ( emerich , df et al , 1999 , cell transplant , 8 , 47 - 58 ) ( alkermes , inc . cambridge , mass . ); and loaded nanoparticles , such as those made of polybutylcyanoacrylate , which can deliver drugs across the blood brain barrier and can alter neuronal uptake mechanisms ( prog neuropsychopharmacol biol psychiatry , 23 , 941 - 949 , 1999 ). other non - limiting examples of delivery strategies for the nucleic acid molecules of the instant invention include material described in boado et al ., 1998 , j . pharm . sci ., 87 , 1308 - 1315 ; tyler et al ., 1999 , febs lett ., 421 , 280 - 284 ; pardridge et al ., 1995 , pnas usa ., 92 , 5592 - 5596 ; boado , 1995 , adv . drug delivery rev ., 15 , 73 - 107 ; aldrian - herrada et al ., 1998 , nucleic acids res ., 26 , 4910 - 4916 ; and tyler et al ., 1999 , pnas usa ., 96 , 7053 - 7058 . the invention also features the use of the composition comprising surface - modified liposomes containing poly ( ethylene glycol ) lipids ( peg - modified , or long - circulating liposomes or stealth liposomes ). these formulations offer a method for increasing the accumulation of drugs in target tissues . this class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system ( mps or res ), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug ( lasic et al . chem . rev . 1995 , 95 , 2601 - 2627 ; ishiwata et al ., chem . pharm . bull . 1995 , 43 , 1005 - 1011 ). such liposomes have been shown to accumulate selectively in tumors , presumably by extravasation and capture in the neovascularized target tissues ( lasic et al ., science 1995 , 267 , 1275 - 1276 ; oku et al ., 1995 , biochim . biophys . acta , 1238 , 86 - 90 ). the long - circulating liposomes enhance the pharmacokinetics and pharmacodynamics of dna and rna , particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the mps ( liu et al ., j . biol . chem . 1995 , 42 , 24864 - 24870 ; choi et al ., international pct publication no . wo 96 / 10391 ; ansell et al ., international pct publication no . wo 96 / 10390 ; holland et al ., international pct publication no . wo 96 / 10392 ). long - circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic liposomes , based on their ability to avoid accumulation in metabolically aggressive mps tissues such as the liver and spleen . the present invention also includes compositions prepared for storage or administration , which include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent . acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art , and are described , for example , in remington &# 39 ; s pharmaceutical sciences , mack publishing co . ( a . r . gennaro edit . 1985 ) hereby incorporated by reference herein . for example , preservatives , stabilizers , dyes and flavoring agents can be provided . these include sodium benzoate , sorbic acid and esters of p - hydroxybenzoic acid . in addition , antioxidants and suspending agents can be used . the present invention also includes compositions prepared for storage or administration that include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent . acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art , and are described , for example , in remington &# 39 ; s pharmaceutical sciences , mack publishing co . ( a . r . gennaro edit . 1985 ), hereby incorporated by reference herein . for example , preservatives , stabilizers , dyes and flavoring agents can be provided . these include sodium benzoate , sorbic acid and esters of p - hydroxybenzoic acid . in addition , antioxidants and suspending agents can be used . a pharmaceutically effective dose is that dose required to prevent , inhibit the occurrence , or treat ( alleviate a symptom to some extent , preferably all of the symptoms ) of a disease state . the pharmaceutically effective dose depends on the type of disease , the composition used , the route of administration , the type of mammal being treated , the physical characteristics of the specific mammal under consideration , concurrent medication , and other factors that those skilled in the medical arts will recognize . generally , an amount between 0 . 1 mg / kg and 100 mg / kg body weight / day of active ingredients is administered dependent upon potency of the negatively charged polymer . the nucleic acid molecules of the invention and formulations thereof can be administered orally , topically , parenterally , by inhalation or spray , or rectally in dosage unit formulations containing conventional non - toxic pharmaceutically acceptable carriers , adjuvants and / or vehicles . the term parenteral as used herein includes percutaneous , subcutaneous , intravascular ( e . g ., intravenous ), intramuscular , or intrathecal injection or infusion techniques and the like . in addition , there is provided a pharmaceutical formulation comprising a nucleic acid molecule of the invention and a pharmaceutically acceptable carrier . one or more nucleic acid molecules of the invention can be present in association with one or more non - toxic pharmaceutically acceptable carriers and / or diluents and / or adjuvants , and if desired other active ingredients . the pharmaceutical compositions containing nucleic acid molecules of the invention can be in a form suitable for oral use , for example , as tablets , troches , lozenges , aqueous or oily suspensions , dispersible powders or granules , emulsion , hard or soft capsules , or syrups or elixirs . compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more such sweetening agents , flavoring agents , coloring agents or preservative agents in order to provide pharmaceutically elegant and palatable preparations . tablets contain the active ingredient in admixture with non - toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets . these excipients can be , for example , inert diluents ; such as calcium carbonate , sodium carbonate , lactose , calcium phosphate or sodium phosphate ; granulating and disintegrating agents , for example , corn starch , or alginic acid ; binding agents , for example starch , gelatin or acacia ; and lubricating agents , for example magnesium stearate , stearic acid or talc . the tablets can be uncoated or they can be coated by known techniques . in some cases such coatings can be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period . for example , a time delay material such as glyceryl monosterate or glyceryl distearate can be employed . formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent , for example , calcium carbonate , calcium phosphate or kaolin , or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium , for example peanut oil , liquid paraffin or olive oil . aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions . such excipients are suspending agents , for example sodium carboxymethylcellulose , methylcellulose , hydropropyl - methylcellulose , sodium alginate , polyvinylpyrrolidone , gum tragacanth and gum acacia ; dispersing or wetting agents can be a naturally - occurring phosphatide , for example , lecithin , or condensation products of an alkylene oxide with fatty acids , for example polyoxyethylene stearate , or condensation products of ethylene oxide with long chain aliphatic alcohols , for example heptadecaethyleneoxycetanol , or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate , or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides , for example polyethylene sorbitan monooleate . the aqueous suspensions can also contain one or more preservatives , for example ethyl , or n - propyl p - hydroxybenzoate , one or more coloring agents , one or more flavoring agents , and one or more sweetening agents , such as sucrose or saccharin . oily suspensions can be formulated by suspending the active ingredients in a vegetable oil , for example arachis oil , olive oil , sesame oil or coconut oil , or in a mineral oil such as liquid paraffin . the oily suspensions can contain a thickening agent , for example beeswax , hard paraffin or cetyl alcohol . sweetening agents and flavoring agents can be added to provide palatable oral preparations . these compositions can be preserved by the addition of an anti - oxidant such as ascorbic acid . dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent , suspending agent and one or more preservatives . suitable dispersing or wetting agents or suspending agents are exemplified by those already mentioned above . additional excipients , for example sweetening , flavoring and coloring agents , can also be present . pharmaceutical compositions of the invention can also be in the form of oil - in - water emulsions . the oily phase can be a vegetable oil or a mineral oil or mixtures of these . suitable emulsifying agents can be naturally - occurring gums , for example gum acacia or gum tragacanth , naturally - occurring phosphatides , for example soy bean , lecithin , and esters or partial esters derived from fatty acids and hexitol , anhydrides , for example sorbitan monooleate , and condensation products of the said partial esters with ethylene oxide , for example polyoxyethylene sorbitan monooleate . the emulsions can also contain sweetening and flavoring agents . syrups and elixirs can be formulated with sweetening agents , for example glycerol , propylene glycol , sorbitol , glucose or sucrose . such formulations can also contain a demulcent , a preservative and flavoring and coloring agents . the pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension . this suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above . the sterele injectable preparation can also be a sterile injectable solution or suspension in a non - toxic parentally acceptable diluent or solvent , for example as a solution in 1 , 3 - butanediol . among the acceptable vehicles and solvents that can be employed are water , ringer &# 39 ; s solution and isotonic sodium chloride solution . in addition , sterile , fixed oils are conventionally employed as a solvent or suspending medium . for this purpose , any bland fixed oil can be employed including synthetic mono - or diglycerides . in addition , fatty acids such as oleic acid find use in the preparation of injectables . the nucleic acid molecules of the invention can also be administered in the form of suppositories , e . g ., for rectal administration of the drug . these compositions can be prepared by mixing the drug with a suitable non - irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug . such materials include cocoa butter and polyethylene glycols . nucleic acid molecules of the invention can be administered parenterally in a sterile medium . the drug , depending on the vehicle and concentration used , can either be suspended or dissolved in the vehicle . advantageously , adjuvants such as local anesthetics , preservatives and buffering agents can be dissolved in the vehicle . dosage levels of the order of from about 0 . 1 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above - indicated conditions ( about 0 . 5 mg to about 7 g per subject per day ). the amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration . dosage unit forms generally contain between from about 1 mg to about 500 mg of an active ingredient . it is understood that the specific dose level for any particular subject depends upon a variety of factors including the activity of the specific compound employed , the age , body weight , general health , sex , diet , time of administration , route of administration , and rate of excretion , drug combination and the severity of the particular disease undergoing therapy . for administration to non - human animals , the composition can also be added to the animal feed or drinking water . it can be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet . it can also be convenient to present the composition as a premix for addition to the feed or drinking water . the nucleic acid molecules of the present invention can also be administered to a subject in combination with other therapeutic compounds to increase the overall therapeutic effect . the use of multiple compounds to treat an indication can increase the beneficial effects while reducing the presence of side effects . in one embodiment , the invention compositions suitable for administering nucleic acid molecules of the invention to specific cell types , such as hepatocytes . for example , the asialoglycoprotein receptor ( asgpr ) ( wu and wu , 1987 , j . biol . chem . 262 , 4429 - 4432 ) is unique to hepatocytes and binds branched galactose - terminal glycoproteins , such as asialoorosomucoid ( asor ). binding of such glycoproteins or synthetic glycoconjugates to the receptor takes place with an affinity that strongly depends on the degree of branching of the oligosaccharide chain , for example , triatennary structures are bound with greater affinity than biatenarry or monoatennary chains ( baenziger and fiete , 1980 , cell , 22 , 611 - 620 ; connolly et al ., 1982 , j . biol . chem ., 257 , 939 - 945 ). lee and lee , 1987 , glycoconjugate j ., 4 , 317 - 328 , obtained this high specificity through the use of n - acetyl - d - galactosamine as the carbohydrate moiety , which has higher affinity for the receptor , compared to galactose . this “ clustering effect ” has also been described for the binding and uptake of mannosyl - terminating glycoproteins or glycoconjugates ( ponpipom et al ., 1981 , j . med . chem ., 24 , 1388 - 1395 ). the use of galactose and galactosamine based conjugates to transport exogenous compounds across cell membranes can provide a targeted delivery approach to the treatment of liver disease such as hbv infection or hepatocellular carcinoma . the use of bioconjugates can also provide a reduction in the required dose of therapeutic compounds required for treatment . furthermore , therapeutic bioavialability , pharmacodynamics , and pharmacokinetic parameters can be modulated through the use of nucleic acid bioconjugates of the invention . alternatively , certain sirna molecules of the instant invention can be expressed within cells from eukaryotic promoters ( e . g ., izant and weintraub , 1985 , science , 229 , 345 ; mcgarry and lindquist , 1986 , proc . natl . acad . sci ., usa 83 , 399 ; scanlon et al ., 1991 , proc . natl . acad . sci . usa , 88 , 10591 - 5 ; kashani - sabet et al ., 1992 , antisense res . dev ., 2 , 3 - 15 ; dropulic et al ., 1992 , j . virol ., 66 , 1432 - 41 ; weerasinghe et al ., 1991 , j . virol ., 65 , 5531 - 4 ; ojwang et al ., 1992 , proc . natl . acad . sci . usa , 89 , 10802 - 6 ; chen et al ., 1992 , nucleic acids res ., 20 , 4581 - 9 ; sarver et al ., 1990 science , 247 , 1222 - 1225 ; thompson et al ., 1995 , nucleic acids res ., 23 , 2259 ; good et al ., 1997 , gene therapy , 4 , 45 . those skilled in the art realize that any nucleic acid can be expressed in eukaryotic cells from the appropriate dna / rna vector . the activity of such nucleic acids can be augmented by their release from the primary transcript by a enzymatic nucleic acid ( draper et al ., pct wo 93 / 23569 , and sullivan et al ., pct wo 94 / 02595 ; ohkawa et al ., 1992 , nucleic acids symp . ser ., 27 , 15 - 6 ; taira et al ., 1991 , nucleic acids res ., 19 , 5125 - 30 ; ventura et al ., 1993 , nucleic acids res ., 21 , 3249 - 55 ; chowrira et al ., 1994 , j . biol . chem ., 269 , 25856 . in another aspect of the invention , rna molecules of the present invention can be expressed from transcription units ( see for example couture et al ., 1996 , tig ., 12 , 510 ) inserted into dna or rna vectors . the recombinant vectors can be dna plasmids or viral vectors . sirna expressing viral vectors can be constructed based on , but not limited to , adeno - associated virus , retrovirus , adenovirus , or alphavirus . in another embodiment , pol iii based constructs are used to express nucleic acid molecules of the invention ( see for example thompson , u . s . pat . nos . 5 , 902 , 880 and 6 , 146 , 886 ). the recombinant vectors capable of expressing the sirna molecules can be delivered as described above , and persist in target cells . alternatively , viral vectors can be used that provide for transient expression of nucleic acid molecules . such vectors can be repeatedly administered as necessary . once expressed , the sirna molecule interacts with the target mrna and generates an rnai response . delivery of sirna molecule expressing vectors can be systemic , such as by intravenous or intra - muscular administration , by administration to target cells ex - planted from a subject followed by reintroduction into the subject , or by any other means that would allow for introduction into the desired target cell ( for a review see couture et al ., 1996 , tig ., 12 , 510 ). in one aspect the invention features an expression vector comprising a nucleic acid sequence encoding at least one sirna molecule of the instant invention . the expression vector can encode one or both strands of a sirna duplex , or a single self complimentary strand that self hybridizes into a sirna duplex . the nucleic acid sequences encoding the sirna molecules of the instant invention can be operably linked in a manner that allows expression of the sirna molecule ( see for example paul et al ., 2002 , nature biotechnology , 19 , 505 ; miyagishi and taira , 2002 , nature biotechnology , 19 , 497 ; lee et al ., 2002 , nature biotechnology , 19 , 500 ; and novina et al ., 2002 , nature medicine , advance online publication doi : 10 . 1038 / nm725 ). in another aspect , the invention features an expression vector comprising : a ) a transcription initiation region ( e . g ., eukaryotic pol i , ii or iii initiation region ); b ) a transcription termination region ( e . g ., eukaryotic pol i , ii or iii termination region ); and c ) a nucleic acid sequence encoding at least one of the sirna molecules of the instant invention ; wherein said sequence is operably linked to said initiation region and said termination region , in a manner that allows expression and / or delivery of the sirna molecule . the vector can optionally include an open reading frame ( orf ) for a protein operably linked on the 5 ′ side or the 3 ′- side of the sequence encoding the sirna of the invention ; and / or an intron ( intervening sequences ). transcription of the sirna molecule sequences can be driven from a promoter for eukaryotic rna polymerase i ( pol i ), rna polymerase ii ( pol ii ), or rna polymerase iii ( pol iii ). transcripts from pol ii or pol iii promoters are expressed at high levels in all cells ; the levels of a given pol ii promoter in a given cell type depends on the nature of the gene regulatory sequences ( enhancers , silencers , etc .) present nearby . prokaryotic rna polymerase promoters are also used , providing that the prokaryotic rna polymerase enzyme is expressed in the appropriate cells ( elroy - stein and moss , 1990 , proc . natl . acad . sci . u s a , 87 , 6743 - 7 ; gao and huang 1993 , nucleic acids res ., 21 , 2867 - 72 ; lieber et al ., 1993 , methods enzymol ., 217 , 47 - 66 ; zhou et al ., 1990 , mol . cell . biol ., 10 , 4529 - 37 ). several investigators have demonstrated that nucleic acid molecules expressed from such promoters can function in mammalian cells ( e . g . kashani - sabet et al ., 1992 , antisense res . dev ., 2 , 3 - 15 ; ojwang et al ., 1992 , proc . natl . acad . sci . usa , 89 , 10802 - 6 ; chen et al ., 1992 , nucleic acids res ., 20 , 4581 - 9 ; yu et al ., 1993 , proc . natl . acad . sci . u s a , 90 , 6340 - 4 ; l &# 39 ; huillier et al ., 1992 , embo j ., 11 , 4411 - 8 ; lisziewicz et al ., 1993 , proc . natl . acad . sci . u . s . a , 90 , 8000 - 4 ; thompson et al ., 1995 , nucleic acids res ., 23 , 2259 ; sullenger & amp ; cech , 1993 , science , 262 , 1566 ). more specifically , transcription units such as the ones derived from genes encoding u6 small nuclear ( snrna ), transfer rna ( trna ) and adenovirus va rna are useful in generating high concentrations of desired rna molecules such as sirna in cells ( thompson et al ., supra ; couture and stinchcomb , 1996 , supra ; noonberg et al ., 1994 , nucleic acid res ., 22 , 2830 ; noonberg et al ., u . s . pat . no . 5 , 624 , 803 ; good et al ., 1997 , gene ther ., 4 , 45 ; beigelman et al ., international pct publication no . wo 96 / 18736 . the above sirna transcription units can be incorporated into a variety of vectors for introduction into mammalian cells , including but not restricted to , plasmid dna vectors , viral dna vectors ( such as adenovirus or adeno - associated virus vectors ), or viral rna vectors ( such as retroviral or alphavirus vectors ) ( for a review see couture and stinchcomb , 1996 , supra ). in another aspect the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the sirna molecules of the invention , in a manner that allows expression of that sirna molecule . the expression vector comprises in one embodiment ; a ) a transcription initiation region ; b ) a transcription termination region ; and c ) a nucleic acid sequence encoding at least one strand of the sirna molecule ; wherein the sequence is operably linked to the initiation region and the termination region , in a manner that allows expression and / or delivery of the sirna molecule . in another embodiment the expression vector comprises : a ) a transcription initiation region ; b ) a transcription termination region ; c ) an open reading frame ; and d ) a nucleic acid sequence encoding at least one strand of a sirna molecule , wherein the sequence is operably linked to the 3 ′- end of the open reading frame ; and wherein the sequence is operably linked to the initiation region , the open reading frame and the termination region , in a manner that allows expression and / or delivery of the sirna molecule . in yet another embodiment the expression vector comprises : a ) a transcription initiation region ; b ) a transcription termination region ; c ) an intron ; and d ) a nucleic acid sequence encoding at least one sirna molecule ; wherein the sequence is operably linked to the initiation region , the intron and the termination region , in a manner which allows expression and / or delivery of the nucleic acid molecule . in another embodiment , the expression vector comprises : a ) a transcription initiation region ; b ) a transcription termination region ; c ) an intron ; d ) an open reading frame ; and e ) a nucleic acid sequence encoding at least one strand of a sirna molecule , wherein the sequence is operably linked to the 3 ′- end of the open reading frame ; and wherein the sequence is operably linked to the initiation region , the intron , the open reading frame and the termination region , in a manner which allows expression and / or delivery of the sirna molecule . the following are non - limiting examples showing the selection , isolation , synthesis and activity of nucleic acids of the instant invention . exemplary sirna molecules of the invention are synthesized in tandem using a cleavable linker , for example a succinyl - based linker . tandem synthesis as described herein is followed by a one step purification process that provides rnai molecules in high yield . this approach is highly amenable to sirna synthesis in support of high throughput rnai screening , and can be readily adapted to multi - column or multi - well synthesis platforms . after completing a tandem synthesis of an sirna oligo and its compliment in which the 5 ′- terminal dimethoxytrityl ( 5 ′- o - dmt ) group remains intact ( trityl on synthesis ), the oligonucleotides are deprotected as described above . following deprotection , the sirna sequence strands are allowed to spontaneously hybridize . this hybridization yields a duplex in which one strand has retained the 5 ′- o - dmt group while the complimentary strand comprises a terminal 5 ′- hydroxyl . the newly formed duplex to behaves as a single molecule during routine solid - phase extraction purification ( trityl - on purification ) even though only one molecule has a dimethoxytrityl group . because the strands form a stable duplex , this dimethoxytrityl group ( or an equivalent group , such as other trityl groups or other hydrophobic moieties ) is all that is required to purify the pair of oligos , for example by using a c18 cartridge . standard phosphoramidite synthesis chemistry is used up to point of introducing a tandem linker , such as an inverted deoxyabasic succinate linker ( see fig1 ) or an equivalent cleavable linker . a non - limiting example of linker coupling conditions that can be used includes a hindered base such as diisopropylethylamine ( dipa ) and / or dmap in the presence of an activator reagent such as bromotripyrrolidinophosphoniumhexaflurorophosphate ( pybrop ). after the linker is coupled , standard synthesis chemistry is utilized to complete synthesis of the second sequence leaving the terminal the 5 ′- o - dmt intact . following synthesis , the resulting oligonucleotide is deprotected according to the procedures described herein and quenched with a suitable buffer , for example with 50 mm naoac or 1 . 5m nh 4 h 2 co 3 . purification of the sirna duplex can be readily accomplished using solid phase extraction , for example using a waters c18 seppak 1 g cartridge conditioned with 1 column volume ( cv ) of acetonitrile , 2 cv h20 , and 2 cv 50 mm naoac . the sample is loaded and then washed with 1 cv h20 or 50 mm naoac . failure sequences are eluted with 1 cv 14 % acn ( aqueous with 50 mm naoac and 50 mm nacl ). the column is then washed , for example with 1 cv h20 followed by on - column detritylation , for example by passing 1 cv of 1 % aqueous trifluoroacetic acid ( tfa ) over the column , then adding a second cv of 1 % aqueous tfa to the column and allowing to stand for approx . 10 minutes . the remaining tfa solution is removed and the column washed with h20 followed by 1 cv 1m nacl and additional h20 . the sirna duplex product is then eluted , for example using 1 cv 20 % aqueous can . [ 0228 ] fig2 provides an example of maldi - tov mass spectrometry analysis of a purified sirna construct in which each peak corresponds to the calculated mass of an individual sirna strand of the sirna duplex . the same purified sirna provides three peaks when analyzed by capillary gel electrophoresis ( cge ), one peak presumably corresponding to the duplex sirna , and two peaks presumably corresponding to the separate sirna sequence strands . ion exchange hplc analysis of the same sirna contract only shows a single peak . the sequence of an rna target of interest , such as a hiv - 1 , is screened for target sites , for example by using a computer folding algorithm . in a non - limiting example , the sequence of gene or rna gene transcripts derived from a database , such as genbank accession numbers shown in table iii , is used to generate sirna targets having complimentarity to the target . such sequences can be obtained from a database , or can be determined experimentally as known in the art . target sites that are known , for example , those target sites determined to be effective target sites based on studies with other nucleic acid molecules , for example ribozymes or antisense , or those targets known to be associated with a disease or condition such as those sites containing mutations or deletions , can be used to design sirna molecules targeting those sites as well . various parameters can be used to determine which sites are the most suitable target sites within the target rna sequence . these parameters include but are not limited to secondary or tertiary rna structure , the nucleotide base composition of the target sequence , the degree of homology between various regions of the target sequence , or the relative position of the target sequence within the rna transcript . based on these determinations , any number of target sites within the rna transcript can be chosen to screen sirna molecules for efficacy , for example by using in vitro rna cleavage assays , cell culture , or animal models . in a non - limiting example , anywhere from 1 to 1000 target sites are chosen within the transcript based on the size of the sirna contruct to be used . high throughput screening assays can be developed for screening sirna molecules using methods known in the art , such as with multi - well or multi - plate assays to determine efficient reduction in target gene expression . the following non - limiting steps can be used to carry out the selection of sirnas targeting a given gene sequence or transcript , eg hiv - 1 . 1 . the target sequence is parsed in silico into a list of all fragments or subsequences of a particular length , for example 23 nucleotide fragments , contained within the target sequence . this step is typically carried out using a custom perl script , but commercial sequence analysis programs such as oligo , macvector , or the gcg wisconsin package can be employed as well . 2 . in some instances the sirnas correspond to more than one target sequence ; such would be the case for example in targeting many different strains of a viral sequence , for targeting different transcipts of the same gene , targeting different transcipts of more than one gene , or for targeting both the human gene and an animal homolog . in this case , a subsequence list of a particular length is generated for each of the targets , and then the lists are compared to find matching sequences in each list . the subsequences are then ranked according to the number of target sequences that contain the given subsequence ; the goal is to find subsequences that are present in most or all of the target sequences . alternately , the ranking can indentify subsequences that are unique to a target sequence , such as a mutant target sequence . such an approach would enable the use of sirna to target specifically the mutant sequence and not effect the expression of the normal sequence . 3 . in some instances the sirna subsequences are absent in one or more sequences while present in the desired target sequence ; such would be the case if the sirna targets a gene with a paralogous family member that is to remain untargeted . as in case 2 above , a subsequence list of a particular length is generated for each of the targets , and then the lists are compared to find sequences that are present in the target gene but are absent in the untargeted paralog . 4 . the ranked sirna subsequences can be further analyzed and ranked according to gc content . a preference can be given to sites containing 30 - 70 % gc , with a further preference to sites containing 40 - 60 % gc . 5 . the ranked sirna subsequences can be further analyzed and ranked according to self - folding and internal hairpins . weaker internal folds are preferred ; strong hairpin structures are to be avoided . 6 . the ranked sirna subsequences can be further analyzed and ranked according to whether they have runs of ggg or ccc in the sequence . ggg ( or even more gs ) in either strand can make oligonucleotide synthesis problematic , so it is avoided whenever better sequences are available . ccc is searched in the target strand because that will place ggg in the antisense strand . 7 . the ranked sirna subsequences can be further analyzed and ranked according to whether they have the dinucleotide uu ( uridine dinucleotide ) on the 3 ′ end of the sequence , and / or aa on the 5 ′ end of the sequence ( to yield 3 ′ uu on the antisense sequence ). these sequences allow one to design sirna molecules with terminal tt thymidine dinucleotides . 8 . four or five target sites are chosen from the ranked list of subsequences as described above . for example , in subsequences having 23 nucleotides , the right 21 nucleotides of each chosen 23 - mer subsequence are then designed and synthesized for the upper ( sense ) strand of the sirna duplex , while the reverse complement of the left 21 nucleotides of each chosen 23 - mer subsequence are then designed and synthesized for the lower ( antisense ) strand of the sirna duplex . if terminal tt residues are desired for the sequence ( as described in paragraph 7 ), then the two 3 ′ terminal nucleotides of both the sense and antisense strands are replaced by tt prior to synthesizing the oligos . 9 . the sirna molecules are screened in an in vitro , cell culture or animal model system to identify the most active sirna molecule or the most preferred target site within the target rna sequence . in an alternate approach , a pool of sirna constructs specific to a hiv target sequence is used to screen for target sites in cells expressing hiv rna . the general strategy used in this approach is shown in fig9 . a non - limiting example of such as pool is a pool comprising sequences having sense sequences comprising seq id nos . 1 - 738 and antisense sequences comprising seq id nos . 739 - 1476 respectively . cells expressing hiv are transfected with the pool of sirna constructs and cells that demonstrate a phenotype associated with hiv inhibition are sorted . the pool of sirna constructs can be expressed from transcription cassettes inserted into appropriate vectors ( see for example fig7 and fig8 ). cells in which hiv expression is decreased due to sirna treatment demonstrate a phenotypic change , for example decreased production of hiv rna or hiv protein ( s ) compared to untreated cells or cells treated with a control sirna . the sirna from cells demonstrating a positive phenotypic change ( e . g ., decreased hiv rna or protein ), are sequenced to determine the most suitable target site ( s ) within the target hiv rna sequence . sirna target sites were chosen by analyzing sequences of the hiv - 1 rna target ( for example genbank accession nos . shown in table iii ) and optionally prioritizing the target sites on the basis of folding ( structure of any given sequence analyzed to determine sirna accessibility to the target ). the sequence alignments of all known a and b strains of hiv were screened for homology and sirna molecules were designed to target conserved sequences across these strains since the a and b strains are currently the most prevalent strains . alternately , all known strains or other subclasses of hiv can be similarly screened for homology ( see table iv ) and homologous sequences used as targets . a cutoff for % homology between the different strains can be used to increase or decrease the number of targets considered , for example 70 %, 75 %, 80 %, 85 %, 90 % or 95 % homology . the sequences shown in table i represent 80 % homology between the hiv strains shown in table iii . sirna molecules were designed that could bind each target sequence and are optionally individually analyzed by computer folding to assess whether the sirna molecule can interact with the target sequence . varying the length of the sirna molecules can be chosen to optimize activity . the sirna sense ( upper sequence ) and antisense ( lower sequence ) sequences shown in table i comprise 19 nucleotides in length , with the sense strand comprising the same sequence as the target sequence and the antisense strand comprising a complimentary sequence to the sense / target sequence . the sense and antisense strands can further comprise nucleotide 3 ′- overhangs as described herein , preferably the overhangs comprise about 2 nucleotides which can optionally be complimentary to the target sequence in the antisense sirna strand , and / or optionally analogous to the adjacent nucleotides in the target sequence when present in the sense sirna strand . generally , a sufficient number of complimentary nucleotide bases are chosen to bind to , or otherwise interact with , the target rna , but the degree of complementarity can be modulated to accommodate sirna duplexes or varying length or base composition . by using such methodologies , sirna molecules can be designed to target sites within any known rna sequence , for example those rna sequences corresponding to the any gene transcript . sirna molecules can be designed to interact with various sites in the rna message , for example target sequences within the rna sequences described herein . the sequence of one strand of the sirna molecule ( s ) are complementary to the target site sequences described above . the sirna molecules can be chemically synthesized using methods described herein . inactive sirna molecules that are used as control sequences can be synthesized by scrambling the sequence of the sirna molecules such that it is not complimentary to the target sequence . an in vitro assay that recapitulates rnai in a cell free system is used to evaluate sirna constructs targeting hiv rna targets . the assay comprises the system described by tuschl et al ., 1999 , genes and development , 13 , 3191 - 3197 and zamore et al ., 2000 , cell , 101 , 25 - 33 adapted for use with hiv target rna . a drosophila extract derived from syncytial blastoderm is used to reconstitute rnai activity in vitro . target rna is generated via in vitro transcription from an appropriate hiv expressing plasmid using t7 rna polymerase . the target rna can also be synthesized chemically as described herein . sense and antisense sirna strands ( for example 20 um each ) are annealed by incubation in buffer ( such as 100 mm potassium acetate , 30 mm hepes - koh , ph 7 . 4 , 2 mm magnesium acetate ) for 1 min . at 90 ° c . followed by 1 hour at 37 ° c ., then diluted in lysis buffer ( for example 100 mm potassium acetate , 30 mm hepes - koh at ph 7 . 4 , 2 mm magnesium acetate ). annealing can be monitored by gel electrophoresis on an agarose gel in tbe buffer and stained with ethidium bromide . the drosophila lysate is prepared using zero to two hour old embryos from oregon r flies collected on yeasted molasses agar that are dechorionated and lysed . the lysate is centrifuged and the supernatant isolated . the assay comprises a reaction mixture containing 50 % lysate [ vol / vol ], rna ( 10 - 50 pm final concentration ), and 10 % [ vol / vol ] lysis buffer containing sirna ( 10 nm final concentration ). the reaction mixture also contains 10 mm creatine phosphate , 10 ug . ml creatine phosphokinase , 100 um gtp , 100 um utp , 100 um ctp , 500 um atp , 5 mm dtt , 0 . 1 u / ul rnasin ( promega ), and 100 um of each amino acid . the final concentration of potassium acetate is adjusted to 100 mm . the reactions are pre - assembled on ice and preincubated at 25 ° c . for 10 minutes before adding rna , then incubated at 25 ° c . for an additional 60 minutes . reactions are quenched with 4 volumes of 1 . 25 × passive lysis buffer ( promega ). target rna cleavage is assayed by rt - pcr analysis or other methods known in the art and are compared to control reactions in which sirna is omitted from the reaction . alternately , internally - labeled target rna for the assay is prepared by in vitro transcription in the presence of [ a - 32 p ] ctp , passed over a g 50 sephadex column by spin chromatography and used as target rna without further purification . optionally , target rna is 5 ′- 32 p - end labeled using t4 polynucleotide kinase enzyme . assays are performed as described above and target rna and the specific rna cleavage products generated by rnai are visualized on an autoradiograph of a gel . the percentage of cleavage is determined by phosphor imager ® quantitation of bands representing intact control rna or rna from control reactions without sirna and the cleavage products generated by the assay . the sirna constructs of the invention can be used in various cell culture systems as are commonly known in the art to screen for compounds having anti - hiv activity . b cell , t cell , macrophage and endothelial cell culture systems are non - limiting examples of cell culture systems that can be readily adapted for screening sirna molecules of the invention . in a non - limiting example , sirna molecules of the invention are co - transfected with hiv - 1 pnl4 - 3 proviral dna into 293 / ecr cells as described by lee et al ., 2002 , nature biotechnology , 19 , 500 - 505 , using a u6 snrna promoter driven expression system . in a non - limiting example , the sirna expression vectors are prepared using the ptz u6 + 1 vector described in lee et al . supra . as follows . one cassette harbors the 21 - nucleotide sense sequences and the other a 21 - nucleotide antisense sequence ( table i ). these sequences are designed to target hiv - 1 rna targets described herein . as a control to verify a sirna mechanism , irrelevant sense and antisense ( s / as ) sequences lacking complementarity to hiv - 1 ( s / as ( ir )) are subcloned in ptz u6 + 1 . rna samples are prepared from 293 / ecr cells transiently co - transfected with sirna or control constructs , and subjected to ponasterone a induction . rnas are also prepared from 293 cells co - transfected with hiv - 1 pnl4 - 3 proviral dna and sirna or control constructs . for determination of anti - hiv - 1 activity of the sirnas , transient assays are done by co - transfection of sirna constructs and infectious hiv - 1 proviral dna , pnl4 - 3 into 293 cells as described above , followed by northern analysis as known in the art . the p24 values are calculated with the aid of , for example , a dynatech mr5000 elisa plate reader ( dynatech labs inc ., chantilly , va .). cell viability can also be assessed using a trypan blue dye exclusion count at four days after transfection . other cell culture model systems are generally known in the art , see for example duzgunes et al ., 2001 , nucleosides , nucleotides & amp ; nucleic acids , 20 ( 4 - 7 ), 515 - 523 ; cagnun et al ., 2000 , antisense nucleic acid drug dev ., 10 , 251 ; ho et al ., 1995 , stem cells , 13 supp 3 , 100 ; and baur et al ., 1997 , blood , 89 , 2259 . these cell culture systems can be readily adapted for use with the compositions of the instant invention . the sirna constructs of the invention can be evaluated in a variety of animal models , including for example a hollow fiber hiv model ( see for example gruenberg , u . s . pat . no . 5 , 627 , 070 ), mouse models for aids using transgenic mice expressing hiv - 1 genes from cd4 promoters and enhancers ( see for example jolicoeur , international pct publication no . wo 98 / 50535 ) and / or the hiv / siv / shiv non - human primate models ( see for example narayan , u . s . pat . no . 5 , 849 , 994 ). the sirna compounds and virus can be administered by a variety of methods and routes as described herein and as known in the art . quantitation of results in these models can be performed by a variety of methods , including quantitative pcr , quantitative and bulk co - cultivation assays , plasma co - cultivation assays , antigen and antibody detection assays , lymphocyte proliferation , intracellular cytokines , flow cytometry , as well as hematology and cbc evaluation . additional animal models are generally known in the art , see for example bai et al ., 2000 , mol . ther ., 1 , 244 . particular degenerative and disease states that can be associated with hiv expression modulation include but are not limited to acquired immunodeficiency disease ( aids ) and related diseases and conditions , including but not limited to kaposi &# 39 ; s sarcoma , lymphoma , cervical cancer , squamous cell carcinoma , cardiac myopathy , rheumatic diseases , and opportunistic infection , for example pneumocystis carinii , cytomegalovirus , herpes simplex , mycobacteria , cryptococcus , toxoplasma , progressive multifocal leuco - encephalopathy ( papovavirus ), mycobacteria , aspergillus , cryptococcus , candida , cryptosporidium , isospora belli , microsporidia and any other diseases or conditions that are related to or will respond to the levels of hiv in a cell or tissue , alone or in combination with other therapies the present body of knowledge in hiv research indicates the need for methods to assay hiv activity and for compounds that can regulate hiv expression for research , diagnostic , and therapeutic use . the use of antiviral compounds , monoclonal antibodies , chemotherapy , radiation therapy , analgesics , and / or anti - inflammatory compounds , are all non - limiting examples of a methods that can be combined with or used in conjunction with the nucleic acid molecules ( e . g . ribozymes and antisense molecules ) of the instant invention . examples of antiviral compounds that can be used in conjunction with the nucleic acid molecules of the invention include but are not limited to azt ( also known as zidovudine or zdv ), ddc ( zalcitabine ), ddi ( dideoxyinosine ), d4t ( stavudine ), and 3tc ( lamivudine ) ribavirin , delvaridine ( rescriptor ), nevirapine ( viramune ), efravirenz ( sustiva ), ritonavir ( norvir ), saquinivir ( invirase ), indinavir ( crixivan ), amprenivir ( agenerase ), nelfinavir ( viracept ), and / or lopinavir ( kaletra ). common chemotherapies that can be combined with nucleic acid molecules of the instant invention include various combinations of cytotoxic drugs to kill cancer cells . these drugs include but are not limited to paclitaxel ( taxol ), docetaxel , cisplatin , methotrexate , cyclophosphamide , doxorubin , fluorouracil carboplatin , edatrexate , gemcitabine , vinorelbine etc . those skilled in the art will recognize that other drug compounds and therapies can be similarly be readily combined with the nucleic acid molecules of the instant invention ( e . g . ribozymes , sirna and antisense molecules ) are hence within the scope of the instant invention . the sirna molecules of the invention can be used in a variety of diagnostic applications , such as in identifying molecular targets such as rna in a variety of applications , for example , in clinical , industrial , environmental , agricultural and / or research settings . such diagnostic use of sirna molecules involves utilizing reconstituted rnai systems , for example using cellular lysates or partially purified cellular lysates . sirna molecules of this invention can be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of endogenous or exogenous , for example viral , rna in a cell . the close relationship between sirna activity and the structure of the target rna allows the detection of mutations in any region of the molecule , which alters the base - pairing and three - dimensional structure of the target rna . by using multiple sirna molecules described in this invention , one can map nucleotide changes , which are important to rna structure and function in vitro , as well as in cells and tissues . cleavage of target rnas with sirna molecules can be used to inhibit gene expression and define the role ( essentially ) of specified gene products in the progression of disease or infection . in this manner , other genetic targets can be defined as important mediators of the disease . these experiments will lead to better treatment of the disease progression by affording the possibility of combination therapies ( e . g ., multiple sirna molecules targeted to different genes , sirna molecules coupled with known small molecule inhibitors , or intermittent treatment with combinations sirna molecules and / or other chemical or biological molecules ). other in vitro uses of sirna molecules of this invention are well known in the art , and include detection of the presence of mrnas associated with a disease , infection , or related condition . such rna is detected by determining the presence of a cleavage product after treatment with a sirna using standard methodologies , for example fluorescence resonance emission transfer ( fret ). in a specific example , sirna molecules that can cleave only wild - type or mutant forms of the target rna are used for the assay . the first sirna molecules is used to identify wild - type rna present in the sample and the second sirna molecules will be used to identify mutant rna in the sample . as reaction controls , synthetic substrates of both wild - type and mutant rna will be cleaved by both sirna molecules to demonstrate the relative sirna efficiencies in the reactions and the absence of cleavage of the “ non - targeted ” rna species . the cleavage products from the synthetic substrates will also serve to generate size markers for the analysis of wild - type and mutant rnas in the sample population . thus each analysis will require two sirna molecules , two substrates and one unknown sample which will be combined into six reactions . the presence of cleavage products will be determined using an rnase protection assay so that full - length and cleavage fragments of each rna can be analyzed in one lane of a polyacrylamide gel . it is not absolutely required to quantify the results to gain insight into the expression of mutant rnas and putative risk of the desired phenotypic changes in target cells . the expression of mrna whose protein product is implicated in the development of the phenotype ( i . e ., disease related or infection related ) is adequate to establish risk . if probes of comparable specific activity are used for both transcripts , then a qualitative comparison of rna levels will be adequate and will decrease the cost of the initial diagnosis . higher mutant form to wild - type ratios will be correlated with higher risk whether rna levels are compared qualitatively or quantitatively . all patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains . all references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually . one skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned , as well as those inherent therein . the methods and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention . changes therein and other uses will occur to those skilled in the art , which are encompassed within the spirit of the invention , are defined by the scope of the claims . it will be readily apparent to one skilled in the art that varying substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention . thus , such additional embodiments are within the scope of the present invention and the following claims . the invention illustratively described herein suitably can be practiced in the absence of any element or elements , limitation or limitations that are not specifically disclosed herein . thus , for example , in each instance herein any of the terms “ comprising ”, “ consisting essentially of ” and “ consisting of ” may be replaced with either of the other two terms . the terms and expressions which have been employed are used as terms of description and not of limitation , and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof , but it is recognized that various modifications are possible within the scope of the invention claimed . thus , it should be understood that although the present invention has been specifically disclosed by preferred embodiments , optional features , modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art , and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims . in addition , where features or aspects of the invention are described in terms of markush groups or other grouping of alternatives , those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the markush group or other group . table i hiv target and sirna sequences seq seq seq sequence id upper seq id lower seq id uuuggaaaggaccagcaaa 1 uuuggaaaggaccagcaaa 1 uuugcugguccuuuccaaa 739 caggagcagaugauacagu 2 caggagcagaugauacagu 2 acuguaucaucugcuccug 740 agaaaaggggggauugggg 3 agaaaaggggggauugggg 3 ccccaauccccccuuuucu 741 guagacaggaugaggauua 4 guagacaggaugaggauua 4 uaauccucauccugucuac 742 acaggagcagaugauacag 5 acaggagcagaugauacag 5 cuguaucaucugcuccugu 743 gaaaaggggggauuggggg 6 gaaaaggggggauuggggg 6 cccccaauccccccuuuuc 744 uuagauacaggagcagaug 7 uuagauacaggagcagaug 7 caucugcuccuguaucuaa 745 uagauacaggagcagauga 8 uagauacaggagcagauga 8 ucaucugcuccuguaucua 746 agcagaagacaguggcaau 9 agcagaagacaguggcaau 9 auugccacugucuucugcu 747 auuagauacaggagcagau 10 auuagauacaggagcagau 10 aucugcuccuguaucuaau 748 auacaggagcagaugauac 11 auacaggagcagaugauac 11 guaucaucugcuccuguau 749 gagcagaagacaguggcaa 12 gagcagaagacaguggcaa 12 uugccacugucuucugcuc 750 agagcagaagacaguggca 13 agagcagaagacaguggca 13 ugccacugucuucugcucu 751 gcagaagacaguggcaaug 14 gcagaagacaguggcaaug 14 cauugccacugucuucugc 752 agauacaggagcagaugau 15 agauacaggagcagaugau 15 aucaucugcuccuguaucu 753 uacaggagcagaugauaca 16 uacaggagcagaugauaca 16 uguaucaucugcuccugua 754 uauuagauacaggagcaga 17 uauuagauacaggagcaga 17 ucugcuccuguaucuaaua 755 gauacaggagcagaugaua 18 gauacaggagcagaugaua 18 uaucaucugcuccuguauc 756 auggaaaacagauggcagg 19 auggaaaacagauggcagg 19 ccugccaucuguuuuccau 757 gucaacauaauuggaagaa 20 gucaacauaauuggaagaa 20 uucuuccaauuauguugac 758 uauggaaaacagauggcag 21 uauggaaaacagauggcag 21 cugccaucuguuuuccaua 759 augauagggggaauuggag 22 augauagggggaauuggag 22 cuccaauucccccuaucau 760 cagaagacaguggcaauga 23 cagaagacaguggcaauga 23 ucauugccacugucuucug 761 caauggccauugacagaag 24 caauggccauugacagaag 24 cuucugucaauggccauug 762 ucaacauaauuggaagaaa 25 ucaacauaauuggaagaaa 25 uuucuuccaauuauguuga 763 aauggccauugacagaaga 26 aauggccauugacagaaga 26 ucuucugucaauggccauu 764 ugauagggggaauuggagg 27 ugauagggggaauuggagg 27 ccuccaauucccccuauca 765 gacaggcuaauuuuuuagg 28 gacaggcuaauuuuuuagg 28 ccuaaaaaauuagccuguc 766 auuuucggguuuauuacag 29 auuuucggguuuauuacag 29 cuguaauaaacccgaaaau 767 cuauuagauacaggagcag 30 cuauuagauacaggagcag 30 cugcuccuguaucuaauag 768 agacaggcuaauuuuuuag 31 agacaggcuaauuuuuuag 31 cuaaaaaauuagccugucu 769 aaaugauagggggaauugg 32 aaaugauagggggaauugg 32 ccaauucccccuaucauuu 770 uaugggcaagcagggagcu 33 uaugggcaagcagggagcu 33 agcucccugcuugcccaua 771 uaguaugggcaagcaggga 34 uaguaugggcaagcaggga 34 ucccugcuugcccauacua 772 gaaaacagauggcagguga 35 gaaaacagauggcagguga 35 ucaccugccaucuguuuuc 773 accaucaaugaggaagcug 36 accaucaaugaggaagcug 36 cagcuuccucauugauggu 774 aaugauagggggaauugga 37 aaugauagggggaauugga 37 uccaauucccccuaucauu 775 uggaaaacagauggcaggu 38 uggaaaacagauggcaggu 38 accugccaucuguuuucca 776 ggaaaacagauggcaggug 39 ggaaaacagauggcaggug 39 caccugccaucuguuuucc 777 gauuauggaaaacagaugg 40 gauuauggaaaacagaugg 40 ccaucuguuuuccauaauc 778 aaaaugauagggggaauug 41 aaaaugauagggggaauug 41 caauucccccuaucauuuu 779 uggaaaggugaaggggcag 42 uggaaaggugaaggggcag 42 cugccccuucaccuuucca 780 aucaaugaggaagcugcag 43 aucaaugaggaagcugcag 43 cugcagcuuccucauugau 781 uggaaaccaaaaaugauag 44 uggaaaccaaaaaugauag 44 cuaucauuuuugguuucca 782 ccaucaaugaggaagcugc 45 ccaucaaugaggaagcugc 45 gcagcuuccucauugaugg 783 agggauuauggaaaacaga 46 agggauuauggaaaacaga 46 ucuguuuuccauaaucccu 784 ggaaaccaaaaaugauagg 47 ggaaaccaaaaaugauagg 47 ccuaucauuuuugguuucc 785 uagggggaauuggagguuu 48 uagggggaauuggagguuu 48 aaaccuccaauucccccua 786 uacagugcaggggaaagaa 49 uacagugcaggggaaagaa 49 uucuuuccccugcacugua 787 cucuauuagauacaggagc 50 cucuauuagauacaggagc 50 gcuccuguaucuaauagag 788 ggauuauggaaaacagaug 51 ggauuauggaaaacagaug 51 caucuguuuuccauaaucc 789 ccaaaaaugauagggggaa 52 ccaaaaaugauagggggaa 52 uucccccuaucauuuuugg 790 auggaaaccaaaaaugaua 53 auggaaaccaaaaaugaua 53 uaucauuuuugguuuccau 791 cagugcaggggaaagaaua 54 cagugcaggggaaagaaua 54 uauucuuuccccugcacug 792 acaauggccauugacagaa 55 acaauggccauugacagaa 55 uucugucaauggccauugu 793 ccaugcauggacaaguaga 56 ccaugcauggacaaguaga 56 ucuacuuguccaugcaugg 794 auuauggaaaacagauggc 57 auuauggaaaacagauggc 57 gccaucuguuuuccauaau 795 aacaauggccauugacaga 58 aacaauggccauugacaga 58 ucugucaauggccauuguu 796 aaaaaugauagggggaauu 59 aaaaaugauagggggaauu 59 aauucccccuaucauuuuu 797 gccaugcauggacaaguag 60 gccaugcauggacaaguag 60 cuacuuguccaugcauggc 798 uagcaggaagauggccagu 61 uagcaggaagauggccagu 61 acuggccaucuuccugcua 799 caaaaaugauagggggaau 62 caaaaaugauagggggaau 62 auucccccuaucauuuuug 800 aagaaaugaugacagcaug 63 aagaaaugaugacagcaug 63 caugcugucaucauuucuu 801 ucuauuagauacaggagca 64 ucuauuagauacaggagca 64 ugcuccuguaucuaauaga 802 gcucuauuagauacaggag 65 gcucuauuagauacaggag 65 cuccuguaucuaauagagc 803 caggcuaauuuuuuaggga 66 caggcuaauuuuuuaggga 66 ucccuaaaaaauuagccug 804 aggagcagaugauacagua 67 aggagcagaugauacagua 67 uacuguaucaucugcuccu 805 aaacaauggccauugacag 68 aaacaauggccauugacag 68 cugucaauggccauuguuu 806 cggguuuauuacagggaca 69 cggguuuauuacagggaca 69 ugucccuguaauaaacccg 807 caacauaauuggaagaaau 70 caacauaauuggaagaaau 70 auuucuuccaauuauguug 808 ucaaugaggaagcugcaga 71 ucaaugaggaagcugcaga 71 ucugcagcuuccucauuga 809 ggaaaggugaaggggcagu 72 ggaaaggugaaggggcagu 72 acugccccuucaccuuucc 810 uuucggguuuauuacaggg 73 uuucggguuuauuacaggg 73 cccuguaauaaacccgaaa 811 ucggguuuauuacagggac 74 ucggguuuauuacagggac 74 gucccuguaauaaacccga 812 acagugcaggggaaagaau 75 acagugcaggggaaagaau 75 auucuuuccccugcacugu 813 augcauggacaaguagacu 76 augcauggacaaguagacu 76 agucuacuuguccaugcau 814 aagccaugcauggacaagu 77 aagccaugcauggacaagu 77 acuuguccaugcauggcuu 815 agccaugcauggacaagua 78 agccaugcauggacaagua 78 uacuuguccaugcauggcu 816 gcauuaucagaaggagcca 79 gcauuaucagaaggagcca 79 uggcuccuucugauaaugc 817 aauuggagaagugaauuau 80 aauuggagaagugaauuau 80 auaauucacuucuccaauu 818 agaaaaaaucaguaacagu 81 agaaaaaaucaguaacagu 81 acuguuacugauuuuuucu 819 gaagccaugcauggacaag 82 gaagccaugcauggacaag 82 cuuguccaugcauggcuuc 820 acaggcuaauuuuuuaggg 83 acaggcuaauuuuuuaggg 83 cccuaaaaaauuagccugu 821 gaagaaaugaugacagcau 84 gaagaaaugaugacagcau 84 augcugucaucauuucuuc 822 uuuucggguuuauuacagg 85 uuuucggguuuauuacagg 85 ccuguaauaaacccgaaaa 823 accaaaaaugauaggggga 86 accaaaaaugauaggggga 86 ucccccuaucauuuuuggu 824 gaagugacauagcaggaac 87 gaagugacauagcaggaac 87 guuccugcuaugucacuuc 825 uucggguuuauuacaggga 88 uucggguuuauuacaggga 88 ucccuguaauaaacccgaa 826 auagggggaauuggagguu 89 auagggggaauuggagguu 89 aaccuccaauucccccuau 827 agaagaaaugaugacagca 90 agaagaaaugaugacagca 90 ugcugucaucauuucuucu 828 auuggagaagugaauuaua 91 auuggagaagugaauuaua 91 uauaauucacuucuccaau 829 ggaagugacauagcaggaa 92 ggaagugacauagcaggaa 92 uuccugcuaugucacuucc 830 aggcuaauuuuuuagggaa 93 aggcuaauuuuuuagggaa 93 uucccuaaaaaauuagccu 831 uuauggaaaacagauggca 94 uuauggaaaacagauggca 94 ugccaucuguuuuccauaa 832 gggauuauggaaaacagau 95 gggauuauggaaaacagau 95 aucuguuuuccauaauccc 833 uagaagaaaugaugacagc 96 uagaagaaaugaugacagc 96 gcugucaucauuucuucua 834 agcucuauuagauacagga 97 agcucuauuagauacagga 97 uccuguaucuaauagagcu 835 guaugggcaagcagggagc 98 guaugggcaagcagggagc 98 gcucccugcuugcccauac 836 cuuaggcaucuccuauggc 99 cuuaggcaucuccuauggc 99 gccauaggagaugccuaag 837 gcaggaacuacuaguaccc 100 gcaggaacuacuaguaccc 100 ggguacuaguaguuccugc 838 ggggaagugacauagcagg 101 ggggaagugacauagcagg 101 ccugcuaugucacuucccc 839 uacaauccccaaagucaag 102 uacaauccccaaagucaag 102 cuugacuuuggggauugua 840 uucccuacaauccccaaag 103 uucccuacaauccccaaag 103 cuuuggggauuguagggaa 841 aagcucuauuagauacagg 104 aagcucuauuagauacagg 104 ccuguaucuaauagagcuu 842 ccuauggcaggaagaagcg 105 ccuauggcaggaagaagcg 105 cgcuucuuccugccauagg 843 aggggaagugacauagcag 106 aggggaagugacauagcag 106 cugcuaugucacuuccccu 844 uccuauggcaggaagaagc 107 uccuauggcaggaagaagc 107 gcuucuuccugccauagga 845 cagcauuaucagaaggagc 108 cagcauuaucagaaggagc 108 gcuccuucugauaaugcug 846 aucuccuauggcaggaaga 109 aucuccuauggcaggaaga 109 ucuuccugccauaggagau 847 agcaggaacuacuaguacc 110 agcaggaacuacuaguacc 110 gguacuaguaguuccugcu 848 gaaaccaaaaaugauaggg 111 gaaaccaaaaaugauaggg 111 cccuaucauuuuugguuuc 849 aaaccaaaaaugauagggg 112 aaaccaaaaaugauagggg 112 ccccuaucauuuuugguuu 850 cagaaggagccaccccaca 113 cagaaggagccaccccaca 113 ugugggguggcuccuucug 851 uagcaggaacuacuaguac 114 uagcaggaacuacuaguac 114 guacuaguaguuccugcua 852 ugcauggacaaguagacug 115 ugcauggacaaguagacug 115 cagucuacuuguccaugca 853 uuaggcaucuccuauggca 116 uuaggcaucuccuauggca 116 ugccauaggagaugccuaa 854 uauggcaggaagaagcgga 117 uauggcaggaagaagcgga 117 uccgcuucuuccugccaua 855 auagcaggaacuacuagua 118 auagcaggaacuacuagua 118 uacuaguaguuccugcuau 856 uagacauaauagcaacaga 119 uagacauaauagcaacaga 119 ucuguugcuauuaugucua 857 cauuaucagaaggagccac 120 cauuaucagaaggagccac 120 guggcuccuucugauaaug 858 cuauggcaggaagaagcgg 121 cuauggcaggaagaagcgg 121 ccgcuucuuccugccauag 859 gauagggggaauuggaggu 122 gauagggggaauuggaggu 122 accuccaauucccccuauc 860 acaauccccaaagucaagg 123 acaauccccaaagucaagg 123 ccuugacuuuggggauugu 861 auucccuacaauccccaaa 124 auucccuacaauccccaaa 124 uuuggggauuguagggaau 862 aaccaaaaaugauaggggg 125 aaccaaaaaugauaggggg 125 cccccuaucauuuuugguu 863 ucuccuauggcaggaagaa 126 ucuccuauggcaggaagaa 126 uucuuccugccauaggaga 864 caugcauggacaaguagac 127 caugcauggacaaguagac 127 gucuacuuguccaugcaug 865 ccuguguacccacagaccc 128 ccuguguacccacagaccc 128 gggucuguggguacacagg 866 caucaaugaggaagcugca 129 caucaaugaggaagcugca 129 ugcagcuuccucauugaug 867 gacauagcaggaacuacua 130 gacauagcaggaacuacua 130 uaguaguuccugcuauguc 868 gaaaggugaaggggcagua 131 gaaaggugaaggggcagua 131 uacugccccuucaccuuuc 869 agugacauagcaggaacua 132 agugacauagcaggaacua 132 uaguuccugcuaugucacu 870 gcagaugauacaguauuag 133 gcagaugauacaguauuag 133 cuaauacuguaucaucugc 871 ggagcagaugauacaguau 134 ggagcagaugauacaguau 134 auacuguaucaucugcucc 872 ccaaggggaagugacauag 135 ccaaggggaagugacauag 135 cuaugucacuuccccuugg 873 gaagcucuauuagauacag 136 gaagcucuauuagauacag 136 cuguaucuaauagagcuuc 874 gggaagugacauagcagga 137 gggaagugacauagcagga 137 uccugcuaugucacuuccc 875 caugccuguguacccacag 138 caugccuguguacccacag 138 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agcagaugauacaguauua 152 uaauacuguaucaucugcu 890 agagaaccaaggggaagug 153 agagaaccaaggggaagug 153 cacuuccccuugguucucu 891 cccuacaauccccaaaguc 154 cccuacaauccccaaaguc 154 gacuuuggggauuguaggg 892 ugacauagcaggaacuacu 155 ugacauagcaggaacuacu 155 aguaguuccugcuauguca 893 uuaucagaaggagccaccc 156 uuaucagaaggagccaccc 156 ggguggcuccuucugauaa 894 aagugacauagcaggaacu 157 aagugacauagcaggaacu 157 aguuccugcuaugucacuu 895 gcaggaagauggccaguaa 158 gcaggaagauggccaguaa 158 uuacuggccaucuuccugc 896 uaggcaucuccuauggcag 159 uaggcaucuccuauggcag 159 cugccauaggagaugccua 897 caaggggaagugacauagc 160 caaggggaagugacauagc 160 gcuaugucacuuccccuug 898 aaagagcagaagacagugg 161 aaagagcagaagacagugg 161 ccacugucuucugcucuuu 899 cuccuauggcaggaagaag 162 cuccuauggcaggaagaag 162 cuucuuccugccauaggag 900 uaucagaaggagccacccc 163 uaucagaaggagccacccc 163 gggguggcuccuucugaua 901 auuaucagaaggagccacc 164 auuaucagaaggagccacc 164 gguggcuccuucugauaau 902 augccuguguacccacaga 165 augccuguguacccacaga 165 ucuguggguacacaggcau 903 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gggauggaaaggaucacca 603 uggugauccuuuccauccc 1341 aauuaaagccaggaaugga 604 aauuaaagccaggaaugga 604 uccauuccuggcuuuaauu 1342 aaaggaauuggaggaaaug 605 aaaggaauuggaggaaaug 605 cauuuccuccaauuccuuu 1343 acuuuccgcuggggacuuu 606 acuuuccgcuggggacuuu 606 aaaguccccagcggaaagu 1344 acagaagaaaaaauaaaag 607 acagaagaaaaaauaaaag 607 cuuuuauuuuuucuucugu 1345 agcaacagacauacaaacu 608 agcaacagacauacaaacu 608 aguuuguaugucuguugcu 1346 uauugucugguauagugca 609 uauugucugguauagugca 609 ugcacuauaccagacaaua 1347 uuaaaagaaaaggggggau 610 uuaaaagaaaaggggggau 610 auccccccuuuucuuuuaa 1348 ugcuuaagccucaauaaag 611 ugcuuaagccucaauaaag 611 cuuuauugaggcuuaagca 1349 caggaagauggccaguaaa 612 caggaagauggccaguaaa 612 uuuacuggccaucuuccug 1350 ccagaugagagaaccaagg 613 ccagaugagagaaccaagg 613 ccuugguucucucaucugg 1351 gauugggggguacagugca 614 gauugggggguacagugca 614 ugcacuguaccccccaauc 1352 aaaugaacaaguagauaaa 615 aaaugaacaaguagauaaa 615 uuuaucuacuuguucauuu 1353 agccaccuuugccuagugu 616 agccaccuuugccuagugu 616 acacuaggcaaagguggcu 1354 gacuuuccgcuggggacuu 617 gacuuuccgcuggggacuu 617 aaguccccagcggaaaguc 1355 ccaguaaaauuaaagccag 618 ccaguaaaauuaaagccag 618 cuggcuuuaauuuuacugg 1356 gcaauguaugccccuccca 619 gcaauguaugccccuccca 619 ugggaggggcauacauugc 1357 aacuuuaaaugcaugggua 620 aacuuuaaaugcaugggua 620 uacccaugcauuuaaaguu 1358 uugggggguacagugcagg 621 uugggggguacagugcagg 621 ccugcacuguaccccccaa 1359 ggacuuuccgcuggggacu 622 ggacuuuccgcuggggacu 622 aguccccagcggaaagucc 1360 cuagaacuuuaaaugcaug 623 cuagaacuuuaaaugcaug 623 caugcauuuaaaguucuag 1361 ucaguacaaugugcuucca 624 ucaguacaaugugcuucca 624 uggaagcacauuguacuga 1362 aaggaauuggaggaaauga 625 aaggaauuggaggaaauga 625 ucauuuccuccaauuccuu 1363 uacccacagaccccaaccc 626 uacccacagaccccaaccc 626 ggguuggggucugugggua 1364 gagacaggcuaauuuuuua 627 gagacaggcuaauuuuuua 627 uaaaaaauuagccugucuc 1365 cugcuuaagccucaauaaa 628 cugcuuaagccucaauaaa 628 uuuauugaggcuuaagcag 1366 aggaagauggccaguaaaa 629 aggaagauggccaguaaaa 629 uuuuacuggccaucuuccu 1367 agacauacaaacuaaagaa 630 agacauacaaacuaaagaa 630 uucuuuaguuuguaugucu 1368 cauguuuucagcauuauca 631 cauguuuucagcauuauca 631 ugauaaugcugaaaacaug 1369 uuggaaaggaccagcaaag 632 uuggaaaggaccagcaaag 632 cuuugcugguccuuuccaa 1370 ggcuguuggaaauguggaa 633 ggcuguuggaaauguggaa 633 uuccacauuuccaacagcc 1371 uaaauggagaaaauuagua 634 uaaauggagaaaauuagua 634 uacuaauuuucuccauuua 1372 aggaagaagcggagacagc 635 aggaagaagcggagacagc 635 gcugucuccgcuucuuccu 1373 aaaaaagaaaaaaucagua 636 aaaaaagaaaaaaucagua 636 uacugauuuuuucuuuuuu 1374 aucagaaagaaccuccauu 637 aucagaaagaaccuccauu 637 aauggagguucuuucugau 1375 agaccccaacccacaagaa 638 agaccccaacccacaagaa 638 uucuuguggguuggggucu 1376 caaguagauaaauuaguca 639 caaguagauaaauuaguca 639 ugacuaauuuaucuacuug 1377 aaagcuauagguacaguau 640 aaagcuauagguacaguau 640 auacuguaccuauagcuuu 1378 ugcugcauauaagcagcug 641 ugcugcauauaagcagcug 641 cagcugcuuauaugcagca 1379 uuuaaaugcauggguaaaa 642 uuuaaaugcauggguaaaa 642 uuuuacccaugcauuuaaa 1380 uuuucagcauuaucagaag 643 uuuucagcauuaucagaag 643 cuucugauaaugcugaaaa 1381 acugcuuaagccucaauaa 644 acugcuuaagccucaauaa 644 uuauugaggcuuaagcagu 1382 ggaaaggaccagcaaagcu 645 ggaaaggaccagcaaagcu 645 agcuuugcugguccuuucc 1383 uguaccaguaaaauuaaag 646 uguaccaguaaaauuaaag 646 cuuuaauuuuacugguaca 1384 gaagaaaaaauaaaagcau 647 gaagaaaaaauaaaagcau 647 augcuuuuauuuuuucuuc 1385 guguacccacagaccccaa 648 guguacccacagaccccaa 648 uuggggucuguggguacac 1386 ggggggauugggggguaca 649 ggggggauugggggguaca 649 uguaccccccaaucccccc 1387 ggaagaagcggagacagcg 650 ggaagaagcggagacagcg 650 cgcugucuccgcuucuucc 1388 gaagcggagacagcgacga 651 gaagcggagacagcgacga 651 ucgucgcugucuccgcuuc 1389 uuaaaugcauggguaaaag 652 uuaaaugcauggguaaaag 652 cuuuuacccaugcauuuaa 1390 aacccacugcuuaagccuc 653 aacccacugcuuaagccuc 653 gaggcuuaagcaguggguu 1391 guuuucagcauuaucagaa 654 guuuucagcauuaucagaa 654 uucugauaaugcugaaaac 1392 ggauuaaauaaaauaguaa 655 ggauuaaauaaaauaguaa 655 uuacuauuuuauuuaaucc 1393 guacccacagaccccaacc 656 guacccacagaccccaacc 656 gguuggggucuguggguac 1394 gauuaaauaaaauaguaag 657 gauuaaauaaaauaguaag 657 cuuacuauuuuauuuaauc 1395 aagccucaauaaagcuugc 658 aagccucaauaaagcuugc 658 gcaagcuuuauugaggcuu 1396 gcaggacauaacaagguag 659 gcaggacauaacaagguag 659 cuaccuuguuauguccugc 1397 cccacugcuuaagccucaa 660 cccacugcuuaagccucaa 660 uugaggcuuaagcaguggg 1398 gggacuuuccgcuggggac 661 gggacuuuccgcuggggac 661 guccccagcggaaaguccc 1399 aucaccuagaacuuuaaau 662 aucaccuagaacuuuaaau 662 auuuaaaguucuaggugau 1400 uagagcccuggaagcaucc 663 uagagcccuggaagcaucc 663 ggaugcuuccagggcucua 1401 gggcuguuggaaaugugga 664 gggcuguuggaaaugugga 664 uccacauuuccaacagccc 1402 uuucagcauuaucagaagg 665 uuucagcauuaucagaagg 665 ccuucugauaaugcugaaa 1403 ugacccaucaaaagacuua 666 ugacccaucaaaagacuua 666 uaagucuuuugauggguca 1404 agaaaaaauaaaagcauua 667 agaaaaaauaaaagcauua 667 uaaugcuuuuauuuuuucu 1405 agaagcggagacagcgacg 668 agaagcggagacagcgacg 668 cgucgcugucuccgcuucu 1406 aagaaaaaauaaaagcauu 669 aagaaaaaauaaaagcauu 669 aaugcuuuuauuuuuucuu 1407 aauggagaaaauuaguaga 670 aauggagaaaauuaguaga 670 ucuacuaauuuucuccauu 1408 gcugaacaucuuaagacag 671 gcugaacaucuuaagacag 671 cugucuuaagauguucagc 1409 aaaaagaaaaaaucaguaa 672 aaaaagaaaaaaucaguaa 672 uuacugauuuuuucuuuuu 1410 gaacaagccccagaagacc 673 gaacaagccccagaagacc 673 ggucuucuggggcuuguuc 1411 gugauaaaugucagcuaaa 674 gugauaaaugucagcuaaa 674 uuuagcugacauuuaucac 1412 gagcccuggaagcauccag 675 gagcccuggaagcauccag 675 cuggaugcuuccagggcuc 1413 aguggggggacaucaagca 676 aguggggggacaucaagca 676 ugcuugauguccccccacu 1414 gccugggagcucucuggcu 677 gccugggagcucucuggcu 677 agccagagagcucccaggc 1415 uggaaaggaccagcaaagc 678 uggaaaggaccagcaaagc 678 gcuuugcugguccuuucca 1416 agcaggacauaacaaggua 679 agcaggacauaacaaggua 679 uaccuuguuauguccugcu 1417 ccuagaacuuuaaaugcau 680 ccuagaacuuuaaaugcau 680 augcauuuaaaguucuagg 1418 aguagauaaauuagucagu 681 aguagauaaauuagucagu 681 acugacuaauuuaucuacu 1419 aaauuaaagccaggaaugg 682 aaauuaaagccaggaaugg 682 ccauuccuggcuuuaauuu 1420 aguaaaauuaaagccagga 683 aguaaaauuaaagccagga 683 uccuggcuuuaauuuuacu 1421 ugugauaaaugucagcuaa 684 ugugauaaaugucagcuaa 684 uuagcugacauuuaucaca 1422 agcccuggaagcauccagg 685 agcccuggaagcauccagg 685 ccuggaugcuuccagggcu 1423 cacugcuuaagccucaaua 686 cacugcuuaagccucaaua 686 uauugaggcuuaagcagug 1424 aaaaaaucaguaacaguac 687 aaaaaaucaguaacaguac 687 guacuguuacugauuuuuu 1425 gagccugggagcucucugg 688 gagccugggagcucucugg 688 ccagagagcucccaggcuc 1426 uuccgcuggggacuuucca 689 uuccgcuggggacuuucca 689 uggaaaguccccagcggaa 1427 gagagacaggcuaauuuuu 690 gagagacaggcuaauuuuu 690 aaaaauuagccugucucuc 1428 gcugugauaaaugucagcu 691 gcugugauaaaugucagcu 691 agcugacauuuaucacagc 1429 ccacagaccccaacccaca 692 ccacagaccccaacccaca 692 uguggguuggggucugugg 1430 caggaagaagcggagacag 693 caggaagaagcggagacag 693 cugucuccgcuucuuccug 1431 uaagccucaauaaagcuug 694 uaagccucaauaaagcuug 694 caagcuuuauugaggcuua 1432 uaaaaaagaaaaaaucagu 695 uaaaaaagaaaaaaucagu 695 acugauuuuuucuuuuuua 1433 gacagaagaaaaaauaaaa 696 gacagaagaaaaaauaaaa 696 uuuuauuuuuucuucuguc 1434 guaccaguaaaauuaaagc 697 guaccaguaaaauuaaagc 697 gcuuuaauuuuacugguac 1435 aaaagaaaaaaucaguaac 698 aaaagaaaaaaucaguaac 698 guuacugauuuuuucuuuu 1436 aaaaaucaguaacaguacu 699 aaaaaucaguaacaguacu 699 aguacuguuacugauuuuu 1437 agagcccuggaagcaucca 700 agagcccuggaagcaucca 700 uggaugcuuccagggcucu 1438 caggggcaaaugguacauc 701 caggggcaaaugguacauc 701 gauguaccauuugccccug 1439 cugcauuuaccauaccuag 702 cugcauuuaccauaccuag 702 cuagguaugguaaaugcag 1440 uaaaugcauggguaaaagu 703 uaaaugcauggguaaaagu 703 acuuuuacccaugcauuua 1441 aaguaaacauaguaacaga 704 aaguaaacauaguaacaga 704 ucuguuacuauguuuacuu 1442 ccacacaugccuguguacc 705 ccacacaugccuguguacc 705 gguacacaggcaugugugg 1443 aguagauuucagagaacuu 706 aguagauuucagagaacuu 706 aaguucucugaaaucuacu 1444 caucagaaagaaccuccau 707 caucagaaagaaccuccau 707 auggagguucuuucugaug 1445 accaguaaaauuaaagcca 708 accaguaaaauuaaagcca 708 uggcuuuaauuuuacuggu 1446 cacagaccccaacccacaa 709 cacagaccccaacccacaa 709 uuguggguuggggucugug 1447 aggggggauugggggguac 710 aggggggauugggggguac 710 guaccccccaauccccccu 1448 ugcauuuaccauaccuagu 711 ugcauuuaccauaccuagu 711 acuagguaugguaaaugca 1449 caauggacauaucaaauuu 712 caauggacauaucaaauuu 712 aaauuugauauguccauug 1450 cugaacaucuuaagacagc 713 cugaacaucuuaagacagc 713 gcugucuuaagauguucag 1451 gccucaauaaagcuugccu 714 gccucaauaaagcuugccu 714 aggcaagcuuuauugaggc 1452 uguacccacagaccccaac 715 uguacccacagaccccaac 715 guuggggucuguggguaca 1453 gaaguaaacauaguaacag 716 gaaguaaacauaguaacag 716 cuguuacuauguuuacuuc 1454 guaggaccuacaccuguca 717 guaggaccuacaccuguca 717 ugacagguguagguccuac 1455 caguggggggacaucaagc 718 caguggggggacaucaagc 718 gcuugauguccccccacug 1456 acccacugcuuaagccuca 719 acccacugcuuaagccuca 719 ugaggcuuaagcagugggu 1457 aaaaauugggccugaaaau 720 aaaaauugggccugaaaau 720 auuuucaggcccaauuuuu 1458 uggggggacaucaagcagc 721 uggggggacaucaagcagc 721 gcugcuugaugucccccca 1459 guacaaauggcaguauuca 722 guacaaauggcaguauuca 722 ugaauacugccauuuguac 1460 aagcuauagguacaguauu 723 aagcuauagguacaguauu 723 aauacuguaccuauagcuu 1461 cagaagaaaaaauaaaagc 724 cagaagaaaaaauaaaagc 724 gcuuuuauuuuuucuucug 1462 aaaugcauggguaaaagua 725 aaaugcauggguaaaagua 725 uacuuuuacccaugcauuu 1463 agccucaauaaagcuugcc 726 agccucaauaaagcuugcc 726 ggcaagcuuuauugaggcu 1464 ccacugcuuaagccucaau 727 ccacugcuuaagccucaau 727 auugaggcuuaagcagugg 1465 aagaagcggagacagcgac 728 aagaagcggagacagcgac 728 gucgcugucuccgcuucuu 1466 aaauggagaaaauuaguag 729 aaauggagaaaauuaguag 729 cuacuaauuuucuccauuu 1467 agccugggagcucucuggc 730 agccugggagcucucuggc 730 gccagagagcucccaggcu 1468 aacaagccccagaagacca 731 aacaagccccagaagacca 731 uggucuucuggggcuuguu 1469 uaccaguaaaauuaaagcc 732 uaccaguaaaauuaaagcc 732 ggcuuuaauuuuacuggua 1470 uucaaaaauugggccugaa 733 uucaaaaauugggccugaa 733 uucaggcccaauuuuugaa 1471 agaagaaaaaauaaaagca 734 agaagaaaaaauaaaagca 734 ugcuuuuauuuuuucuucu 1472 cuguguacccacagacccc 735 cuguguacccacagacccc 735 ggggucuguggguacacag 1473 gccuguacugggucucucu 736 gccuguacugggucucucu 736 agagagacccaguacaggc 1474 caguaaaauuaaagccagg 737 caguaaaauuaaagccagg 737 ccuggcuuuaauuuuacug 1475 uacaaauggcaguauucau 738 uacaaauggcaguauucau 738 augaauacugccauuugua 1476 [ 0262 ] table ii a . 2 . 5 μmol synthesis cycle abi 394 instrument reagent equivalents amount wait time * dna wait time * 2 ′- o - methyl wait time * rna phosphoramidites 6 . 5 163 μl 45 sec 2 . 5 min 7 . 5 min s - ethyl tetrazole 23 . 8 238 μl 45 sec 2 . 5 min 7 . 5 min acetic anhydride 100 233 μl 5 sec 5 sec 5 sec n - methyl 186 233 μl 5 sec 5 sec 5 sec imidazole tca 176 2 . 3 ml 21 sec 21 sec 21 sec iodine 11 . 2 1 . 7 ml 45 sec 45 sec 45 sec beaucage 12 . 9 645 μl 100 sec 300 sec 300 sec acetonitrile na 6 . 67 ml na na na b . 0 . 2 μmol synthesis cycle abi 394 instrument reagent equivalents amount wait time * dna wait time * 2 ′- o - methyl wait time * rna phosphoramidites 15 31 μl 45 sec 233 sec 465 sec s - ethyl tetrazole 38 . 7 31 μl 45 sec 233 mm 465 sec acetic anhydride 655 124 μl 5 sec 5 sec 5 sec n - methyl 1245 124 μl 5 sec 5 sec 5 sec imidazole tca 700 732 μl 10 sec 10 sec 10 sec iodine 20 . 6 244 μl 15 sec 15 sec 15 sec beaucage 7 . 7 232 μl 100 sec 300 sec 300 sec acetonitrile na 2 . 64 ml na na na c . 0 . 2 μmol synthesis cycle 96 well instrument equivalents : dna / amount : dna / 2 ′- o - wait time * 2 ′- o - reagent 2 ′- o - methyl / ribo methyl / ribo wait time * dna methyl wait time * ribo phosphoramidites 22 / 33 / 66 40 / 60 / 120 μl 60 sec 180 sec 36o sec s - ethyl tetrazole 70 / 105 / 210 40 / 60 / 120 μl 60 sec 180 min 360 sec acetic anhydride 265 / 265 / 265 50 / 50 / 50 μl 10 sec 10 sec 10 sec n - methyl 502 / 502 / 502 50 / 50 / 50 μl 10 sec 10 sec 10 sec imidazole tca 238 / 475 / 475 250 / 500 / 500 μl 15 sec 15 sec 15 sec iodine 6 . 8 / 6 . 8 / 6 . 8 80 / 80 / 80 μl 30 sec 30 sec 30 sec beaucage 34 / 51 / 51 80 / 120 / 120 100 sec 200 sec 200 sec acetonitrile na 1150 / 1150 / 1150 μl na na na [ 0263 ] table iii human hiv - 1 sequences accession name subtype af069669 se8538 a af069671 se7535 a af069673 se8891 a af107771 ugse8131 a af193275 97bl006 af193275 a af361872 97tz02 af361872 a af361873 97tz03 af361873 a af413987 98ua0116 af413987 a af004885 q23 - 17 a1 af069670 se7253 a1 m62320 u455 u455a a1 u51190 92ug037 a1 af286237 94cy017 . 41 a2 af286238 97cdktb48 a2 a04321 iiib lai b ab078005 ares2 ab078005 b af003887 wc001 b af003888 nl43wc001 b af004394 ad87 ada b af033819 hxb2 - copy lai b af042100 mbc200 b af042101 mbc925 b af042102 mbc18 mbcc18 b af042103 mbcc54 b af042104 mbcc98 b af042105 mbcd36 b af042106 mbcc08r01 c18r01 b af049494 499jc16 b af049495 nc7 b af069140 dh12 - 3 b af070521 nl43e9 lai iiib / ny5 b af075719 mntq mnclone tq b af086817 twcys lm49 b af146728 vh b af224507 wk b af256204 s61i1 af256204 b af256205 s61d15 af256205 b af256206 s61g1 af256206 b af256207 s61g7 af256207 b af256208 s61i15 af256208 b af256209 s61k1 af256209 b af256210 s61k15 af256210 b af256211 s61dl1 b af286365 wr27 b aj006287 89sp061 89es061 b aj271445 gb8 gb8 - 46r him271445 b ax078307 bh10 b ay037268 arch054 b ay037269 arms008 b ay037270 bol 122 b ay037274 arma173 b ay037282 arma132 b d10112 cam1 b d86068 mck1 b d86069 pm213 b k02007 sf2 lav2 arv2 b k02013 lai bru b k02083 pv22 b k03455 hxb2 hxb2cg hxb2r lai b l02317 bc bcsg3 b l31963 th475a lai b m15654 bh102 bh10 b m17449 mncg mn b m17451 rf hat3 b m19921 nl43 pnl43 nl4 - 3 b m26727 oyi , 397 b m38429 jrcsf jr - csf b m38431 ny5cg b m93258 yu2 yu2x b m93259 yu10 b nc_001802 hxb2r b u12055 lw123 b u21135 weau160 ghosh b u23487 contaminant manc b u26546 wr27 b u26942 nl4 - 3 lai / ny5 pnl43 nl43 b u34603 h0320 - 2a12 ach3202a12 b u34604 3202a21 ach3202a21 b u37270 c18mbc b u39362 p896 89 . 6 b u43096 d31 b u43141 han b u63632 jrfl jr - fl b u69584 85wcipr54 b u69585 wcipr854 b u69586 wcipr8546 b u69587 wcipr8552 b u69588 wcipr855 b u69589 wcipr9011 b u69590 wcipr9012 b u69591 wcipr9018 b u69592 wcipr9031 b u69593 wcipr9032 b u71182 rl42 b x01762 rehtlv3 lai iiib b z11530 f12cg b [ 0264 ] table iv human hiv - 1 sequences accession name subtype ab032740 95tnih022 01_ae ab032741 95tnih047 01_ae ab052995 93jpnh1 01_ae ab070352 nh25 93jpnh25t 93jp - nh2 . 5t 01_ae ab070353 nh2 93jpnh2env 01_ae af164485 93th9021 01_ae af197338 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06_cpx aj288981 97se1078 06_cpx aj288982 95ml127 06_cpx af286226 97cn001 054 07_bc af286230 98cn009 07_bc ax149647 c54a c54 07_bc ax149672 c54d ax149672 07_bc ax149771 cn54b 07_bc ax149898 c54c 07_bc af286229 98cn006 08_bc ay008715 97cngx6f 08_bc ay008716 97cngx7f 08_bc ay008717 97cngx9f 08_bc af289548 96tzbf061 10_cd af289549 96tzbf071 10_cd af289550 96tzbf110 10_cd af179368 gr17 11_cpx aj291718 mp818 11_cpx aj291719 mp1298 11_cpx aj291720 mp1307 11_cpx af385934 urtr23 12_bf af385935 urtr35 12_bf af385936 arma159 12_bf af408629 a32879 af408629 12_bf af408630 a32989 af408630 12_bf ay037279 arma185 12_bf af423756 x397 af423756 14_bg af423757 x421 af423757 14_bg af423758 x475 af423758 14_bg af423759 x477 af423759 14_bg af450096 x605 af450096 14_bg af450097 x623 af450097 14_bg af069669 se8538 a af069671 se7535 a af069673 se8891 a af107771 ugse8131 a af193275 97bl006 af193275 a af361872 97tz02 af361872 a af361873 97tz03 af361873 a af413987 98ua0116 af413987 a af004885 q23 - 17 a1 af069670 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