CA2119927A1 - Duplex-forming polynucleotide conjugates - Google Patents

Abstract

2119927 9306122 PCTABS00021 The invention provides ligand-binding duplexed structures having significantly enhanced stability under physiological conditions.
The structures are provided in the form of polynucleotide conjugates capable of adopting a duplexed structure, in which annealable polynucleotide strands are coupled covalently at one or both ends through a chemical linker which establishes a stabilizing bridge between strands. Various applications for the stabilized duplexed structures are described, including therapeutic applications for example in the treatment of AIDS.

Description

211~927 WO 931W~22 PCI`/CA92/00423 '' 1 DUPL~X-FORMING, POLYNUCLEOTIDE CONJI.JGATES

This invention is in the field of nucleic acid chemistry. Mor~
par~icularly, the invention relates to polynucleotide conjugates that adopt 5 a li~and binding duplexed structure, ~o the produ~tion of such conjugates particularly via automated synthesis techniques, and to their use in therapeutic, di~gnostic and o~her applications.

Backc~ound to the Invent!on ~ :
The ability to regulate cellular processes 2t ~he gene~ic lev~l in a highly selective and therapeutic manner is now offered by various forms o~oligonucleotide-based pharmaceuticals. These Qligonucl~otides are ;~ dssigned according to their nucleic acid sequence to arrest geneticprocesses by binding disruptively to a selected genetic target, usuall~/ a ~ ` -viral gene or a human gene that is associated with a partioulaF disease sta~e such as cancer or a condition such as inflammation. Transcription of an undesired gene can, for example, be arrested by a synthetic oligonucleotide that hybridizes selectively to a control region or coding region of ~hat gene; similarly, translation of an undssired protein can be arrested using an oligonucleotide that hybridizes with a control re~ion or ~ ~:
coding region of the messen~er RNA encoding that protein. Many of the problems associat~d with the practical use of such oligonucleotide-based therapeutics, such as cell uptake, stability, and cos~ of production, have been resolvsd by recent advances in nucleic acid chemistry.
These current strategies contemplate principally ~he use of uligonucleotides which, in order to hybridize to thsir intended nucleic acid -target, are necessarily single-stranded complements of that t3rget. That is, oligonucleotides intended for use as pharm3ceuticals are designed currently to bind as single^stranded entities to other ~ucleic acid ~arg~ts, : `
whether single-stranded messenger RNA or single str~nded DNA tthe so-called "sense" and "anti-sense" approaches, revi~wed for example by SU~Ji~UTE SHEET ~:~

2119927 : ~

-2- ;
example by Uhlmann et al, 1990, Chemical Rev., 90:543) or, as has more ~-recently been proposed, to double stranded DNA (the "tr;plex" approach). These approaches neglect other cellular tarOets that are at ieast equally attractive in the overall development of gene regulating therapeutics. More particularly, it would ~~`

3 be desirable to provide oligonucleotide agents capable of interfering with interactions specifically between nucleic acids and their ligands, particularly their protein ligands, having a role in infectious and other disease states.
. ~.
The feasibility of designing oligonucleotides that interfere with a -10 protein/nucleic acid interaction of therapeutic interest is complicated in tha~, in the rnajority of instances, the protein recogni7es a nucleic acid that is doublestranded in structure; and further in that double stranded oligonucleotides of the small size necessary for pharmaceutical applications, for uptake by the cell, and . .. ..
for stability, are highly unstable and must typicaliy be incubated under, 15 temperatures so cold and/or salt concentr~tions so high as to make subsequent study and use of the duplexed structures impractical.
: ' '' ~ It is a principle object of the preser,t invention to provide polynucleotide conjugates that are capable of adopting a ligand-binding duplexed structure.
It is a further object of the present invention to provide ligand-bind;ng polynucleotide conjugates of enhanced stability, i.e. of enhanced physical or -~
chemical stability. ~ -ît is another object of the present invention to provide a process for preparing such duplexed structures of enhanced stability.

It is another object of the present invention to provide compositions for therapeutic use that incorporates, as an active ingredient, a polynucleotide conju~ate that is capable of adopting a stability~nhanced duplexed structure that binds with SIJBSTITUTF ~HEET

WO 93/116122 2 1 1 9 9 2 7 P~/CA92/00423 ~ .
polynucleotide conjugate that is eapable of adopting a stability-enhanced duplexed structure that binds with a target ligand of therapeutic in~erest.
:: .-Summary of ~he Inventipn There is provided by the present invention a family of duplex- :
forming compounds, herein referred to as polynucleotide conjugates, which comprise a first polynucleotide strand having an end, a second polynucleotids s~rand having an end and which is capable of annealing ~:
with the first polynucleo~ide strand to form a ligand bindin~ structure, and ~: -a chemical linker which is coupled between ends of the strands to form a bridge permi~ting the conjugate to form a ligand^binding duplexed structure.

According to one aspect of the present invention, the stability~
enhanced duplexed structures o~ ~he invention are prs)vided in the form of linear pvlynucleotide conjugates, conforrning to ths general formuia:

:.. . :~
X - L - Y tl) 20 wherein:
X is a polynuoleotide havin~ a 3'terminus;
Y is a polynuoleotide capable o~ annealing with )(, and having a 5'terminus; and L is a chemical linker coupled be~wesn the 3'terminus of X and the 5~terminus of Y to form a bridge permit~ing the conjugate to 25 form a ligand-binding duplexed strue~ure.

C:ompounds conformin~ to the general formula (i) are linear polynucleotide conjugates and are most conveniently produced using automated polynucleotide syn~hesis techniques. Forthis purpose, the 30 present invention further provides analo~ues of the chemical linkers in bifunctional form for incorporation between nucleotide strands using established nucleotide coupling protocols.

SUB~tTUTE SI~EEl~

WO 93/0612 pcr/cA92/oo423 2119~27 4 The stability-enhanced duplexed structures may also be in the form - -of cyclic polynucleotide conjugates, which ccnform either to the general formula: :

~\ ';-''.. '~'' X Y Illa~

L : ~
1 0 "' '' ` '~''' wherein~
X is a polynucleotide having a 5'terminus and 3 3'terminus;
Y is a polynucleotide capabie of annealing with X and having a 3'terminus and a 5'terrninus; H H ~-Z is a polynucleotide coupled covalently be~ween the 5'terrninus of X and the 3'terminus of Y; and L is a chen ical linker cvupled between the 3'terminus of X and the 5'~erminus of Y, to form a bridge permitting the conjugate to form a -;
ligand-bindingduplexed structure;
~0 ' :
or to the general formula:
~ ' X Y Illb) wherein~
3C) X and Y are as defined above; and L1 and L2 are independently selected chemical linkers coupled, ~ :
respectively, bstween ~h~ 3'terminus of X and the 5'terminus of Y and ~ `

SUB~UTE SHEET `~

WO 93/061 22 2 119 9 2 7 pcrlcA92/oo423 the 5'terminus of )C and the 3'terminus of Y, to form chemical bridges permitting the conjugate to form a ligand-binding duplexed structwe.
- ~, The cyclic polyrluoleotide conjugates of the invention, as represented by formulae lla and llb are suitably prepared by synthesizing : -the linear analogue thereof using the automated nucleotid~ coupling ~ -techniques appropriate for linear conjugates of formula (I) and then clssin~ the linear conjugate typically using either chemioal ur enzymatie ~ -means, to form the cyclic polynucleotide conjugate. ~ -In accordance with another aspect of the present invention, there - ~-is provided a pharmaceutical composition which oomprises a li~and-binding polynucleotide conjuga~e of the present invention and a pharmaceutically acceptable carrier. In valuable embodiments of the invention, the poiynucleotide conjuga~e is one capable of adopting a duplexed structlJre that is reco0nized by i.e. binds with, a target ligand that is a protein, for example a protein capable of regulatin~ gene ~xpression. !n a specific embodiment of thc pres~nt inven~ion, the - :
polynucleotide conju~ate is charact~rized by an affinity for binding with protein which regulates viral gene expression e.g. ~he HIV tat protein.
Alternatively, the polynuGleotid~ conjugat~ can be designed by appropriats s~lection of its componen~ polynucleo~ide strands and linker(s) to bind with proteins that regulate oncogene expression, or expression of gen~s implicated in other diseass states or medioal 2S conditions.

According ~o another aspect of the present invention, the chemioal linker component of the polynucleotide conjugate incorporates a ::
functional group which serves as a sit~ of attachment for a reporter molecule, such as a radiolabe! or other diagnostically useful label.
Accordingly, the invention further provides d~tectably labelled analo~ues of the poiynucleotide conjuga~es, for diagnostic use or for use in assays SUB~lTU~E SHEET
~

WO 93~06122 PCr/CA92/00423 11g927 6 designed to measure binding between the duplexed form of the conjugate and a ligand, such as a DNA- or RNA-binding protein. Further, the attachment site within the chemical linker may be exploited to couple the polynucleotide conjugate ~o an affinity column matrix, for use in :
5 extracting ligands from biological sources.

These and other aspects of the present invention are now . ~:
described in greater detail with reference to the accompanying drawings, ~ -in which~

Brief RQferenc~ to the Drawin~
- -Figures 1 and 2 illustra~e duplexed structures of various confsrmations and configurations that can be stabilized in acsordance with the present invention. Solid lines illustrate polynucleotide structure - ` :
and.hatchin~ identifies th~ nucleotide components. The symbol "." is ~ -used to indicate hydrogen-bonded base-pairing within anr)ealed regions of th~ polynucleotide strands, and the symbol L is used ~o indicate loca~ion ~ -of ths chemical linker;
Figure 3 shows incorporation of a specific linker of ~he present-~ -invention between polynucleotide strands;

Figures ~7 illustrate the structure of specific polynueleotide 25 conju~ates of the present invention; and Figures 8 and 9 illus~rate graphically the cellular up~ake of specific polynucleotid~ conjugates of the invention.

30 Detailed DescriD~i~n Qf the InventiQn The presen~ invention provides- polynucleo~ide conjugates characterized by the prsperties of ligand bindin0 and enhanced stability.
~. ~

SUBSIIIUTE SHEET ` ~
: -; .

. ` 7 In the present specification, the term "enhanced stability" refers unless otherwise stated to the superior thermal stability o~ a polynucleotide conjugate relative to its unlinked counterpart, as measured using melting ~ ~ -temperature lTm) assays established in the art. The term "ligandnis used 5 herein with reference to 2gents that bind measurably, in the cont~x~ of an assa~/ appropriate for that measurment, to nucleic scid structures, principally double stranded struc~ures but aiso single stranded struc~ures.
The term ligand is thus intended to embraee such agents as proteins.
including proteins that regulate genetic processes such as transcription 10 and translati~n, as well as non-protein en~ities ineluding but not limited tointercalating agents and nucleic acid binding antibiotios as well as other nucleic acids. The term "ligand-bindin~" is thus used with reference ts ~ -polyhucl~otide eonjugates that adopt a structure that is bound measurably by a ligand to which ~he conjugate is targetted.

In providing duplexed structures of enhanced ~tability, the pre~ent invention permits the use of dou~le stranded polynucleotide structures in a wide variety of applications no~ previously possible, because of prior stability problems. B~cause the chemically link~d duptexed structures of 20 ~he pfssent invention are substantially more stable than their unlinked counterpafts under physiological conditions, for example, therapeu~ic applications ~or duplexed structures ars now fea~ible. In addition, it will ~ -~e appreciated that the stability-enhancing effect of ~he chemical linker can be exploited to eliminate polynucleotide regions that are otherwise 25 requir~d to p~rmit formation and maintenance of the desired duplexed structure in vitro and in vivo. Thus, duplexed structures $hat are much ~ -smallQr in molecular weight and accordingly more acceptable for therapeutic use, can be produced. Furthermore, ~he chemical linkers exploited in the present invention are substarltially resistant ~o nuclease 30 di~es~ion, and thus further contribute to duplex stability.

.. . .

SUBSTITUTE SHEET
~ ; ~,, WO 93/06122 P~/CA92~00423 To stabilize polynucleotides, the present invention applies the strategy of incorporating a chemical linker b~ween one or both ends of polynucleotide strands capabi~ of forming a dupl~xed structure. It will be understood that in order to form a duplexed structure, such strands will 5 share at least a re~ion of sequence complementary sufficient to permit annealing of the strands. The individual polynucleotide strands forming the duplex may consist of RNA or DNA monophospha~s or synth~tic -~ :~
analogues thereof, or mixtur~s thereof. Synthetic analogues include for example those incorporating variations in the base cQnstituent, such as thio- and aza-substituted bases; in the sugar consitituent such as alkyl-and halo-substitu~ed riboses and arabinose equivalents; and analogues incorporatin~ variation in the monophosphate group, such as phosphorothioates and dithioates, methyl phosphate and methyi phosphonates, phosphoramidates and phosphorarnidites and the like. As is h~rein described, a polynucleotide strand may also incorporat~ a non~
nucleic acid component, to the extent that duplex formation and ligand - binding ar~ not substan~i311y impaired.
- ~:
The polynucleotid2 strands forming the duplex may be of ~he same or different lengths, and each may incorpora~e any number of nucleotides in the range from 2 ~o a maximum tha~ is dictate~ largely by the limits of automated gene synthesls techniques. Strands consistin~ of not more than ~bout 200 nucleotides, for example not more tt an abou~ 100 nucleotides, will derive the most benefit from th~ stabilking effect of ~he chemical bridge, however. Preferably each of the polynucleotid0 strands : ~ -consists of from 3 to 100 nucl~otides, and more pr~ferably, from about 4 ~o 50 nucleotides. Polynucleotide strands that are capable o~ annealin~
and which can ~hus benefit from ~he linker strategy herein described, include those strands that anneal in their anti-parallel orientation i.e.
consist of beta nucleotides, and strands that consist of alpha nucleotides in one s~rand and beta nucleotides in the other strand, and thus can anneal in the parallel orientation. In the simples~ case, ~he polynucleotide SUB~TITUTE SHEET

WO 93~06122 2 1~9 9 2 7 P(~/CA92/00423 strands will be precisely complementary and equivalent in length, and will anneal along their entire length, to form a completely double stranded duplexed structure. It will be appreciat~d however, that with the aid of a chemical linker, duplexed structures having a variety of conformations 5 and conffglJrations oan be stabilized, in accordance with ~he present in~ention. Some of the dupl~xed structures currently cDntempla~ed are illustrated schematically in Figures 1 and 2, to which referen~ is now ~ -made. Other structur~s or combinations may also b~ stabilked in accordance with th~ pr~s~nt invention, of course.
As shown schematically in Figure 1, duplexed structures that can be gen~rated as linear polynucleotide conjugates of ~he g~neral formula (l~
comprise a single chemical linker incorporated at one end of the duplex struc~ure. Figure 1 (a) illustrates the simplest cas~ which, as described 15 above, incorporates a linker at one end of precisely complementary polynucleotide strands, which anneal along their entire len~th to form a fully double stranded duplex structure. FiQur~ 1 ~b) illustra~e~ the case in -which the annealable strands incorpofate a termina~ mismatch, which ~ -results in a non-annealing "fork" structure at one end of ~he duplex.
20 Figure 1 (c) illustrates the situation in whieh one polynucleotide strand ineorporates an internal, mismatched re~ion resu!ting in a non-anneaied bulge. Figure 1~c~ further illustrates that polynucleotide s~rands of diff~rent len~h can also be linked, according to ~he present inv~ntion, as is furth~r shown by the struc~urs of Fi~ure 1 Id~
2~
Similarly, dupi~xed structures that can be yenerated as cyclic polynucleotide conju~ates of the formula (lla) and (llbi may also adopt various conformations and configurations. As shown in Figure 2(a), the simplest case is again the situation where precisely complemen~ary 30 strands are coupled using chemical linkers at both ends. Similarly, th2 forked structure shswn in Figure 2(b) can also b~ linked at both ends, as may the bul~ed structure shown in Figur~ 2(c~. The forked structur~ of ~UBST~TUTE SHEET ~

WO 93/06122 PCI`/CA92/00423 21199~7 tO
Figure 2(b~ also illustrates that chemical linkers of different length may be used to bridge polynucleotide strands in the annealing relationship desir~d for duplex formation. Duplexes that are more elaborate in structure can also be stabilized if desired, as shown for example in Figures 2(d~ and 2(e). The duplexed structures appearing in Figures 2(a) - (e~ are intended to be embraced by the general formula Illb) recited hereinaboYe.
.
The duplexed structure illus~rated in Figure 2(f) represents a special but important case, in which a cyGlic duplexed struc~ure is created by 11) incorporation of a single chemical linker, as embraced generally by the formula Illa) recited hereinaboYe. In this case, Z is r~presented by the polynucleotide 'loop' brid~ing the annealed polynucleotide strands. As will be described herein, such structures exist naturaily in the unlinked fs~rm, occurring predominantly in the form of RNA nhairpins" that re~ulate the expression of certain viral and other genes through a protein-bindir~g interaotion. Such duplexed structures are accordingly ideal as tar~ets for therapeutic ~pplication, when in their chemically linked form.

As noted ~bove, the linking of duplex-~rming polynucleo~id2 strands is achieved by covalently coupling the chemical linker between neighbouring termini of the polynucleotide strands, either b~tween the 5't~rrninus of one strand and the 3'terminus of the other, or vice ~ersa.
As is shown in Fi~ure 3, linkers are msst suitably incorporated by coupling between the rnonophosphate or analogous groups borne at the termini. It is to be understood that the chemical linkers used in ~he pre~ent inventisn are synthetic chemical linkers as opposed ~o pslynucleotide-based linkers of the type represented b~/ substituent ;Z in ~ -Formula (lla). ~ ~ ~
'`:~ '."
The chemical linker has a length selected ideally ~o preserve the desired annealing r~lationship between strands at the l~cation of the linker. Since numerous duplex conformations oan be stabili~ed using the SUBS~JTE SHEET

WO 93~06122 ` 211 9 9 2 7 PC~/CA~2/00423 1 '1 linker, linkers of similarly various lengths can be incorporated for ~his purpose. (ienerally, the length of the linker will correspond to the length of a linear chain alkane Gomprising from about three carbon atoms ~C3) to about 30 carbon atoms (C30). It has b~en found in particular that a chemical linker havin~ a length equivalent to a linear chain alkane consisting of from 7 to 20 carbon atoms, suitably 8 to 15 carbon atoms and desirably 9-12 carbon atoms, is appropriate to link polynucleotide strands at an annealed location. For coupling of strands at a mismatched, non-annealed location, a chemical linker having a len~th equivalent to greater than about 10 carbon atoms, for example having a length in the range from about 10 car~on atoms ~o ~bout 20 carbon atoms, is suitabl~ for incor,~oration. Since functional groups are al~o - incorporated at the ends of the linker ts:~ permit coupling with nucleotides, ~~ as described below, determination of desired linker length should be made with this in mind. ::

The chemical composition of the linker can vary widely, provided that consideration is given to the nsed for stability under physiolo~ical :~:
conditions and under the conditions encountered durin~ nucleotide couplin0 protocols. The linker may con~ain functional yroups, for example to s~rve as a~achment sites for other molecular entities, providsd that suitable protecting groups are employed durin~ synthesis of -the polynucleotide conju~ate. A key requiremen~ in choosin~ a linker composi~ion is to rc~ain the length appropria~e for duplex formation. in this connsction, it will be appreciated that sid~ chains are acceptable, .par~icularly in the c~ntral region of the linker. Moreover, the desir~d len~th of the linker can be achieved using carbon atoms or carbon atoms in combination with heteroa~oms, including oxygen, sulfur, phosphorus, nitro~en, etc. Also, cyclic structures ean be incsrpe~ra~ed, including benzene and heterocycles such as piperidine, piperazine or pyridine coupled within the linker chain either through a carbon center or a heteroatom. It will also be apprsciated that tlle chemical compositiorl of :

SVB~ T~ SI~ET

WO 93/0Sl22 PCr/CA9VU0423 the linker can be manipulated through component selection to alter hydrophobicity or hydrophilicity, if desired, particularly for the purpose of altering solubility, cellular uptake, and to facilitate dosage Formulation where therapeutic applications are being considered.

For incorpora~ion between nucleo~ides, the chsmical linkers ar~
- provided in th~ form of bifunctional analogues, bearing terminal functional ~roups that, desirably, are amenable to protection and derivatization that adapts them for coupling using the same protocols applied conventionally - ~
~or automated nucleotide coupling. Such bifunctional linker analogues ~ -conformto ~he general formula, - R - linker- R' , 15 wherein, most suitably, R and R' are independen~ly s~lected from among the group consis2ing of -OH, -SH, -N~l and functional ~quivalents of th~se groups. So that the linkers can be incorporated, and ths polynucl~otide conju~ates synthesiz~d, using the currently most practical phosphorarnidite approach, the linker is preferably on~ in which at least 20 one of R and R' is OH. Most preferably, both R and R' are OH.
,` '~' ''~
Bifunctional linkers suitable for use in coupling polynwl~otide strands ~t an annealed location are exemplified by, and include~

HO-(CH2)n-OH, n= 6-18 HO-(Ctl2CH2-O)n-(CH2)2-OH, n = 2-10 HO-lCH2)mCH=CH-lCH2)n-OH, m,n= 2-îO
HO-(CH2)m-Ph~nyl-(CH2)n-OH, m,n= 2-10 HO-lCH2)m-Phenyl-Phenyl-lCH2)m-OH, m,n-2-10 5 lO-lCH2)m-C_C-l ::H2)n-OH, m,n = ~-10 HO-lCH2)m-piperazinyl-lCH2)n-OH, m,n = 2-10 --SUBS,;U~UTE SHEET

wo 93,0~l2? 2 1 1 9 9 2 7 P~r/CA~2100423 HO~((~W2)m~0~P~~0)2~~CH2)n~0H~ m,n = 2-10 It should be appreciated that linkers of appropriate len~th may also be formed in situ i.e. during conjugate synthe~is, by couplin~ sslected linkers sequentially to extend link~r length as desirsd.

The polynucleotide conjugates of general forrnula ~1), which are linear molecules capable of forming duplexed structures can be ~-~
synthesized by applying now conven~ional techniques of polynuoleotide 10 synth~sis, particularly in csmbination with the commercially available polynucleotide synthesizing devices, or "gsne machinesn. Various ~ `~
strategies of solution and solid phase synthesis can be used, of course, including the phosphotries~er method, the solid phas~ H-phosphonate E
method or the solid phase phosphoramidite method. The latter is ~ ~ -15 curr~ntly th~ method of choice, for synthesls of polynucleotide-based compounds of the invention. In th~ phosphoramidite approach, nucleotides that are fully pro~ected ~re coupl~d sequerltially, in th~ 3' -->
5' direction, to a first nucleotide that is coupled relea~ably to a solid supp~r~, such as aminopropyl controlled pore ~lass or polystyrene re~in.
20 Nucleotid~ protec~ing groups include, for nucleophilic amino functions on the bases, either isobutyryi (N-2 of guanine) or ben~oy1 ~N-6 of adenine snd N-4 of cytidine) tha~ are r~movable upon completion of synth~sis by ` :~
ammoniolysis. In the case of deoxyribonucleotides, the 5' prim~ry hydroxyl of the deoxyribose su~ar is proteoted with an ether moiety, 25 either dimethoxytrityl fDMT) or monomethoxytri~yl (MMT~, which is removed by mild protic acids a~ the beginning of each coupling cycls.
The 3' secondary hydroxyl functisn of the deoxyribose su~ar is derivatized with the highly reactive phosphoramidite ~roup, either methyl ;
phosphorarnidite or ~cyanoethyl phosphoramidits, which is activated for 30 coupling by a weak acid.

SUBSl;JIL)TE SI~ET

WO 93/06122 pcr/cA92/oo423 For incorporation into such an automated synthesis procedure, the bifunctional linker analogues of the present invention can be similarly protected and deprotected for coupling. Thus, in the case where the linker analogue bears terminal hydroxyl groups, these may be protected in the same manner as the 5' and 3' hydroxyls of the nucleotides selscted for coupling. In other words, one hydroxyl is protected with the ether moiety, such as DMT, and the other is derivatized to provide the phosphoramidite group, ~o yield a compound of the general s~ructure, I~MT-0-linker-0-phosphoramidite. This permits unidirectional incorporation of the linker into the linear polynucleotide, at a desired -`-position alon~ its length.

.
Techniques for obtaining linkers sui~ably adap~ed for nucleDti~e coupling reac~ion are provided in Example 1 herein. Briefly, for dimethoxy or monomethoxy trityla2ion, the trityl halide and a slight molar Bxcess of th~ diol are reacted in pyridine at room temperature, and the product is recovered after mixing with methanol, rssuspension in chloroform and - -th~n washing and drying, with solvent removal. The tritylated produot can then be phosphitylated, to protect the remaining hydroxyl group, by reac~ion with 2-cyanoethyl-N,N-diisopropylchorophosphoramiditcin the ;~
conventional manner. The so-protected diol linker can then be incorporated into an automated nucleotide synthesis protocol in the same rnann~r as would any protected nucleotide.

Thus, to produce a linear polynucleotide conjug~te, the resin-bound firs~ nucleotide is treated with prstic acid to remove the trityl pro~eçting group at the 5'hydroxyl, the 3'hydroxyi phosphoramidite ~roup of the next nucleotide is activated to allow 3' to 5' couplin~, and then oxidized ~ -to complete coupling. At the desirsd poin~ in the sequence, the protected linker is incorporated using the same deprotec~ion/astivation strate~y and ~ ~ -the couplin~ continues until the linear form of ~he d~ubls s~randed oligonucleotide is produced. This is then released from the solid support SUBS;I;LTUT~ SllEET

~YO 93/0612? 2 1 19 9 2 7 pcr/cA92~oo423 and treated tO deprotect bases, isolated and then purified usin~ well established protocols.

Figure 3 provides the chemical structure resulting from the covalent 5 coupling of a speci~ic triethylene glycol-derived linker, between polynuclsotides. It will be noted that the linker is coupled to the termini of the nucleotides through ~he monophosphates borne on the respective ~' and 3' hydroxyl groups. ;

Substantially the same synthesis protocol can b~ employed for synthesis of RNA-based, linear polynucleotide conju3at~s, but with use of - -a b!ockin~ sroup for the 2'hydroxyl, such as the tert-butyldimethylsilyl :- group (TE~OMS) or ~he triisopropylsilyl group (TiPS), and op~ionally with - ~
.. ~
use of the MMT or DMT e~hers for 5'hydroxyl protsc~ion.

For the production of cyclic polynuclsotide conju~ates of the - :-present invention i.e. those of ~ormulae (llal and (llb), a lin~ar analogue of the cy~lic mole~ule is first produced using the procedure described above for linear polynucleotide conjugats production. The lin~ar analogue is produced such that the ends of the resulting finear conjuga~e can be closed either by chemical reaction or by enzymatic ligation. C:hemical ~-closure can be achieved using various available t~chniques. On~
convenient approach requires fully-deprotected linear precursor s~quen~es and use of chemical condensation reagents, such as cyano~en bromide as described by Prakash, G. et al ~1992) J. Am. Chzm. Soc., 114, 3523-35Z7, water-scluble sarbodiimide as described by A~hiey, G.W. et al, ~ î 991 ) Bioch2mistry, 30, 2927-2933, and N-cyanoimidazole as described by Luebke, K.J. et al, ~1992) Nucleic Acids Res., 2:)i 3005-30Q9. An -alternative approach requires a fuliy-protsct~d linear pl!~curssr with only a ~:
free 5'-OH and a 3'-phosphate selectively deprotected for cycli2ation (see Rao, M.V. et al, (1989) Nucleic Acids Res., 17, 8221-8239. These lin~ar precursors can be prepared in solution Yia the phosphotriester approach. :

SUB~ UT~ S~EET

W O 93/06122 - P(~r/CA92/00423 The typical condensation reagent in this case is t-~2-Mesitylenesulfonyl)-3-Nitro-1,2,4-Triazole ~MSNT). After the post-synthesis coupling, the cyclic oligonusleotides are trèated according ~o standard procedures of deprotection and purification. Another alterna~ive approach generates a 5 fully-protected cyclic oli~onucleotide directly on the polymer-support (see Barbato, S. et al., ~1989) Tetrahedron, 45:4523; and Capobianco, M.L.
etal., 11990) Nucleic Acids Res.,18:2661~. This phosphotriester approach does not require a post-synthesis cyclization, and r~sults cyclic ~`
molecules with hi~h e~ficiency.

Cyclization of the polynuGleotide conjugate can also be achieved by enzymatic ligation of the free ends of a linear conjugate. The ends to be ligated correspond preferably to an annealing si~e in the duple)ted -structure, to f~cilitate action of the enzyme, preferably an RNA or DNA
15 ligase, as appropriate. To anneal DNA ends, the linear conju~ate is preferably incubated first under annealing conditions and then treated with either RNA or DNA ligase. RNA ends can be annealed in similar ~ -fashion, by treatment with RNA ligase in particular. The cyclic polynucleotide conjugates resulting from the reaction can be recovered 20 and purified using techniques established generally for polynucleotides, and as described in the examples herein.

To provide duplexed structures that, in accordan~ with the present invention, exhibit not only enhanced stability but also a ligand 25 binding property, the polynucleo~ide strands ~o be linked during synthesis are selected in terms of their nucleic acid sequence, and based on knowledge of ths particular nucleic acid sequence to which 3 target ligand binds. It will be appreciated that selection of strands appropriate for desired ligand binding can be guided by the vas~ scientific li~erature 30 dealing with protein/nucleic acid interactions. In those instances where a binding domain of specific interest remains to be identified, it will be appr~ciated that the mapping of that domain can be achieYed using SUBSll~lJTE SI~EET

WO 93~0612? 21 1 9 9 2 7 PC~/CA92/00423 conventional approaches, so that a specific binding sequence can be elucidated. The strategy hereindescribed can in fact facilitate such mappin3, by permitting the syn~hesis of a series of stabiliz~d duplexed structures representing putative !igand binding domains that can then be 5 screened for ligand binding activity using for example the mobility shift assays of the type hereindescribed.

In a preferred aspect o~ the present invention, the polynueleotide conjugates are employed as mimics of naturally occurrin3 duplexed ~ ~-10 structures, and the polynucleotide strands in the conjugate are aceordingly selected to correspond in sequence to a naturally occurring -duplex eounterpart. Conceivably, any dupiexed region of 3 naturally oocurring gene or other genetic element can be duplicated in stability~
enhanced form, in aceordance wi~h the present invention. ~ - -Ligands of potential interest inslude thos~ proteins which on bindin~ to their natural, nucleic acid target, directly or indirec~ly, influence ~ - ~
the u~ilization or fate of that nucleic acid tar~et. Examples of such ~ ;
proteins include: ribo- and deoxyribonucleopro~eincornplexes; ~ene regulatory proteins such as repressors, activat~rs and transac~iva~ors, -~
etc.; proteins involYed in the modifications and fate of mRNA molecules, including splicing, polyadenylation, capping, nuciear export, translation, degradation, etc.; proteins involved in the assembly and utiliza~ion of other RNA or ribonucleoprotein structures such as ribozymes, tRNA
synthetases, splicing complexes, etc. In 311 cases, the essen~ial featur~
of such proteins is that they recognise particular nucleic acid ~tr~ctures on the basis of their conformation and/or sequ~nces; embodimen~s of this invention would provide effective analogues when they maintain ~-some or all of such re~uirements.
3û
Also of interes~ ars polynuc}estide conju~ates that bind li~ands other than protein ligands, 8.g. chemical ligands such as interealating SUBSlTUT~ SHEET ;; ~

WO 93/OS122 P~/CA92/00423 agents le.g. psoralen and ethidium bromide), nucleic acid-binding antibioties (e.g. distamycin and ne~ropsin) and other nucleic acid struc~ures. `

In a partieuîarly preferred embodiment of the present invention, the p~lynucleotide conjugates comprise polynucl~otidf~ strands whicll, in their duplex~d farm, exhibit binding affinity for the tat prot2in of the human immunodeficiency virus, HIV. Throu~h interaction wi~h the RNA hairpin structur2 known as Tar, the tat protein mediatas a rapid increase in the produc~ion of the viral componen~s required for HIV replica~ion, which in turn leads to the onset of AIDS. It has been sug~sted that agents capable of int~rfering with the tat/Tar interaction will b~ useful in : -arresting HIV replication, and thus ~fficacious in the $reatment of All)S.
,, .--Th~ present invention accordingly provides a polynucleotide conjugate which is capable of adopting a duplexsd strueture havinQ a bindin3 :`
affini~y for tat. Such binding affinity is revealed using standard mobility shi~ assays, in ~at/Tar complexes, and thus ~at-bindin~, is revealed by alter~d migration relative to tat and Tar alone ~see Roy ~t ai, infra).
According ~o a specific embodiment of the inven~ion, the polynucleotide conju~ate has a chemical stnJcture described in the examples herein. It wiJI b~ apprecia~ed, however, that sequence variation can be tol~ratQd -without loss of tat binding affinity, and such variations which retain ~at bindin0 are within the scope of this embodiment of the present inventjon.

Other viral processes can also be tar~ettsd for therapeutic interfer~noe usin~ the stabilized duplex structures of the prssen~
invention. For example, in HIV, besides the TAR structure, th~ duplex~d RP~E RNA structure re~uired to regulate splicing and the duplexed ~RNAL~",3 structure used as a primer for reverse transcription can be mimicked using the present strategy. There may also be produced duplexed struotures which bind oth~r re~ulatory protein ligands, for example those known to ~xist in human pathogenic virus~s, including: the P protein ~f Hepatitis B `~

SUBS~LUTE SHEET ~-Wl~ 9~/06122 ` PCl`/CA92/00423 virus (HBV); the vp16 protein of HSV; the E1 and E7 prot~ins of Papilloma virus (HPV); the BZLF1 and EBNA^1 proteins of Epstein Barr virus (EBV); as well as additional proteins in these and other viruses.

Many other protein ligands and their corresponding nucleic acid ~ h targets are known in microbial, plant and animal speci~s. Of particular note are regulatory proteins known such as that which interacts with the ~ :-h~at shock el~ment ~HSE~, those which mediat2 inflammatory prncesses such as the interleukins, and ~hose known to be involved in transforming processes giving rise to oancer, such as the jun and fos oncogene families, which bind preferentially to particular DNA ~arge~s, and such targets can also be reproduced in stabilized duplexed form in accordance - with the pr~ent invention.

Formulation and administration of the compounds herein.described, -;
and indeed any annealed polynucleotide strucnlres having pharmaceutical utilit~, can be accomplished in accordance with procedures routinely ~:
appJied to aqueous-soluble compounds. Thus, for parenteral administration, buffered saline solutions are acceptable. Where a ~ ~
reduction in administration frequency is desirable, timed-release pslymeric - :-compositions which do not unfavourably chemically rnodi~y the compounds are acceptable. Modification of pharmacokine~ic properties, ~specialiy distribu~ion, are achieved, for instance, through the use of liposomal or cationic lipid formulations.
In an alternative embodiment of the present invention, the polynucleotid~ conju~ates comprise polynucieotide strands which in their duplexed form present nucleic acid epitopes of interest, for example as immunosens suitable for raising antibodies. The raising of such ;: ~
antibodies can be achieved in the manner conven~ional for polyclonal ~: -. .
antibody production, or for monoclonal antibody procluG~ion. Such ~ ~
an~ibodies will find utility in assays designed to detect ~ptiopes against ~ :
."~'~;.'-.

.. . ' ,:

SU~ TE SHEET

WO 93/06122 PCr/CA92/00423 which the antibodies were raised, especially when conjugated to a suitable reporter molecule; and may also be useful in protecting a region of a polynucleotide while manipuiating that polynucleo~ide at another site.

The polynucleotide conjugate may be coupled via an a~achment site incorporated within the chemical linker, to a desired agent such as a ~ ~
cross-linking agent, reporter rnolecule, cell uptake enilancer such as lipid - - -:
or cholesterol, alkylating groups, chromatographic beads and other functional groups . A vari~ty of chemioal ~roups may serve as lû attachment sites, provided of course that such groups permit couplin~ of ~ :
the polynucleotide strands as desired, and can be protected during polynucleotide synthesis. Ideally, the attachment sits is cs)nstituted by a chemical entity that o~n be protected by a base-labile protecting group removabls by ammoniolysis. One suoh group is the Fl~lOC ~roup used in t 5 conventional peptide synthesis protocols. In another embodiment, the attachmsnt site may be constituted by a phosphats ~roup incorporat~d wi~hin the linker, which can be derivatized following oxidation from either H-phosphonate to phosphate ~riester, or phosphite tries~er to phosphate triester When coupled with a reporter, suoh as a radiolabel, the conjugates of the invention can may also be used diagnostically e.g. as a competing ligand, to assay specimens for the presence of target 3igand in a qualita~ive or semi-quantitative fashion, for example using a compe~itive binding assayformat.

ExamDle 1 - Dimethoxytritylation of linkers As a first step in adapting linkers bearing terminal diol groups for incorporation via automated polynulceotide synthesis, one of ~he two dioi ~roups was first protected using the dimethoxytrity! group. The procedur~ is generally applicable for any diol lînker, and proceeds a~cording to the reaction scheme provided below:
-:

`,.: ~', SUBSl~TE S~IEET

WO 93/06122 2119 9 27 PCr/CA92/00423 2'~
DMTr-CI + HO-~linker)-OH ----> DMTr-O-(linker)-OH :~
. ~.

(i) 10-30mmol o~ the diol compound was co-evaporated with anhydrous pyridine (3 x 20 ml). The residue was then dissolved in fresh dry pyridine (50-150 ml) to yield a final diol concentration of about 1 mmol/5 ml~

(ii) 4,4'-dimethoxy~rltyl chloride (6.7-20 mmo~) was then added in small portions. The ratio between DMTr-CI and dioi was 1:1.5 eq. . .: .
liii) The reaction was followed at room tempera~ur~ by thin layer chromato~raphy (TLC) (MeOH/CHCI3, 1:9, v/v) untii the app~arance of a produet spot that was intense relative to remainin0 DMTr-CI. The reaction - ~ :
was usually complete a~er 2-4 hours. The DMTr derivatives were visualized as red-orange spots using an acidic spray (6t3% aqueous ~ :.
perchloric acid/ethanol, 3:2, v/v).

~iv) When the reaction was comple~e, 20-30 ml of MeOH was added to ~:
quench excess DMTr and the mixtur~ was stirred for an additonal 15 minutes.
~ ., .

~v) The solution was then concentrated to a syrup and the residue was ~ ~
resuspended into 50-150 ml of CHCI3. The chioroform phase was th~n -: .
washed once with 5% NaHCO3 ~25-75 ml), and twice wi~h satura~ed NaCI solution. The aqueous phase was back-extrac~d with CHCI3 125-75 : -ml). The organic phas~s were combined and dri~d over anhydrous sodium .- .
sulphate. After fil~ration, the solution was evaporated down to an oily -:
. :. -residue under reduced pressure.
'~,"'.~';-'' (vi) The residue was purified by flash chromatography on silica gel. The ~`
column was first eluted with petroleum ether/EtoAc (5:1, v/v) and then :~
wi~h petroleum ether / EtoAc ~2:1, v/v). ~ ~
". .:- ..~ ..

'`"''';'' SUBS;~:UT~ SHEET

WO ~3/06122 PCI /CA92/00423 (vii) Fractions containing the final product vvere combined together and the solvent was removed to yield a residue that was dried overnight under vacuum. Yieids, based on the amount of DMTr-CI used, ranged from 60 to 80 %. Products are characterized by standard methods, such 5 as NMR spectroscopy andlor eiem~ntal analysis.

In this manner, ~he following tritylated diol linkers were obtained from the reagents noted below:
.
10 ~: DMTr-O-(CH2)9-OH, yield 76.4% from 1,9-nonanediol 13.6 9 (22.5 mmol)l; l:)MTr-CI 15.0 9 (15 mrnol)l; and pyridine lt~SO mll.

~ ~: DMTr-O-(CH2~2-O-(CH2)2-O(CH2)2-OH, yield 68.2% from triethylene Qlycol 13.4 9 (22.5 mmol)l; DMTr-CI [5.0 y (15 mmol~]; and pyridin~ [100 1 5 mll.

(t:I: DMTr-O-~CH2)3-OH, yield 67.0% f~om 1,3-propanediol [1.7 g ~22.5 mmol~]: DMTr-CI 15.0 9 (15 mmoi)l; and pyridine 1100 mll.

LDI DMT-O-ICH2CH20)5-CH2CH2-OH, yield 68.4% from hexaethylene glycol 16.359122.5 mmol)l, DMT-CI [5 9 ~15 mmol)], and pyridine (100 ~
ml).

ExamDle 2 - Phosphitylation o~ tritylated linkers - -The tritylated linker prepared as described in Example 1 was next derivatized at the remaining hydroxyl group to incorporate a phosphoramidite group, according ~o the reaction scheme illustrated ~.
below~

3~ N~iPr)2 NliPr)2 . ~ ~:
DMTrO-llinkerl-OH ~ Cl-P~---------> DMTrO-llinker]-O-P\
OCH2CH2CN Q~H2CH2CN -: :

SUIBST~ SHEET

''' '~:':

WO 93/0~122 2 ~ 19~ 7 PCl/CA92/00423 (i) The tritylated product obtained from previous preparations (1-5 mmol) was dissolved in dry THF (tO-50 ml). Anhydrous diisopropylethylamine IDIPEA) (4-20 mmol, 4 eq.) was injected under a weak flow of argon.

5 (ii) The phosphitylating reagent 2-cyanoethyl-N,N-diisopropylchlorophosphorarnidite (2-10 mmol, 2 eq., Aldrich Ch~mical Co.) was then added with a syringe over a period of 2-5 minutes. A white precipitate was quickly~ormed.

10 ~iii) The reaction mixture was stirred at room ~emperatur~ for 1-2 hours and monitored by TLC (EtOAc/CH2Cl~llEA, 45:45:10, v/v~.

(iv) When the reaction had gone to completion, th~ exoess - ~ -phosphitylating reagen~ was quenched by adding s~veral ice cubes. Ths mixture was dilut~d with ethyl as~tate ~50-250 ml~ and triethylamine (1-5 mlJ. The solution vvas th~n ~ransferred to a separatory fLInnel and extrac~ed twice wi~h 10% aqueous sodium carbonate and ~wice with : ~ -saturated aqueous s~dium chloride.

20 Ivl Th~ or~anic phase was dried ov~r anhydrous sodiurn sulphate, filter~d, and then evaporated to dryness under reduced pressurs.
. ' ~.
tvi) Ths residue was purified by flash chromatography on silica ~el using -:
petroleum ether /EtOAc/TEA 120:10:1, vlv) as eluant. -(vii) Fractions containin0 pure product were cornbined, ~vaporat~d and then dried ovemi~ht under high vacoum to remove tr~ces of ~riethylamin~. The produot was stored at -20 C. Yield of the isolated :
product varied from B5 ~o 80 %. Product is characteri2sd by standard 30 methods, such as 1H-NMR 31P-NMR, and ~lemental analysis.

~UBSI~UT~ S~EET

21~992~
Wo 93/0~12~ ~ PCr/CAg2/00423 In this manner, and using the tritylated products of example 1 as starting mat~rial, there were prepared the followins linkers sui~able for c~upling betwsen nucleotides via the phosphoramidite approach:

1 ink~r A: I)MT-O-~CH2~9-O-phosphoramidite -TLC (silica gel, p~troleum ether/EtOAc/TEA, 50:10:1, v/vJv): R, 0.84,1H-NMR (CDCI3, 50t:~ MHz): ~1.1~1.62126H, m, CH2, CH(C6)21, 2.63 It, 2Ht J = 6.5 Hz, CH2CNI; 3.02 (t, 2H, J = 6,5 Hz, DMTOCH2); 3.5~3.88 12m with one s centred at 3.78, 1 2i~1, 0~3, CH20P, POCH2CH~CN, N~:HlCH3)2]; 6.79-6.84 ~m, 4H, arom. H ortho of OCI-13); 7.17-7.45 (m, 9H, arom. H). 3~P-NMR
ICDCI3, 121 MHz~: 122.4 ppm.

Linker B: C)MT-O-(CH2CI 120)2-CH2CJ 12-O-phcspho-amidite Tl C (silica gel, petroleum etherlEtOAclTEA, ~0:~0~ h~v~: Rt 0.48,1H-NMP~ lCDC:13, 500 Mltz): ~1.13-1.18 112H, 2d, CH~C~3)21:
2.61-2.64 lrn, 2H, C~2CN~; 3.23 (t, 2H, ~l = 5 Hz, DM~OCH2);
3.56-3.86 lm, 20H, OC~3, OC~I2CH20, C:H20P, POCH2CH2CN, NCH~CH3)2~; 6.77-6.86 Im, 4H, arom. H ortho of OCH3); 7.18-7.47 ~rn, 9H, arorn. H). 3lP-NMR (CDCI3, 121 MHz): 148.6 ppm.

Linker C: DMT-O-lCH2)3-O-phosphoramidite ~ ~.
TLC (silica ~el, petrol~um ~ther/EtOAc/TEA, 50:10:1, v/vlv): R~ ;- .
0.79,~H-NMR ~CDCi3, 5QO MHz~: ~1.0~-1.3~ 12H, 2d, CH(C~b~2]; -1.89-7.97 (m, 2H, C:H2CH2CH2); 2.44-2.51 lm, 2i1, C:H2CN); 3.14-3.19 ~m, 2H, I:)MTOCH2); 3.5~3.88 lm, 12H, OC:~b, CH2C~P, POCH2C:H2CN, NCH(CHJ2J; 6.7~6.84 ~m, 4H, arom. H ortho of .
OCH3); 7.1~7.47 ~m, 9H, arom. H). 31P-NMR (CDCI3, 121 MHz~
147.3ppm.

Linker D: DMT-O-~CH2CH20)5-CH2Ctl2-O-phosphoramidite `''' ~' ' " ~' ' SUBST,~UTe S~lE~T ~::

WO 93/06t22 2 1 1 9 9 2 7 PCl ~CA92~004~3 Linker D: DMT~ CH2CH20)5-CH2CH2-O-phosphorami~ite Tl C (silica gel, petroleum ether/EtOAo/TEA, 50:10:1, v/vlv): R, 0~12~1H-NMR (CDCI3r 500 MHz): ~ 1.15-1.?1 ~12H, 2d, CH~C~3)2];
2.57-2.66 (m, 2H, CH2CN); 3~23 It, 2H, J - 5 Hz, DMTOCH2);
3.56-3.91 [m, 32H, OC~3, OCH~CH20. CH20P, PQCH2CH2CN~
NCH(CHJ~]; 6.76-6.85 ~m, 4H, arom. H ortho of OC:H3); 7~16 7~48 (m, 9tl, arom. H). 3lP-NMR ICDCI3, 121 MHz): 148.6 ppm.

10 Ex~mDlQ3 - General procedure for linear polynucleotide conju~ate synth~sis Controlled pore glass (CPG! was used as the sQlid support matrix .. -- ,.
for both DNA & RNA synthesis. PoJydeoxvribonucleotides ~DNA) were prepared by the CE-phosphoramidite method on an Applied Biosystems ::
391 EP syn~hesizer (0.15 micromole scale). . Cleavage and d~,oro~ec~ion -were eff~cted by standard ammonia tre~tmerlt. Oligoribnnucleotides : -(5~NA) w~re prepared according to ~he m~thod of Usman ~t al, 1987, J. .:
Am. Chem. Soc., 109, 7845-7854, employing 5'-dime~hoxy~rityl-2'-t-ZO butyldimethoxysilyl ribonueleosid~-3'-CE- phosphoramidi~e~ ~Peninsula . :.
Labs, CA or ChemGenes Corp., MA). Syn~heses were carried out on an Applied Biosys~ems 380E~ synthesi~er using a modified 0.2 micromole cycle. Gleavag~ from the support, base ~ phospha~e deprotection, and .`.
removal of the ~'-TEiDMS groups were performed by established `~ `;
procedures ~Scarin~e et al. 1 990, Nuol . Acids Res ., 1 8, 5433-5441 ) . The . ~ -crude oligonucleotide in TBAF solution was desal~ed vn a Cla Sep-Pak ~: ~
cartridge prior to purification. ~:

The linker phosphoramidite ~dissolved in dry ac¢toni~rile, 0.2-9.:3 M) was coupled to the suppsrt-bound polynucleotide at th~ desirsd location, usiny the synthesis cycle conventionai for stalldard nucleoside phosphorarnidites. :

.:

SU~S~UTE SHEET.

WO 93/Olil ?2 PCI`/~A9?/00423 In one synthesis cycle, the DMTr protecting groups were removed from the extended oligom~r with 2.5 % dicholoroacetic acid I
dichloromethane. AXer several washes 5ac~tonitrile is the only solvent 5 used for all washes), cyanoethyl protected nucleoside phosphoramidites (0.12 M in dry acetonitril~) were coupled to the support in the pr~sence of 0.5 M ~etrazole. The coupling time for DNA oligomsrs was 15 sec IABI 391 EP) and 2 x 6 minutes for RNA oligomers (ABI 380B). Double couplings were used for RNA synthesis since these phosphoramidit~s are 10 much less reactive than their DNA homologs. This is followed by cappin~
of the unreacted hydroxy ~roups (Ac20/DMAP), and oxidation of the ph~sphite triest~rs to the phosphates (121H20~. The CyciRs were repeat~d until th~ desired polynucleotids conjugate was obtain~d. The conj~Jgate was then cleaved from the CPG support by treatm0nt with concentrated amrnonia for one hour at room temper~ure. Depro~ec~ion of DNA -~
conjugates and of RNA conjugates was achieved by incubation in ammonia at 55~C for 6-16 hours. For RNA conjugates specifically, deprot0ction was performed with ammonia in ethanol (3:1), and a final trea~ment involved incubation in 1M TBAF at room ~emperature~ The 20 avera~e coupling yield, as assayed by trityl measur~ment, was 97-99 %
for DNA oligos, and 95 - 97 % for RNA oligos.

A summary of th~ protocols used in RNA conjugate synthesis is provided in Table 1 ~eiow, for convenience~
TABLE 1: Synthetic cyçle for th~ preparation of linker-derivatized TAF~ -o3igoribonucleotides STEP REAGENT OR SOLVENT PURPOSE TIME ~ ~;
Isecl '.. ' 31:) -Dichloro~cetic acid in Detritylation 5 x 20 -CH2CI2 (2.5:97.5; v/v) SUBSIIIUTE SHEET ~:

wo 93~06122 2 ~1 9 9 2 7 P~/CA92/00423 2t 2 Anhydrous CH3CN Wash 90 3 . Activated phosphoramidites Coupling 2 x 360 in anhydrous CH3CN

4 Anhydrous C:H3C:N Wash 20 HPLC grade CH2C12 Wash 20 -6 Anhydrous CH3CN Wash 20 7 DMAP/THF 16.5 g: 94 ml) Capping 60 ---- Ac20/Lutidine/THF , ~
; V/V/Y) ... '.,,' 8 t).l M 12 in THFiLutidine/H20 Oxidation 60 -~
(1 60:~0:4; vlvlv) - -9 Anhydrous CH3CN Wash 3 x 2t~
?0 ~ :-~ The coupling reactions were carried out by pre-mixing 0.5 M te~razole `: -with 0.15 - 0.30 M standard or modified phosphoramidites in anhydrous --. ~ ....
CH3CN.
The crude deprotec~ed polynucleotide conju~a~ were purified by standard electrophor~sis methods (Atkinson 31 Smith, in 11984~
"Oligonucleotide Synthesis: A Practical Approach" ~iait, M.J.; ~d.) IRL
Press, OxfordJWashington, D.C.)using 15-20 % polyacrylamide / 7M urea gels. The bands were visualized by UV shadowing and the product vvas cut out and eluted from the gel. The eluted conju~at~ was finally -3() desalted on a C18 Sep-Pak and quantitated by OD2~,0.

"'"' ~

SUBSU~UTE SHEET ~

~706122 28 PC~/CA92/~0423 Each oligonucleotide linker conjugate was checked for homogeneity and "sized" by 5'-32P-end labeling / analytical PAGE against the crude material and oligonucletide markers. These RNA oligomers were further characterized by ~nzymatic RNA sequencing lDonis-Ksller, H. 11980)

5 Nucleic Acids Res., 8, 3133-3142} or base-composition analysis [Seela, F. & iC~iser, K. ~1987) Nucleic Acids Res., 15, 3113-3129l.-Exam~le 4 - Polynucleotide conjugate synthesis To eva!uat2 the eWect of the chemical linker on the stability and 10 ligand binding propertiss of a naturally occurring duplex structure, th~re was first employed a model system comprising DNA strands capable of duplexing to form an EcoRI recogni~ion/cleavage site. As shown below in - structure 1, unlinked oligomers consti~uting the EcoRI si~e were examin~d ~~ for comparison.
16 : :
5' - GGAArrC:C - 3' ~:
3' - CCrrAAGG - 5' ';
5' - GGAATTCC - linker B- ~;GAArrCC - 3' 2 5' - GGAArrCC - linker C - GGAATTCC - 3' 3 :

- . .:
For comparison, there was prepared linsar polynucleo~ide conju~ate -2 which contains triethy!ene glycol-derived linker B, having th~ structure .
-0-ICH2)2-0-~CH2)2-0-(CH2)2-0; and linear polynucleotide conju~ate ~
which contains pr~panediol-derived linker C, havin~ the structure -0-~ .
(CH2~3-0-. If the length and nature of linker B has besn s~lec~sd ~ ~:
appropriately, potynucleotide conjugate 2 should adopt a duplexed ~-structure that is digested more rapidly by EcoRI than the unlink~d control 30 molecule 1. Ths linker C in conjugate 3 is expected to be too short to permit functional annealling of the strands, which shsuld translate into slower EcoRI digestion relative to conjugate 2. The conjugates were SUBSIlI~TE SHEET

wo 93/06122 2 1 1 9 9 2 7 pcrfcA~2/oo4~3 2~
prepared using the protocols described above in example 3 and then radiolab~lled, and ~he EcoRI digestion reaction was monitored, in the following manner: `
.
5 Polvnuci~Qtide labellin~: oligonucleotides (5 pmol) were dissolved in 70 mM Tris-HCI lPH 7.0, 10 mM MgCI2, 10 mM KCI and 5 mM dithiothreitol (Drr) and incubated with 9 pmol of y-32P-labelled ATP and 10 units of T4 - .
polynucleotide kinase (New England Biolabs) at 37C for 1-2 h. The -~
reaction was terminated by heating th~ mix~ure to ~O~C for 10 min, and . ~:
10 then was slowly cooled to room temperature. The solution was desalted by passage through a Bio^spin column IBIO-RAD, Bio-spin 6 for the unlinked control, and Bio-spin 30 for the polynucleo~ide conjugates). An alt~rhative method for purification of labelled polynucleotide involved one~
time extraction wi~h an equal volume of phenol solution ~nd precipitation 15 using two volumes of ethanol/acetate (1~ /v) at -20C overnight, with coll~ction and dryiny under high speed vacuum.
~'."...`~,.
~QeL~9~: 1 pmol of the selected, 32P-lab~lled substrat~ was -: -~
incubated with 20 units o~ EcoRI ~Pharmacia) in 1û mM Tris-HCI ~pH 7.5), 20 :100 mM NaCI, 10 mM MgCI2, 1 mM ~ME and 1t30~y BSA/rnl (~otal volume: 2~ . The reactions were carried out at room temperature, and -2,u1 of sampl~ was removed at different time int~rvals. The samples were -analyzed on a 20% denaturing polyacrylamide gel.

Undcrthese conditions, 60% of the unlinked csntrol was diges~ed ~ ~
after Z4 h whereas conjugate 2 was complet~ly di~ested within 11 hours.: :`
The increase in digestion rate is approxirnately 6-8 fold fas~er with conjugate 2, owing to enhanced stability of the duplex~d structure. As ~ :
~xpected, di~estion of conju~ate 3 was very slow (only abou~ 5% of starting material was digested after 24 h incubation). These results ~ ~
indicate clearly that a linker of appropriate len~th can si~nificantly -~::

SUBSIIIUTE SHEET

WO 93/1)6122 pcr/cA92/oo423 enhance ~he stability of and retain the function of duplexed structures, including those having protein binding affinity.

Examp!e S - Produetion of RNA polynucleotide conjugatss The RNA structure known as Tar consists of 59 bases in mos~ HIV-1 isolates, arranged in a s~em-loop structure with two or three bulges in the stem. Previous studies have shown how2Yer that the ~ull length Tar s~ructure can be reduced significantly in size to a 27-mer ~Fig.4~ while retsining full tat-binding activity (Surnner-Smith et al, J. Virol., 1991, ~ ~
~O 65:5196. ~ -Various linear polynucleotide conjugates, repr~senting ana~ogues of ~` :
. a 27-mer truncated version of Tar ~Figure 4) were synthesized and evaluated. All were prepared using the synthesis proeedures previously described hereinabove. As Figur~ 4 illustrates, the linear polynucleotide conjugatestested comprised two classes; one class in which the 6-mer loop in ~he Tar analogue (4) was rsplaced by each of four different linkers -(conjugates 5A, 5B, 5C and 5D) and another class in which ~he ~mer Ioop was replaced by two ccupled link~rs (5BB and 5CC). The stabili~y and tat blnding properties of th~se oli~onucleotides were determined and compared, and the resul~s are shown in Table 2 below.

Melting temperagure ~Tm) measurements wers carried out in 100 mM NaCI110 mM sodium phosphate buffer (pH 7.û). Sarnples were heated from 25 t~ 85C in 1 C increments using a HP 8459 UV/VIS
spectrophotometer and a HP ~911)OA temp2rature controller. The concentration of nucleic acid was 2.5-3.0 ,uM, and absorbance was monitored at 260 nm. Tm values were determined by a first-derivative plot : :~:
of absorbance vs temp2rature. Each experiment was performed in duplicate and the average reported as the thermal denaturation tempera~ure. ;

SUB5~UTE SHEET

WO 93/06122 2 1 19 9 2 7 pcr/cA~2/oo423 Ligand binding o~ Ihe oligonucleotides was assessed by gel electrophoresis and RNA mobility shift assay. Linker-derivatized oligoribonucleotides (5A-5CC) and ~he csntrol sequences (4, 6 and 7, Fig.4j were 5'-32P-labeled with T4 polynucleotide kinase and [y-32PlATP.
5 The labeled oligomers were then purified by phenolichloroform sxtraction/EtOH precipitation or spin-column filtration IBio-Rad, Bio-Spin - ;:
30). Prior to binding assays, the RNAs were dissolved in 20 mM Tris-HCI
IpH 7.5)/ 100 mM NaCI, heated to 85C for 3 min, then slow-cooled to room temperature. Binding assays were carried ou~ in 20 ~I reaction mixtures containing 10 mM Tris-HCI (pH 7.5), 50 mM NaCI, 1 mM l)TT, 1 mM EDTA, 0.5 lJ/ml RNAsin tPromeg3), 0.09 Jug/ml BSA, 5% ~v/v) ~ ~
glycerol, Q.1 nM 32P-labeled RNA 1200~5000 cpm) and ~ither peptide ; :: :
,,.,. derived from the HIV-1 Tat protein RKKRRQRRRPPQGS (amino acids 49- `
62 of HiV LAI isolate) (Weeks et al., Science, 1990, 249:1281; Delling et :
~/., Proc. Natl. Acad. Sci., 1991, 88:6234) (American Peptide Co., San~a :
Clara, CA~ or full-length Tat protein ~American Bio-Technologies, Inc.) at a concentration of 0.5 pM to 1000 nM ~Roy et a/.,Genes Dev., 1990, 4:1365). The reactions wsre inclJbated at 23~C: for 25 min, chilled on ice fot 5 min, ~hen loaded on 5% native polyacrylamide gels ~acrylamide:bis~
2û acrylamide - 30:~).8, w/w) containing 5% glycerol. The ~e~s were pre~
run for 15 min prior to loading, then run ~or 2.5 h at a cons$ant current of 30 mA at AC in 0.5X TBE buffer. The gels were dried onto DEAE pap~r (Whatman DE81) and exposed to Kodak X-Omat X-ray film with an intensifying screen overnight at -70C. Competition binding experiments --25 were carried out as dsscribed above excep~ ~hat the corlcentr~tion of Tat protein was kept constant at 100 nM and unlabeled comps~itor R3`JA was ~ -added in a concentration range of 0.9 nM ~o 5000 nM.

SUBSIII~lTE SH~ET :~

21~927 -WO 93J061~2 PCr/CA92/00423 TABLE ll: Thermodynamic and binding properties of TAR analogues QLIGOMER SUBSTITUTION Tm (C) tKd) BINDING(%I
, 4 6-nt loop (v~ sequence) 60 + (0.41) 45.
g `
5A linker A / loop 61 + (0.71~ 40.
- ;:
5B linker B I loop 58 + ~0.95) 42. -~

5C linker C / loop 56 5D linker D / loop 63 + ~0.66) 56.
,~.- ' ' ''`'" ~''' 5BB 2 X linker B I loop 59 + (1.13) 38.

5CC 2 X linker C t loop 56 ~ ~0.43) 17.

6 + 7 without connection 32 - ~:

2Q ^^-K~ values are expressed in nanomolar concentrations : ~ -+ strong binding - no binding Binding capacity indicates ~h~ % of active RNA molecules capable of bindingto peptide upon sa~uration : :
The thermal denaturation experiments indicated that every linker-derivatized TA~ analogue had some secondary stmcture. With the exception of structure 5C which incorporates a linker expected to be too short to allow proper duplex formation, binding assays revealed tat-binding function in the conjugated duplexes versus the unlinked controls. :
Similar binding was also confirmed in experiments usin3 the full length tat protein, SUBSI~IUTE SHEET ~ ~
. .:

wo 93/06122 211 9 9 2 7 PCr/CA92/00423 Further evaluation of linker incorporation has indicated that relatively short linkers can be used to advantage, to replace nucieotides resident in the polynucleotide strands, e.g. to replace nucleotides in the ~ :
bulge o~ TAR. In particular, a Tar conjugate was produced in which the - :
5 bulge 5'-U-C:-U-3' was replaced by the structure 5'-U-LC-LC-3', to yield structure 8 ~Kd = 0.51nM, Tm=~0C)) shown below~
Ioop ..
C G
G C -.
A U ~ . ~
LC G C : :
~) A U
G C -~
b~'- G C ~:
G C :-where LcLc is -o-(cH2)3-o-po2-o-(cH2~3-9-~

Tat-binding analysis of ths resulting structure has shown that 25 replacement of nucleotides within ~he bulge preserved the ta~-bindin~
structure of TAR. Thus, in certain instances, linkers equivalent in length --to C3 can be used, particularly within the so-called bulge structures which ~ :
form at non-annsaled sites of duplex structures.

Moreover, studies with a short un-linksd duplex (oligomer 6 ~ 7) of same length have shown that this duplex has a significant ~ower Tm (32C) when compared to its linked counterparts ~56 - 63C~, and it also failed to form any effective complexes with Tat-d~rived pep~ide, probably due to its thermal instability. This provides strong evidence that synthetic linkers add subs~an~ial stability ~o the un-linked duplex structures ~v a such degree tha~ their normal biological functions, such as binding to proteins, can be maintained. :

SUBS~UTE SHEET ;

2119g27 Wo Q3~06122 pcr/cAs2/oo423 In another experiment, ~here was successfully generated a particular Tar analog where the linker was incorporated at the bottom of the duplex (oligomer 9, below).
:~''' `' C G ~ :
G C ~ ~:
A U

10 CU ; ` ~
G C -~ -A U : ~
G C ~ ~ .
G (~
LD ` ~
~.
.~ . 20 Both Trr, m~asurem~nts (T,~, = 61 C) and binding assays (Kd = 2.20nM~
indicated that this analog also-retains the physical and bindin3 properties of the wild-type Tar structure.
'~
~xample 6 - Binding assays with fuil-length Tat and competition 25 experiments To evaluate possible differenc~s in bindin~ a~finity for th~ short Tat-peptide and full length native Tat proteinl the binding affinities of the Tar conjugat~s for full-length Tat protein (B6 amino aeids~ wer~ assessed using the mobility shift assay. By this method, Ths Kd value for ~he fu!l- -30 len~th Tat (1.17 nM~ was slightly hi3her than that for the Ta~-derived peptide tO.71 nM). When Tar conjugate 5B was add~d ~o 3 pre-form~d complex between the 27mer fragment of the wild-type Tar stsm-lDop (oligomer 4) and full-length Tat protein, stron~ curnp~tition with the TAR
sequ~nce was observed. The complex was totally compe~d away wh~n 35 the ratio between the Tar oonjugate and the Tat protein was 1:1.

ExamDle 7 - Synthesis of cyc!ic polynucleotide conjugates To synth~size cyclic polynucleotide conju~a~es, th~rQ w~s a~pli~d ~:
ths general apprsach of (a) synthssizin~ th~ corresponding lin~ar ~:

~UBS~IlJTE SHEET ~: `

WO 93~06122 2 1 1 9 9 2 7 PCI ~CA92/00423 polynucleo~ide eonjugate in the manner described previously herein, and then (b) cyclizing the linear polynutceotide conjugate either via enzymatic Jigation (DNA or RNA ligase~ or by chemical closure. In particular, the en~ymatic ligation approach has been applied ~o conv~r~ linear Goniugate 5 ~, to the cyolic TAR conjugate 1 t/ as shown below~

C G C ~ G . :~
G C G C -A ~ A
C ~ C
5 ' - U
3 ' - C ~ iga59 C
~ U ~ --> ~ lJ `: ~
G C G C ~ ~
b-' l Si;C G C
~~ C G~

-~
To prepare the cyciic analogue, the linear conjugate 10 was ~irst - :
radiolabelled with yamma 32P-ATP as described previously h~rein. The heated T4 polynucleotide mixture was then cooled slowiy to room ~ ~:20 t~mpera~ure, and 1~ul (10 units~ of T4 RNA ligase wer~ then mixed with 10ll1 of radiolabelled con)ugate, 2,u1 of ATP (1ûmM1 and 7 ul of lX ligase ~ -buff~r consisting of 66mM Tris-HC:I (pH 7.5~, 6.~ mM MgCI2, î mM DTT, : ~ -and 1 mM ATP. The ligation reaotion was pursued for four hours a~ room temp~ratur~.
2~ :
The ligated product was then purified on a 2t)% denaturing ;~
polyacrylamide gel. Th~ band correspondin~ to the cyclic conjugate (~vident from its faster migration relative to linear conjugates) was cut out and extracted from the gel with 0.3 M hJaOAc at r~om 1:ernp~rature : ~:
ov~rnight. The sodium acetate solu~,on containing ~h~ cyclio conjugat~ ; ;
was then w~sh~d wi~h zn equal volume of phenol solution in ord~r to :eliminate any proteinaceous contamination. After this step, two volumes SUIBSTITUTE 5~EET

2i~9927 WO 93/06122 PCriCA92~00423 of ethanolJacetone (1:1, v/v) solution were added to the aqueous phase, and the mixture was stored at -20C overnight. The cyclie conjugate 11 was ultimately collected and was dried under high speed vacuum.

5 Example 8 - Liga~ion site optimi~ation for generating cyolic conjugates To cyclize the polynucleotide conjugates as efficientJy as possible ~ i number of potential ligation sites ~a-e) were examined using structur~ 12 ~Fig. 5). To prepar~ ~his cyclic polynucleotide conju~ate, the linear ~:
conju~ates (one for each ligation site chosen) were synthesized and 1C) radiolabelled with gamma 32P-ATP as described pr~viously h~rein. 10 Jul of each radiolabeJ~ed conjugate was added to 2ul of ATP (1OmM~, 2,ul of DMSO(1C)C)%), ~/l of 10X ligase buffer consisting of 500mM Tris-Ht~l ~pH 7.8). 100mM M9CI2 tOOmM ~-mercaptoethanol, 10mM ATP, and 1~1 (10 UNITS) of RNA Ligase. The ligation reaction was pursued for 4 hours 15 at 37C:. The ligated pro~ucts (2~1 of eaçh~ were examined by separation on 20% denaturing polyacrylamide and compared directly to an equivalent amount of unli~ated lin2ar radiolabelled polynucleo~ide conjugate on the sarn~ gel.

From these resul~s, it was de~e~mined ~hat ligation site c ~between ~he A and G residue) on the front strand imrnedia~ely beneath th~ -UC:U-bulge gave the best conversion of linear to cyclie conjugate.

Exam~ - Bindin~ properties of cyclic poiynucl~otide conju~a~es Using the best li~ation site identified from the previous example, there was successfully genera~ed a series of Ta~ conjuga~es; two of them are illustrated in Fi~ure 6. E~oth of these constnlcts (14 ~ 151 ar~ 21-mers and differ only in the chemical linker used to replacs the nucleotide loops at the top and bottom of the duplex. Oli~omer 14 contains Linker A and oligomer 1 5 contain~ linker D. All three cyclic p~lynucleo~ide ~:
conju~ates were subjected to the binding assay as described pr~iously. ~ ~

SUI~ST~T~ SI~EET - :-~vo 93/061~2 2 1 1 9 9 2 7 PCI /CA92f00423 It was found that the 31-mer (oligomer 13) as well as the linker D
cyclic conjugate (oligomer 15) bind efficiently to both peptide and the ~ull-length Tat protein, although, for reasons that are not clear, n~ binding ~ `
was seen with oligomer 14. It is possible tha~ while the length of ~he : ~ ~
chemical linker used does not appear to be significant in the linear series, - ~ -it may be significant for proper functioning of cyclic polynucleotide conjugates that bind to Tat protein. This sugg~sts ~hat synthetic duplex~
stabilizing linkers should have some flexibility in order to allow ~he mini^
duplexes to adopt possible conformational changes up~n protein - - ;
1 0 recognitions.

ExamD!~ 10 - In Vitro Stability and cell uptake of polynucleotide conjugates -,~
A number o~ different polynucleotides were used in a comparative analysis of the relative stability o~ linear Yersus cyciic c~njugates. For thess studies the following conjugates were used IFig. 7). To evaluate conjugate s~ability further there was also generated a 21-mer RNA
oligomer ~5'-CUUC:GCAGUAUGUUAGCCGGU-3') which has the same base composition as ~he cycl~c oligomers 14 & 15, but should remain in single-stranded open-circle form due to the non-c~mplernentarit~ between ~he bases (Figure 7, oligomer 16). Each of the polynucleotide conjugates u~i2d was synthesized, radiolab~lled, and ligated as previously described herein.
After radiolabelling and/or ligation, the pslynucleo~ides were purified on ~ .
2S 20% denaturing polyacrylamide gels as previously described. For each of th~ various conditions, the same amoun~ of radiolab~ d gel-purified ;
polynucleotidc was used 1300,000 CPM). The conditions used for each of ~he reactions ar~ described below. ~:
Exounuclease lll: 300,000 CPM of gel-purified polynucieotide was incubat~d in the presence of 20 units of Exonuclease 111 11~1~ and 1~1 of 10X buffer which csnsisted of 500mM Tris-HCI pH 8.û, 50mM Mgt:12, ~ .

SUBSII~ SHEET -:

~t-199~7 wo 43/06l22 pcr/cAs2/oo423 1 OOmM i3-mercaptoe~hanol. Enzymatic treatment was pursued for 6 h at 37C and a sample was removed for analysis a~ this time.

Munq Bean Nuclease: 300,ûO0 CPM of gel-purified polynucleotide was incubated in the presence of 5 units of Mung Bean Nuclease (t,ul) and 1/ul :~
of 10X buffer which consisted of 50CmM sodium ac~tate pH 5,0, 300rnh/1 NaC:I, 1 mM ZnSO4. Enzymatic treatment W35 pursued for 6 h at 37C and a sample was removed for analysis at this time.

C~!f Intestinal Alkaline Phos~: 300,000 of CPM g~J-purified -polynucleotid~ was incubated in the presenoe of 5 units of calf intestinal alkaline phoshatase and i~l of 10X buffer which consisted of 50C)mM
Tris-HCI pH 8.5, and lmM EDTA. Enzymatic treatment was pursued for : -20 h at 37C and a sample was removed at this time.
~:e71 Extr~ct an~lu~lear Extracts: C@ll and nuclear extracts were prepared essentially ~y the method of Dignam et. al., 1983, Nucl. Acids. Res., 1 1:1475. The amount of protein in sach extract was ~etermined using Bcvine Serum Albumin as a standard. 300,ûO0 CPM of g~l-purified polynucleotide was incubated in the pr~s~nce of 8ug cell extract protein, or 6/ug nuclear ex~ract protein at 37C. Equivalent sarnples from both eell and nuclear extract di~estions were removed at various ~imes ~8 and 24 h).

6 Samples from all ~reatments were applied to 20% denaturin~ ;
polyacrylamide gels and exposed t~ Kodak X-Omat AR film. The band of ;
interest was excised from the gel and the amount of radioactivity was determined. The relative stability of each treatment was determined by cornparing the amount of radioactivity of eaeh sample to the amount of radioactivity of a control sampie which was not treated wi~h the same ~:~
enzyme. Results of thes~ stability studies are pr0sented b~low: ~

.. ..
SUB~TU~E ~HEET ~;

wo 43/U612~ 2 1 1 9 9 2 7 PC~ICA92/00423 Table 111: Stability studies of TAR conjugates TREATMENT' TIME ~h) #4 #5A #16 #13 #14 #15 EXONUCLEASE 111 6 8.0% 19% 14% 46% 7~% 84%
-MUNG BEAN 6 3.0% 27% 8.5% 35% 49% 54% -~

CIAP 24 + + - - - - -.~--CELL EXTRACT 84.8% 1.7% 2.4% 72% 73% 86%

240.2% 0.3% 0.5% 34% 55% :37%

NUC. EXTRACT B1.0% 1.5% 33% 83% 88% 84%
2~0.1% ~).3% ~).5% 5~% 31% :~2% :~
.. ~ -:
All treatments were carried ou~ at 37 C. Cellular and nuclear extracts :-wer~ obtained from H P-~ cells (liver cells).
+ Sensitiv~ to dephosphorylation by CIAP treatment.
- Not sensitive to d~phosphorylation by CIAP treatment.
% # of full length molecules remaining These results demonstrat~ that Tar conjugate 5A has a similar -:
stability as thB wild-typ~ sequence loligomer 4) in cellular and ns~clear ~:extracts althou~h th~ conjugate appears far more stable a~ains~ slngle strand-specific nucleases such as mung bean nucleases. The duplex~
forming cyclic linker molecules ~oligomer 14 & 15) ar~ much more stable than both the linear conjugates and the single-strand~d cyciic Gnntrol : :
(oligomer 16). ~

~UBST,~UT~ S~EET ~-

Claims (23)

WE CLAIM:

1. A duplex-forming, polynucleotide conjugate, comprising a first polynucleotide strand having an end, a second polynucleotide strand which is capable of annealing with the first polynucleotide strand and having an end, anda chemical linker which is coupled covalently between said ends to form a bridgepermitting the first and second polynucleotides to form a ligand binding, duplexed structure.

2. A duplex-forming, polynucleotide conjugate according to claim 1, selected from among the group consisting of:
(i) a linear polynucleotide conjugate of the formula;
X - L - Y (I) wherein:
X is a polynucleotide having a 3'terminus;
Y is a polynucleotide capable of annealing with X, and having a 5'terminus; and L is a chemical linker coupled covalently between the 3'terminus of X and the 5'terminus of Y to form a bridge permitting X and Y to form a ligand-bindingduplexed structure;
(ii) a cyclic polynucleotide conjugate of the formula:
(IIb) wherein:
X is a polynucleotide having a 5'terminus and a 3'terminus;
Y is a polynucleotide capable of annealing with X and having a 3'terminus and a 5'terminus;

Z is a polynucleotide coupled covalently between the 5'terminus of X and the 3'terminus of Y; and L is a chemical linker coupled covalently between the 3'terminus of X and the 5'terminus of Y, to form a bridge permitting X and Y to form a ligand-binding duplexed structure; and (iii) a cyclic polynucleotide conjugate c)f the formula:

(IIB) wherein:
X and Y are as defined above, and L1 and L2 are independently selected chemical linkers coupled, respectively, between the 3'terminus of X and the 5'terminus of Y and the 5'terminus of X and the 3'terminus of Y, to form chemical bridges permitting X
and Y to form a ligand-binding duplexed structure.

3. A polynucleotide conjugate according to claim 2, which in duplexed form binds a protein ligand.

4. A duplex-forming polynucleotide conjugate according to claim 3, which in duplexed form binds a protein that regulates gene expression.

5. A duplex-forming polynucleotide conjugate according to claim 4, which in duplexed form binds a protein that regulates viral gene expression.

6. A duplex-forming polynucleotide conjugate according to claim 5, which in duplexed form binds to the HIV tat protein.

7. A duplex-forming polynucleotide conjugate according to claim 3, which in duplexed form presents an immunogenic epitope.

8. A duplex-forming polynucleotide conjugate according to any one of claims 3 to 7, wherein said conjugate is a linear polynucleotide conjugate of formula (I).

9. A duplex-forming polynucleotide conjugate according to any one of claims 3 to 7, wherein said conjugate is a cyclic polynucleotide conjugate of formula (IIa).

10. A duplex-forming polynucleotide conjugate according to any one of claims 3 to 7, wherein said conjugate is a cyclic polynucleotide conjugate of formula (IIb).

11. A duplex-forming polynucleotide conjugate according to claim 8, wherein said chemical linker is equivalent in length to an alkane having from 7 to 20 carbon atoms.

12. A duplex-forming polynucleotide conjugate according to claim 11, wherein X and Y comprise a region of mismatched nucleotide sequence forming a bulge structure when in duplexed form.

13. A duplex-forming polynucleotide conjugate according to claim 12, which binds with the HIV tat protein.

14. A duplex-forming polynucleotide conjugate according to claim 13, wherein X and Y are polynucleotides having a precisely complementary nucleic acid sequence.

15. A duplex-forming polynucleotide conjugate according to claim 14, wherein said chemical linker is equivalent in length to an alkane having from 7 to 20 carbon atoms.

1 6. A duplex-forming polynucleotide conjugate according to claim 15, wherein X and Y comprise a region of mismatched nucleotide sequence forming a bulge structure when in duplexed form.

17. A duplex-forming polynucleotide conjugate according to claim 16, which binds to the HIV tat protein.

18. A duplex-forming polynucleotide conjugate according to claim 9, which binds the HIV tat protein.

19. A duplex-forming polynucleotide conjugate according to any preceding claim, wherein X and Y are polynucleotide monophosphates.

20. A duplex-forming polynucleotide conjugate according to claim 19, wherein X and Y are both polydeoxyribonucleotides.

21. A duplex-forming polynucleotide conjugate according to claim 19, wherein X and Y are both polyribonucleotides .

22. A process for preparing a cyclic, duplex-forming, polynucleotide conjugate as defined in claim 17, which comprises the steps of synthesizing a linear analogue thereof in which two chemical linkers permitting the conjugate to form a duplexed, ligand-binding structure are incorporated between polynucleotides X and Y, and then cyclizing the resulting linear polynucleotide conjugate by chemical or enzymatic means.

23. A pharmaceutical composition comprising a duplex-forming, polynucleotide conjugate according to any one of claims 3 to 21, and a pharmaceutically acceptable carrier.