CA2185918A1 - Determination and identification of active compounds in a compound library - Google Patents

Abstract

Libraries of compounds such as nucleic acids or peptides are contacted with a target molecule and libraries that have at least one compound that binds with at least a minimum activity are determined by a reiterative process in which a change in the rate of recorvery (elimination) of compounds that bind to the target indicates that the library contains such a compound. The procedure may also be used to determine indirectly the sequence of such compound by employing sublibraries, each of which has a known entity at a known position of the compound.

Description

WO 95127D72 2 ~ 1 8 PCrNS95104098 r ~ ~TION OF
ACTI-JE ~ Ou J5 IN A _ _ LIBBARY
' Ql~ the Inventio~
Thi6 invention relates to det~-rm;n;n~ whether or not there is em active _ ~ in a ~ , _ u..d library . This invention further relates to det~orm;n;nri the identity of an active , u-.d present in a library of , _llds.
Various procedures have been L/lU~OSed for identifying compounds present in ~ library of ~ , u~lds that are artive with respect to a sperif;~d target. Such activities have generally been directed to identifying nucleic acids or peptides which bind to a protein or other target molecule.
For example, one such procedure for identifying a nucleic acid present in a nucleic acid library that binds to a target molecule involves contacting a library with a target molecule, by a reiterative ~LU~.-dUL~I which includes ampl;f;r~t;r~n. Thus, for example, Rinzler & Vogelstein (Nucleic Acids Res . 17, 3645, 1989 ) SU13SrlTUrE SHEET (RULE 26) Woss/27072 2~ 2~ /u. _Q~
lmd Tuerk and Gold (Science 249, 505, 1990) have disclosed an in vitro procedure in which the nucleic acids in the library are exposed to target molecules under competitive binding conditions.
Those nucleic acids capable of binding to the target molecule are L- cuv~L~d in preference to those that are not bound, and the recovered, active, nucleic acids are subjected to an amplification uC~dur e BUCh aB the polymerase chain reaction ~PCR) . ~he lif;~r1 nucleic acids are re-exposed to target, isolated and then , l; f; Pd once again . The value of the ampl; f; r~t; ~n step is that with each iteration of the procedure the target is exposed to a mixture of nucleic acids that is progressively enriched for molecules that bind with the highest affinity to the target. This is considered to be a ` ini~torial approach because it is the property of the entire active nucleic acid molecule that contributes to its ability to bind to the target and leads to its selection to the exclusion of other molecules in the library that bind to the target with lower af f inity . The procedure avoids a signal (active nucleic acids) -to-noise (non-active nucleic acids) problem by its process of reiteration. Each time the library is exposed to the target in early iterations, the amount of bound highly active nucleic acids may be very small compared to the background of those bound or recovered that ~Lre not active, but eventually the active nucleic acids are abundant enough because of the reiteration and amplification that they can be easily measured and i ~nt i f; ed . For these reasons, very large libraries can be used and even single lr~rlllr~ of highly active members can be identified. The reiterative procedure, when taken as a whole and coupled with amplification, has a low enough background and eventually a high enough signal from active nucleic acids that it uvc:I~ ~ the usual limitations from signal-to-noise ratio that re8trict the useful complexity of most screening ylu~edllLes. While this is impressive, this class of procedures effectively and dramatically limits the r~lrTn;ci-~1 diversity allowed in the libraries. This essentially means that only nucleic acids or SUBSTITU~E SHEET (~UI E 26) 2 7 ~
W0 95l27072 1 ~ v5 slightly 'if;~-d nucleic acids czm be used because these methods rely upon amplification and discrimination by biological methods in order to allow both completion of the reiteration as well as direct and accurate irl~nt;f;rat-;nn of the active members of the library at the end of the ~JLUCédULe.
Screening procedures for peptide libraries are not reiterative. In vitro techniques such as PrR that will amplify peptides are not currently available. Accordingly, different strategies have been implemented. In one such approach, peptides of random sequence elre displayed on bacteriophage, the phage are contacted with a target and those phage that interact with the target are isolated, recloned and the S~ nc~R ~ncndin~ the active peptides are detPrmin--d (Devlin et al., Science 2~, 404, 1990; Scott and Smith, Science 249, 386, 1990). In another approach, referred to by some as "Encoded Synthetic Libraries" (ESL) , (Dower et al., Patent WO
93/06121; Brenner ~ Lerner, Proc. Natl. AcAd. Sci., U.S. 89, 5381, 1992; Needels et al., Proc. Natl. Acad Sci. U.S. gQ, 10700, 1993) peptides are bound either directly or via a bead to nucleic acids in such a way that as an amino acid is added to a growing peptide, one or more nucleotides ~-nrn~l i n5 the added amino acid is added orthn~nn~l ly to the bead or to the peptide. ~he advantage of the ESL approach is that high _-lF'Yi ty synthetic peptide libraries can be Assayed in a combinatorial fashion. All scree~ling strategies that use codes to indirectly identify the active members of a library ( such screens have been described for other kinds of synthetic libraries in addition to peptides) are ~ cirJn~d to allow combinatorial selection of libraries to identify rare, highly active library members. The signal-to-noise problem is UVer~- by brute-force approaches such as using cell sorting terhnnlngy to examine individual beads each cnn~;n;nrJ multiple copies of a single library member and its l.:ULL q~ ;n~ code. In this sense, such ~L.-cedures are, in effect, methods for quickly screening large numbers of ~ _ ''-, one by one, with advanced terhnnlogy, as SUBSrlTUTE SHEET ~RULE 26~

W0 95/27072 2 1 ~ ~ 9 1 ~ P~ o ~
opposed to screening pools or mixtures simultaneously. The coding technology facilitates the identificatioP of the active ~ ~ u-.db because in such methods the amount of material isolated i6 usually very small (i.e., one "bead's worth" of peptide). By u6ing an amplif iable nucleic acid code, the direct identi~ication of the code (and, therefore, the iPdirect identification of the active compound) is possible. There are, however, limitations to these kinds o~ technologies, including (1) the need for complex chemistry to couple the library members to their respective codes, ( 2 ) the need in some ~ c to have the peptides attached to a comparatively large bead which, even though it might be relatively iPert, might nevertheless impede interaction between peptide and target, (3) possible interaction between the target and the nucleic acid code or between peptides and codes and ( 4 ) in some of these :S the u-.ds of the libr~ry are not in solution so that the selection conditions are dissimilar from those iP which an active compound would normally be expected to function, namely binding in solution to the target.
There are serial approaches for screening peptide libraries (Houghten et al., Nature 354, 84, 1991) that do not have the complications of encoded library methodologies. Typically, a library consists o~ pools of peptides each containing the same number of amino acids; for example, the library might contain 400 pools o~ hexameric peptides such that each pool has one of the 20 amino acids in the first and second position of the peptides (thus 20x20=400 pools) and the L~ ;nin~ 4 positions o~ the peptides are random in every pool. The target is contacted with each of the 400 pools and each pool is me~sured for activity; for every active pool (for example, ala.his.x.x.x.x), 20 new sub-pool6 are synthesized wherein the f irst two amino acid6 of the active pool are conserved (i.e., ala.his), the third amino acid is fixed (i.e., ala.his.gly.x.x.x in the first pool, ala.his.ala.x.x.x in the second pool and so on) and the fourth, fifth and sixth positions S~ SH~ (RU~ 261 2~85 W095l27072 9 ~ 8 PCrlUS95/04098 are rAn~' i 7ed so that the 20 pools are distinguished by the identity of the amino acid in the third position of the peptide.
This serial "unrAn~` i 7Ation" ~LuceduLe is continued until active peptides have been selected in which all six positions ~re identified. Such a procedure has also been described ~or olirJnnllrleotides (Ecker et al., patent W093/04204) . This kind of procedure avoids the rhPm;cAl limitations of the ol ;rjnnllrleotide procedures that use ~ lifirAtion (i.e., the library members do not need to be ~ lifiecl), as well ag the rh~mirAl c lirc~tions of the encoded-libr~ry pLuceduLos, but these limitations are replaced by the signal-to-noise problem because these procedures are essentially no more than seri~l pooling strategies that do not effectively address the signal-to-noise limitation. Therefore, these approaches are A~7~1itive rather thAn, ;nAtorial~ whereby pools containing less active but more abundant s~lhrlA~sPs of _u--ds will be cho6en whereas pools containing less abundant, more active u--ds are likely to be ignored. Thus, in the example given above, the pool with the best averaqe activity might have been ala.his.x.x.x.x but there might have been inrl;vi~llAl members in other pools (e.g., val.leu.x.x.x.x) th~t had higher activity than any ;n~iividual members in the selected pool but these would not be identif ied .
In summary, there are three common kinds of procedures for screening synthetic libraries: (1) For ol jrjnnllrleotides, there are reiterative ylu.:.:duL.:s th~t include 1 i f i r~tion of the nucleic acids. These ~1OC~dUL~S are ;nAtnrial and minimi7~ the signal-to-noise problem. Without i lifirRtinn~ the direct ident; f; rat; nTI of the active compounds present in large libraries by these yLu~ eduL~s would not be possible because the amount of material recovered from the reiterative ~lLU~_edULe would be too low to use in known procedures for identifying nucleic acid sequences.
Because of the requirement for ~ lificRtion~ however, these pLUCOduL~S are restricted to a very limited set of u..ds, SUB~S~ITUTE S~IEET ~RIJI E 2ffl Wo9s/27072 P~ r n~mely nucleic acids that can be amplified. (2) Coding procedures f re used to facilitate identi~icltion of non-ampli~iable ~ Jul.d6 via amplification of nucleic acid code6 that are associated with, or bound to, each compound of a library. These ~Lu.~duL~s are com~binatorial to the extent that the signal-to-noise problem is reduced by the use of screening procedures that m;nimi-e the noise and r ~;m; 7e the sign~l genernted by even ~ 6ingle ~ctive library member. Such }fLuue:.luL~_6t which usually involve physical separation of a solid support to recover copies of those individual, ~ ullds that are active, result in low yields of material, but the amplification of the code allows direct identification by known methods of nucleic acid sc~qllGnr;nrJ. These ~LuceduL~s are generally more cumbersome, do not allow sampling in solution phase, and present rhP11~n~-os in chemistry that stem from the need to have a unique nucleic ~cid code attached to each ~ ulld in the library.
(3) Seri~l "UIIL ` ;7~tion" procedures are used to screen synthetic libraries for active compounds. These procedures are very f lexible with regard to the chemistry that can be used and avoid some of the ~1; r~tiong of the other procedures, but they are not combinatorial because they do not address the signal-to-noise problem. There~ore, yLU- e:duL~:8 (1) and ~2) limit the kind6 of libraries that can be screened for terhn;r-f~l (chemistry) re~sons, where~s ULU~._.lUL~ (3) is likely to yield relatively abundant, less active ~ ullds in preference to the rarer, most active ~ _ ~. A procedure that would enable the use of a wide variety of chemistries and synthetic libraries in solution, as in ,uLuceduL~ (3), but that was also truly combin~torial, effectively m;n;m;7inS or eliminating the signal-to-noise problem, as in ~LuceduLe8 (1) and (2), would be a very powerful screening approach. The invention described below is a description of one such ~Lu- éduL~:.
~UBSTITUTE SHEET (RU~E 26 -7 I ~ ~ o of the In~rentian In accordance with one aspect of the present invention, there is provided a ~)L~JCeduLé for det~nmin;nr whether or not there is present in a library of compounds an active ~ ulld.
In accordance with another aspect of the present invention, there is provided a procedure of identifying an active ~ , ulld which is present in a library of such, ~ u-.ds.
In accordance with a further aspect of the present invention, there is provided a yL~ceiuLe which is ArplirAhle to a wide vAriety of di~ferent , u.lds, and which does not require lif;rAtion~ and which is capable of dist;nrJ~l;Rh;nr between those libraries that contain a unique or very rare compound(s) having at least a desired activity and those libraries that include a high representation of compounds with some lower level t 5 ) of activity .
The present invention ~lso provides An indirect method for identi~ying the , u..d or _ u--ds present in a library which has a sper;fi~ activity for a target ~ec~
Description of Drawings The present invention will be further described with respect to the Drawings wherein:
Figure 1 gr~phically Le~L~c.-Ls chi~nges in le~veLy for eAch round in a reiterative process for a library rontAin;ns~ only " losers ";
Figure 2 grArh; c~l ly represents changes in recovery for each round in a reiterative process for a library containing a single "winner ";
SUôSTlTUTE SHEE~ (P~U-E 2 W0 95/27072 2 1 ~ P~
Figure 3 graphically Lcy~e_..Ls changes in rc_uvcLy for each round in a reiterative proces6 for a library containing "loser6'~
and a library containing one winner;
Figure 4 graphically represents changes in 1CUUVCLY for each round in a reiterative process for different libraries; and Figure 5 depicts various bPn7n~liA7e~ine structures that might be used in formulating a libr~ry.
Det~; led Descrintion Qf the Invention Nore particularly, in accordance with an aspect of the present invention, there is provided a process for scree~ing a library of _ -c to dPt~rminp whether or not there is present in such librr~ry at least one _ ' ~hereinafter referred to as a "winner" ) that binds to a target molecule with at le~st a ~pe--;fi--d level of affinity, Kd ~iqsn~iAtion const~nt) or ~ctivity wherein the library is contacted with a target molecule under conditions at which those u.-ds present in the library having at least the sper; f i ~rl level of activity bind to the target molecule. As a result of such contacting, there is bound to the target any u-.ds which have the sper; ~; P~l level of activity in ~A~ rtllre with compounds which do not have such activity.
There~fter, those ~ h~ving less th~n the spe~if;~rl level of affinity or activity ~hereinafter referred to as "losers" ) are ,1 imin~ted from the mixture at a faster rate than winner -c.
The presence in the library of at least one winner u-ld is detr~rm;n~ci by detecting a change in the rate at which _u.lds are eliminated ~leuuvcred) from the mixture ~which can be detected by either mecsuring elimination of compounds or mcrlsuring Le uvcLy of _ tlc), with such change in rate being reflected as a change in the percentage of the compounds eliminated ~LcuuvcLcd) during such elimination ~ recovery ) .
q'hus, for example, if there is to be det~rm;nr~d whether of not a library contains a winner . ' or compounds which bind to SllBSr'rUTE SY'rT 'R'Jl E 2`'~;1 Wo gv/27072 2 ~ r~ o ~
a target molecule with at least a sp~~if;~d affinity (or Rd), the library ifi contacted with the target molecules under condition6 appropriate to recover by partitioning f rom the library winner compounds that are bound to the target. Under such conditions, some loser ~u--d6 will also be recovered. The recovery of 8uch loser compounds can be due to the background in the ~ssay and/or binding of abundant _ u--ds with low activity. Upon repeating of the ~LuuedULe wherein compounds Le~ uveled in the first contacting are again contacted with the target, losers will be eliminated at P f aster rate than winners . Thus, the proportion of winners to losers increAses rapidly with e~ch iteration of e.~o~,uLe to target.
At some point in the reiterAtion process, the proportion of winners to losers becomes great enough that winner u--ds pLe' ;nAte in the population. Accordingly, if one plots recovery of total u.~ds per round of iteration, there is a decrease in the rate of elimination of '~, and ultimately, in an ;r~1i7ed situation, a plateau is reached and the amount of compound obseLved binding to target with further iterations is constant. The presence of winner ( 8 ) in a library can be readily detected by de~rTn;n;ng a change in the rate of the ~1 iTn;n~tit~n of the u,.ds that bind to the target, or a change in the rate of .uv~Ly of unbound _ ~ from the mixture.
For example, suppose library C has 1000 copies of 1000 unique _u--ds, wherein all compounds are losers except u--.ls #C1-lOo, which have a Rd for the target of 1 micromolar, and compound #C101, which has ~ 0.1 micromolar Rd and i6 therefore ten times mor~ ~ctive theLn any other compound in the libr~ry. Upon e~o~uL.3 of 0.1 micromolar target to the library in the first round o~ a reiterative procedure, 50%, or about 500 copies, of #C101 will ~e Le~.uve~ed (because the target conc~ntration is equal to Rd for this compound) whereas only about 10%, or 100 copies of #C1-100 will be le~:uve~ed. In addition, suppose that the Ie~uvery procedure has a background Le~uv.=Ly of 1%, or about 10 copies of SUBS~I~LrTE SHEET (RULE 26) WO 9S/27072 2 ~ P~ o ~
lo-the other 899 ullds ~ #C102-1000 ) . In the second round of reiteration, 50% of the ,~ ;n;n~ #C101, or about 250 copies, are L~._uvc~L~d along with lOt~ or 10 copies, of #Cl-100, nnd 1% or ~
random assoL i L of about 90 of the inactive compounds . To summarize, after two rounds there are 250 copies of #C101, 10 copies of the 100 less active compounds #C1-100, and 90 unique inactive ~ _ u--ds. After a third round, there would be a`oout 125 copies of #C101, only one copy of each of the 100 less active u.lds, and virtually no loser __ -c LC..,vCICd. #ClOl would now be in a majority in the ~ ;nin5 library even though originally it was uui ` `cd 100:1 by r~R that were only 10 times less active.
Thus, the !?1~l in~nr~e of ~ _ ' C101 would result in a change in the rate of ~limination of the u--ds ( or ~ change in the rate of rc_uvcLy of the compounds) in subse~uent iterations, and such change in the rate of r~_uvCLy or rate of elimination indicates that the library contains a ~ _ ' that has at least the spe~ifier1 activity (a winner).
The preaent invention takes account of the f act that if a library contains rare u..ds that interact spe~-if;~-~l ly with a tarqet, after contacting said librAry with target and isolating active _ u--ls, some small percentage of inactive compounds, or compounds with lower activity, will also be isolated. The Le-uvcLy of inactive, u--ds can be due to the background in the assay and/or the binding of abundant ul-ds with low activity. When the process of contacting '~ with target and isolating nctive ~ u--ls is reiterated, the snme small percentage of inactive or less active compounds will be ~ ~ _uvcLcd with the most active u--ds. Because the percentage LC~.VCLY of most active u-.ds will be much greater than the percentage Lc~lvcly of inactive or less active ,u.,ds per round, there will be an ever SUBSTITUTE SHEET (RULE 26~

W0 95/27072 ~ 3 ~ r ~ .
widening difference in the cumulative percentage of active and inactive compounds recovered through the reiterative procedure.
The underlying principle of the present invention i8 in the dif f erence in signal between a library that contain6 a highly /~ctive compound and a library that doe6 not. Suppose that library D is identicAl to library C in composition but does not contain a ~ u,-d equivalent to #C101. That is, #Dl-100 all have low activity with Rd - 1 micromolar, and the other 900 compounds all have no activity ( losers ) . Suppose libraries C and D were to be measured for ~signal ' ~e.g. the L~ inirl5 number of molecules) after each round. After the first round, library C would have a~out 500 copies of #C101, 100 x 100 = 10,000 of the less active #Cl-loO, and about 900 x 10 - 9000 losers for a total of 19,500 molecules. Library D would have 100 x 100 = 10,000 of #Dl-100, and about 900 x 10 - 9000 inactive ullds for a total of 19, 000 molecules. Note that library C has o~ly 500 more molecules due to the highly active #C101. Measured as a percentage of the library put into the first round of reiteration, library C would have Le~ UVe:LI,I 1 . 9596 ~ ed to 1. 9o% L~ /V~ for library D . This very small difference would very likely be within the experimental uncertainty ~or "noise" ) of the binding and meaDuL~ t methods.
After two rounds, libr~ry C would have 250 copies of #C101, 100 x 10 = 1000 of #Cl-100, and 89 of #C102-1000 for a total of 1,339 molecules compared to library D that would have 1089 molecules.
The difference in Le-;UVe:Ly between the two rounds, more than 2096, might well be d~t~ctAhle. After round 3, however, library C would have 125 copies of #C101 and 100 x 1 = 100 of, _ 's #Cl-100 for a total of 225 molecules, - ed to library D that would only have 100 x 1 = 100 of , _ ~c #Dl-100. In other words, what w~s a minor difference in the first round, and a moderate difference in the second round, is now almost a 2-fold difference in percent recovery at round 3, with library C returning 225/1339, or nearly 17% recovery compared to library D returning 100/1089 or only about Su~sTlTuTE '`.~!EET IRt~LF ~

W0 95/27072 2 ~ 8 5 q 1 ~ r~ . o l~ --94 of the input into the third rourd. This example underscores how Il reiterative procedure can dis~in~;~h between two nearly identical libraries even when the difference between the two is only a single r~re but highly active member.
Thus, it should be ~ L from the above illustration that as compounds originally bound to the target are eliminated f rom library C, there is a ~ tectAhl~ change in the rate of elimination or rate of LeuvV~ly between rounds two and three for library C
which has a winner, whereas in libr~sry D there is not a signific~nt change in the rate of elimination or rc~uv~Ly of such ~ ullds.
Thus, whether or not a library rontn;nC a winner can be det~-nmin~d without amplification by detecting a change in the rate of elimination of those compounds that originally hound to the target.
As a ~urther illustration of the process of the present invention for identifying whether or not a library contains a compound that binds to a target with at least a sperifi~d activity, there may be def ined as a winner, a _ ~ in the library that binds to a target with a Kd value in the nanomolar range or le66, whereas losers might bind to the target with a Etd in the micromolar r~nge or higher.
The binding of losers is commonly ref erred to as 'noncpec;f;c" because it is a characteristic shared by all library members. If a target can rern~n;7e and bind to a particular member or subclass ~winners) with much higher affinity, such binding is referred to as "specific" because it is specific to those winners r~ther th~n to the other members of the libr~ry. For example, DNA-binding proteins typically have nnncper;fi~ binding affinity for any DNA sequence in the micromolar range whereas they bind specif ic DNA sequences with nanomolar or lower ~d -UTE SHEE~ (RULE 26) WO 95127D72 ~ I 8 5 9 1 8 PCTIUS95/0409~
If a library cnntA;nin~ 1014 molecules cnntA;nq no winners,it will have a low background value if appropriate conditions are chosen for assay. For example, in an appropriately designed binding ~ssay, generally a total of 1% or less of losers is measured as interacting with the target. ~i~ a library containing only losers has a ba-hyL-,u--d value of 1% and those , Ulld8 associated with the target are L~_~v~ and ~c _~,oq~d to target, once Again only 1% o~ the u--ds will be associated with target.
Thus, if one were to rnnt;nll~ this process of recovery of those compounds associated with target and re-exposure to target and to plot the results, one would predict a linear result in ~ semi-log plot as shown in Figure 1.
If a library contains one or more winners, under ideal conditions all winners will interact with the target at each iteration provided that the concentration of winners is less than the target ,n~ Lion, and the target concentration is greater than the R~ for its ARsoriAt;nn with the winners. Thus, the proportion of winners to total u--ds associated with target increa~es rapidly with each iteration of exposure to target. At some point in the reiteration process the proportion of winners to total ~ u--ds becomes great enough that winner compounds ,Ull ' n~te in the population. Accordingly, in the semi-log plot, the curve visibly departs from linearity; ultimately, in an ideAl;~ed situation, a plateau is reached and the same amount of u.ld is observed binding to target with further rounds of iteration. For examplc, an irl~Al;7ed binding experiment is shown in Figure 2 in which a library rnntA;nR 106 copies of 10~ different nucleic acid sequences of which only one sequence is a winning sequence. By the fifth iteration, the curve departs visibly from linearity. Figure 3 demonstr~tes a superimposition of the plots from Figures 1 and 2. By the fifth iteration 100 times more ' is reouv.:L~:d from a library contA;n;n~ a winner (hereinafter L~ Ll~:d to as a "winner library" ~ than from a library SUBSrlTUlE SHEET ~RULE 26) WO 95/27072 ~ r~
-1~
containing no winners ~hereinafter a "loser library"); by the sixth iteration, 10,000 times more compound is recovered from a winner librrry than from a loser library.
Consider the case of a library that cont~ins two compounds that can interact with target, such that, _ u--d A interacts with higher affinity for the target th~n _u--d 3. If the assay is carried out such that the concentration of target is at or below the Rd for binding to _ ' B but above the Rd for binding to /1 A, after several iterations, most of the observed interaction with target will be due to ~ _u--d A. For example, if A interact6 with target with twice the af f inity of ' B, after 4 iterations, there i8 approximately 2~, or 16, times more compound A interacting with target than compound B.
Similarly, if compound A interacts with target with three times the affinity of ' B, ~fter 4 iteration6, there is 34, or 81, timcs more ~ ' A L~_V~r~L~ ~ from the libr~ry th~n _ Il--d B.
Thus, libraries are likely to be identified as "winner libraries~
because of a single best member compound, unless they contain numerous _ u--ds that interact with a target with very similar affinities. Furthermore, for the same reasons, if two libraries are -- ~d and library A contains a winner with just marginally higher af f inity than any winners in libr~ry B, the library A will cle~rly be scored ~8 a winner libr~ry relative to library B. This is important to the procedure because the ability to ascertain that particular library contains winner ( 8 ) of higher af f inity than other libraries will allow indirect elucidation of the winner(s) identity in the f inal screening steps of the overall procedure .
If a library contains two winner compounds that interact with a t~rget with the same high affinity, then the plateau level in the semi-log plot will be twice as high as the plateau level in a library containing only one winner (Figure 4). Thus, under ir~ i7ecl conditions, one can not only distingnif~h r winner library S~ !Tt 5~EE~ F 261 Wo95127072 r~ x. ~o.

from a loser library but al60 estimate whether there i5 more than one winner in a library.
Thus, as should be Ppparent, in accordnnce with the present invention, if a library contains no winner6, a plot of the material r~:c.uveLe:d per round (or material eliminated per round~ will be, monotonic with ever dQcreasing amounts of material L_cuvel~d each round, usually according to a relatively fixed percentage recovery (fixed percentage of el;minnt;~n of losers) each round. A library containing one or more wi~ners will follow the same plot until a round is reached where the winning u.-ds that are L~Cuv~l~ d at a higher percentage per round (eliminat~d at ~ lower percentage per round), become a m2jority of the L~ ;n;ng library mixture. In all subse~uont rounds, the plot will reach a "plateau" because a relatively high recovery of material will be seen every round ( a relatively low elimination of material in each round) once the majority of the L. ;nin~ compound6 are winners. Thus, by such a plot, the presence or absence of a winner in a library can be readily ~l~t~m; npd.
Although in accordance with one: ' -';r ':~ the so-called pl;m;n~tinn of logers is accomplished by recontacting of L~:~uv~Ied -_ ~c with the target in a reiterative ~LuceduL~:~ the prese~t invention also contemplates other methods for eliminating so-called losers in order to identify whether or not the library cnnt~;nC a winner .
Thus, for example, instead of recovering bound , u..ds and recontacting the target with the L~:~Ov~:L~d, ul~ds, after the i~itial binding, the target could be treated in any manner that would preferentially eliminate from the target those _ ~c that bound to the target at lower affirity. As a non-limiting example, this could be ~ccomplished by a dialysis yLU~ ULt: in which series of buffer (dialys~te) changes into which the library is SUBSTITUTE SHEET (RULE 26~

WO95n7072 ~ 3 r_l,.a_'C

dialyzed would preferentially contain losers that cross the dialysis membrane whereas the target would ~-nnt;nlle to preferentially contnct the winners that cannot cross the membrane while bound to the larger target . If, f or example , the target-library incubation sample is placed in a dialysis chamber with a ten-fold excess of dialysate across a dialysis membrane that has a molecular weight cut-off such that a relatively large target could not cross the membrane but the relatively small library members can, then approximntely 909~ o~ the unbound library members will migrate across the dicllysis membrane into the dialysate. Each time the dialy~ate is F`~ hAn~ed with fresh buffer, another 9096 of the unbound members (losers) will migrate across the membrane, thereby lowering the total number of losers in the sample. The winners, in contrast, will be prefere~tially retained in the sample due to their interaction with the target.
The dialysate changes could even be achieved by a cnntinl-nu~
flow of buffer into which the losers ~LaLe~ ILially diffuse. After a period of time, the target-containing sample, or a portion thereof, could be assayed for the amount of library L~ ;n;ng. A
library containing high affinity winners would retain more material a$ter longer periods of time than a library with lower affinity compounds that would diffuse across the membrane at a much higher rate. Such a cnnt;nllnu~ time-b~lsed ~L~ ULe is essentially & very rapid multi-round procedure wherein the rounds consist of the constantly "reset" initial dialysis condition as the dialysate is changed .
Another ~L.~ceduL~ for eliminnting losers to thereby enrich winners after contact of a library with a target molecule which may be employed for screening a nucleic acid library i~volves the generation of "sense" and "antisense" strands from the nucleic acids that bound to the target molecule . Cycleg of hybri~ At; nn and melting of the strands, with elimination of those strands that SUBSTITUTE SHE'T (RULE 261 W09sl27072 2 1 8 5q l 8 r~l,s.. ~'0 do not hybridize, as described in U. S . Patent Serial No . 079, 677, filed on June 18, 1993 result in elimin~tion of losers, and by using a plot as hereinabove described, there can be det~rmi n~d whether or not such library contains a winner sequence.
Thus, as should be apparent, any one of a wide variety of ol eduL~s that will elimin~te the so-called losers at n rate greater that the elimination of wiD~ers will permit a A~t~rm;nAt;r-T~
of whether or not a library contains a winner in that the elimination of the losers will result in a rate of change with respect to recovery of the ~ ~ u.lds which change in rate indicates that the library contains a winner.
Thus, in accord~nce with the present invention, a change in the rate of elimination of ul.ds that are bound to the target (or a change in the rate of LeOuVt:Ly of compounds ~ indicates whether or not a winner is present in a library. Thus, for example, the rate can be a two-fold change or greater in the rate of LeCUVe:Ly of ~ u..~s or in the rate of ~l imini-ti~n of _ ' . Thusr as should be apparent, the rate of el ;minAtion of '~ for the ~JU~uUSe3 of the present invention is related to the rate of LecuveLy of ~ u--ds, and a change in the rate of either is i nA i CAt i ve of the presence of a winner in a library .
In accordance with the present invention, one can c~lclllate the initial size of a library (N = total number of molecules ) required to screen for winners of a particular rarity, and the number of iterative rounds (R) required to detect a 100% incre~se in the number of Le inin~ molecules in a winner library versus a loser library. ~ winner has an initial frequency (f) equal to the ratio of winners to lo~ers in the library and can be recovered from each iteration with a certain percent yield (y). The losers are LecuveL~d with a certain background percentage (b). Let d = the smallest number of molecules that can be detected (by fluoresce~ce SUB511~!T~ v~lE'~ ~R~ 2'i~

WO 95/27072 2 1 ~ PCTIUS95104098 or a variety of other method6). Then, the number of rounds required to result in a two-fold ~or 1009r ) increase in the total molecule number L~ -in;n5 i6 defined by the equation R =
log~f )/log~b/y) . And, the library must be of size N
d/[~f)~y/lOO)R] to have enough molecules left at round R to be above the detection threshold. For example, a rF-A~l-nAhly large library in practice can contain about N=1015 molecules ~ about nanomole), with a detection threshold of about d=10,000 molecules, a background of b=0.19s, and a yield/iteration of winners of about y=2096. Given these parameters, these two equations can be solved together for R and f to show that about one winner in 105 total molecules would be detectable after 4 rounds of selection as a 2-fold increase in the ~bse~ved number of molecules recovered.
These Gre r~-A~-n~hle numbers for an actual experiment, but are not necessarily limiting because detection threshold, bac:~yLuulld, initial library size, and yield of winners could all be even more advantageous in practice, allowing for even rarer winners to be detected. Note that in this example the library could contain 107 copies of 105 unique sequences and the procedure could detect one unique s~qu~nr~ that has the highest affinity for the target.
Alternatively, the library could h~ve as many as 10l5 unique B~qlllrnrF.~ and the P1U~6lUL~: could det~ct a subset ~or ~amily) of sequences of which there are as few aB 107 in the library.
The compounds that are used in formulating a library which is tested in accordance with the present invention may be any one of a wide variety of '~, particularly since in accordance with the present invention it is not required to amplify compounds that initially bind to a t21rget molecule. Thus, for example, the library may be a nucleic acid library which may be formed f rom either single .LLelllded and/or doubl~ ~LL~.Ided nucleic acids, and such singl~s ~,LLc,~ded nucleic acids may be either DNA or RNA.
Similarily, when employing nucleic acids or oligonucleotides, such nucleic acids may be ~ifiF~d or, ~;fi~-l nucleic acids.
SU~ST~TUTE SHEET (RUI E 26) 218~8 WO 95/27072 r~ C'C ~ _ The term "nucleic acid" as used herein means that the nucleic acid may be a r;honllrleir acid, i.e. an RNA; a - deoxyrihnnl~r~lr-ic acid, i.e. a DNA; or a mixed rihon~rt~;r/deoxyrihnnllrlr~;c acid; i.e., the nucleic acid may include ribose or deoxyribose sugars, 2 '-O-methyl ribose or other 2' substituted or conjugated sugars, or ~ mixture of such sugars.
Alternatively, the nucleic acid may include other 5-c~rbon or 6-carbon sugars, such as, for example, arabinose, xylose, glucose, galactose, or deoxy derivatives thereof or ~ny mixture of sugars.
One or more of the phnsFhorus-containing moieties of the nucleic acids may be ';f;e~ or I ';f;~d. The phos~lluLus-containing moiety may be, for example, a phosphate, phosphonate, alkylphosphonate, aminoalkyl phosphonate, alkyl-thiophosphonate, phosphoramidate, phosphorodiamidate, phosphorothioate, phosphorodithio~te, phosphorothion~te, rhc srhnrothiolate, E~ho~uhuLGlllidothiol~te or phnsrhnrimid~te. It is to be understood, however, that the scope of the present invention is not to be limited to any sFerif;r rhnsrh~rus moiety or moieties. Also, one or more phosphorus moieties may be ';f;~d with a cationic, anionic, or zwitterionic moiety. The nucleic acid may also contain one or more bAckh~m~ l;nk~c which do not contain phosphorus, such ~s carbonates, ~LLu~ ' hyl esters, AretAm;~l~t~C, carbamates, ~cet~ls, ~nd the like. The nucleic ccids m~ly clso contllin one or more b~ckhon~ linkage of peptide nucleic acids (PNA). (Egholm et al., J. h~. Chem. Soc.. ~, 1895, (1992).
The nucleic acids also include any natural or unnatur~l, substituted or unsubstituted, purine or pyrimidine base. SUch purine ~d pyrimidine bases include, but are not limited to, n~Ltural purines and pyr;l~ ;n~s such as adenine, cytosine, thymine, guanine, uracil, or other purines and pyr;m;~;n~s, or analogs thereof, such as isocytosine, 6-methyluracil, 4,6-di-lly.lLu,~y,uyLimidine, l~y~uù~ Lhine~ Y~nth;n--~ 2~6-rl;Fnn;nnpurine~ 5-SUBSTITUTE SHEET (RULE 26~

WO gsl27072 2 ¦ 8 5 ~ 1 8 ~ 'O IDSv azacytosine, 5-methyl cyto6ine, 7-deaza-adenine, 7-deaza-guanine, and the like.
The nucleic acid6 may be '; f; ed 6uch that at lea6t one nucleotide unit of the nucleic acids may include a conjugate group.
Such conjugate groups include, but are not limited to, (a) amino acid6, including D-amino acid6 and L-amino acid6; ~b) peptide6, polypeptides, and protein6; (c) dipeptide mimic6; (d) 6ugar6; (e) 8ug~r pho6phate6; (f ) n_ .LuL~ 6mitter5; (g) ~ ~~; (h) poly ( l-y dL U~y~lL U~Jy lmethacrylamide ); ( i ) po lyethylene imine; ( j ) dextrans; (k) polymaleic anhydride; (1) cyclodextrin6; (m) starches; ( n ) steroids, including sterols such as, but not limited to, chole6terol; (o) acridine; (p) vitamin6; and (q) polyalkylene glycols, such as polyethylene glycol. guch moieties may make the nucleic ~cids more re6i6tant to degrad~tion in cell6 and in the circlllAt;nn, and/or make the nucleic acid6 more r ~ to cell6.
The conjugate moiety may be attached to the 3 ' terminal nucleotide unit and/or the 5~ t~rm;n~l nucleotide unit and/or to an internal nucleotide unit(s), or conjugate moieties may be attached to two or more nucleotide unit6 elt the 3 ' end ~nd/or the 5 ~ end of the nucleic acid. In one . `; L, substituted nucleotide unit6 m~ly altern~te with un6ub6tituted nucleotide unit6. In another - L, all of the nucleotide unit6 are sub6tituted with a conjugate moiety.
The conjugate moiety may be attached to the nucleic acid at the purine or pyrimidine ba6e, ~t the pho6phate group, or to the sug~r. When the conjugate moiety i6 attached to the ba6e, it i6 preferably attached at certain po6itions of the bage, ~ r~n~l;ng upon the base to which the moiety is attached. When the moiety is attached to adenine, it may be attached at the C2, N6, or C8 positions. When the moiety is attached to guanine, it may be attached at the N2 or C8 positions. When the moiety is attached to cytosine, it may be attached at the c5 or N4 positions. When the SUI~STITUTE SI~EET (RULE 26~

~t W095127072 7~ r~l~o c ~-~
moiety is attached to thymine or uracil, it may be attached at the C5 position.
-In one: -'; L, the nucleic acid includes from about 5 to about 100 nucleotidc units, preferably from about 8 to about 60 nucleotide units.
In yet another ; , the nucleic acid represents a portion of a larger molecule which contain6 non-nucleic acid components, such ~1S, for example, peptides or proteins, or simple cArbohydrates, and lipids.
The nucleic acids may be in the form of a single strand, a double strand, a stem-loop structure, a pseU~inknnt~ or a closed, circular structure.
The nucleic acids may be synthesized by a variety of accepted means known to those skilled in the art. For example, the nucleic acids may be synthesized on an automated nucleic acid synthesizer. Alternatively, the nucleic acids may be synthesized enzymatically through the use of flFlnkin~ or primer 8~ nr~C at the 5 ' and 3 ' ends . In another alternative, the nucleic acids may be synthesized by solution phase chemistry. It is to be understood, however, that the scope of the present invention iB not to be limited to any particular meAns of synthesis.
The compounds used in forming a library in accordance with the present invention may be peptides.
Alternatively, the compounds may be organic _ ~ ullds, such ~8, for ex~mple, olirJnsarrhArides, b~-n7o~ 7epines, etc. The selection of a 8uitable type of compound for forming a library is deemed to be within the scope of those skilled in the art from the t~nrh; n~': herein .
~il~l IME S/ !EE~ ULE 26 Wo95127072 2 ~ ~9 ~ o 1 ~
Similarly, the target molecule may be ~my one of a wide variety of target molecules to which compounds in the library may bind. Although the preferred target molecule is a polypeptide or protein, the present invention is not limited to such targets.
Similarly, the library screened in accordance with the present invention may be comprised of mixed ~ _ ul~ds. For example, the library ~iA may be comprised of ol; ~, -- D
containing a mixture of amino acids and nucleotides. Thus, in accordance with the present invention, the library can be formed from any one of a wide variety of _ u~-ds, and can be screened by contacting the library with any one of a wide variety of target molecules. Also, in accordance with the present invention, there need only be in the library ul.ds that will bind to a target, with the screening being ~ h~ 80 as to effect binding to the target molecule of those UlldD that have at least a Sp~r; f; ed activity.
Although in a preferred 'i L, such predet~rmined activity is def ined in terms of u~lds that bind to a target with ~t least a spPr;f;~-cl affinity ~or E~d), the present invention also contemplates de~-rm;n;ns~ whether or not a library contains one or more ~ that bind to a target with a sper; f; ~1 activity in which such activity is me~sured in terms of in2ctivating or activating a target molecule.
For example, if a u.-d binds to a site on the target in a way th~t blocks the ~ctivity of th~t target sr~irh;~ ically, one can ~ t~rm; nQ the presence of winners in the library by measuring the reduction of activity of the target. In this - ~; L, the inhibitory activity of the library would be tested after each iteration under conditions where all starting libraries exhibit activity. As material is lost from each library, thereby lowering the total concentration of ~ u.-ds in the inhibition SUBSTITUTE SHÉET (RULE 26) Wo gsl27072 -23- r ~ s,,~ ~ IC5 1i assay, loser libraries will lose their inhibitory activity. Winner libraries, however, will maintain a level of inhibition as long as - the initial winner concentration exceeds the IC50 for that compound.
For example, suppose that ICso of winners is 1 nanomolar, the library has a nonspecific IC50 of 1 micromolar, and the initial library concentration is 100 micromolar. If the frequency of winners in the libr~ry is one per 10~ compounds, then after 2 rounds o s~1ec~;nn recovering 509~ of the winners ~nd 19~ of the losers each round, the losers would be present at a rnn~ntration of only 10 nanomolar, and would therefore exhibit no nonspecific ;nhih;1-;~ n. Libraries nnnt~;n;n~ winners, however, would still contain 2 . 5 nanomolar winner and would inhibit >5096 through at least 3 rounds. Note that without reiteration the winners would not be detect~ble bec,luse of the nonspecific inhibitory activity of the library as a whole. In general, if winners have an IC50 x-fold lower than the losers but are rarer than l:x in the library, then only a reiterative yLuc~duLc will allow them to be identified.
It is not a prerequisite of this invention to determine the amount of r~ . uv~ d ~ _u~-d after each iteration, or to prepare e semi-log plot, although such evaluations are ~L~LL~:d. As a non-limiting example, a b~n70~ 7~rin~ library (Bunin & Ellman, J.
Am. Chem. Soc. ~, 10997, 1992 ) is tested for the presence of winners by contacting the library compounds with a target, separating bound from nu~l bu~i~d compounds by any of a number of separ~tion t~rhn;q~ b~sed upon the diference in molecular weight of the unbound b~on7o~l;n7~l in~c vs. those bound to target, such techniques being well-known in the art, extracting bound b~n7orliA7~r;n--c, con~nct;n~ such extrncted c with target once again and ~ rmt;nll;n~ such iterations for as many cycles as is desired or possible. After the last iteration, one quantitates the presence of extracted ~n7nrl;~7er;n~c~ for example by accelerator mass spectroscopy (Vogel & Turteltaub, Trends Analyt. Chem. 11, 142, 1992 ) . By running in parallel a sample of one or more SUB~TITUTE SHEE~ (RULE 26~

W095/27072 ~ '0l -2~
ban7o~ 7ep;nPs previously det~rm;nad to be loser(s), one can establish background levels for such loser(s) and by comparison deduce whether the experimental library contains winners.
As should be apparent to those skilled in the art, the term "iteration" or "reiteration" is not limited to recontacting those compounds that originally become bound to the target with the target and such term broadly -n~ ses a variety of procedures for treating those _ ~ that bind to the target, some of which have at least the spe~i f i acl activity and some of which have less thnn the spe~i f i ~ QCtiVity in such a manner that those _ u-.ds that bound to the target and did not have the spe~; f; Pd activity would be eliminated at a faster rate than those __ '~, if any, which bound to the target and had at least the specified activity.
Thus, for example, such reiteration to eliminate at a faster rate u-.ds that do not have a speri f j ad activity may involve a series o~ rounds or steps in which the ~ ~ ul~ds that bind to the target ~re rG~ L~d and recontacted with the tarqet or may involve ~/L~.eé-lULeS as hereinabove described, for example, dialysis or, in the case of nucleic acids or ol;gnn~ antides, melting of sense and antisense strands of those nucleic acids that bind to the target ecule in a manner such that those having a lower binding af f inity are eliminated at a f aster rate .
The amount of material Le_uveL~ in the reiterative procedure employed in the present invention may be detarminad in a wide variety of ways. Thus, for example, every member in a library can be radioactively labeled or tagged with a f luorescent dye. A8 a non-limiting example, a nucleic acid library is labeled with "P by enzymatic end-l~helin~ te-hn;~es well known in the elrt.
After each iterntion the nucleic acids associated with the target are rc~)veL~.l and all or part of the r~ e~ nucleic acids are measured for Cerenkov disintegrations. Since such determinations are carried out in a scintillation bl,e~:Ll ~ar without addition of ~E S~,E~7 ~R~r ~6) ~ Wo gs/27072 2 ~ ~ ~ 9 1 ~3 r ~ o io~
fluor, the total amount of the evaluated 6ample i5 preserved for _urther iterations of e..~oDu.e to target. The number of Cerenkov disintegrations measured at each iteration determines the feasibility of carrying out a further iteration. Another non-limiting ex~mple iB a library containing compounds each of which is covalently attached to a defined nucleic acid sequence ~a "tag sequence" ) such that the quantity of compounds can be measured by any of the methods known in the art that use hybr;rli ,~i nn of a nucleic acid probe to a target sequence, in this case the tag sequence .
Alternatively, as another non-limiting example of the instant invention, each member of a peptide library is labeled with a relatively small fluor such eLs fluorescein or rhn~i~min~. After each iteration of ~u~ule to target, the quantity of recovered peptides is measured with a fluorometer. Once again, all or p~rt of the e-_uveLéd peptide population can be used for a further iteration if it is det~rmin~ that there is sllffiri~nt fluorescent material to permit another round of s~lec~inn and quantitation.
The present invention is also directed to prescreening in order to determine whether or not certain types of u--ds or a certain size of a _ u-ld may be best suited for interacting with a target molecule.
Thus, for example, in searching for a peptide-based therapeutic, it may be desirable to rl~t~rmin-- the smallest peptide that intereLcts with a t~rget since larger ~ _ ullds are more expensive to produce and could be less bioavailable.
Accordingly, ûne can generate peptide libraries of different sizes and rl~t~rmin~ whether any library cnn~inR a winner. Thus, for example, if libraries of dipeptides, tripeptides and teLlc~ye~Lides ~re det~rmin~cl not to cont~in winners where~s SUBSTITUTE S~EET (RULE 26~

Woss/27072 2 ~ ~59 ~ ~ F~llu~ 01~50 pentapeptide and hexapeptide libraries are determined to contain winners, subsequent determination of winner peptide ~eq~Pnce~ can be carried out with pentapeptide libraries since these librarie6 are ~l~t~rminDd to be the smAllest molecular weight libraries containing winners. Alternatively, both the pentapeptide and hexapeptide libraries (but not smaller molecular weight libraries) can be further analyzed for winning sequences.
As another non-limiting example of the instant invention, 6ingle 6LL~nded nucleic acids i~lDnt;f;ed as interacting best with a target are often determined to as6ume digcrete re~ n7n;7:1hlD
structures such as stem-loops or pseudoknots. These structures are often sequence no~ e~ific but are prefiumably essential for providing a scaffolding to present the selected sequence-specific rrgion of the nucleic acid to the target in such as way as to optimize interaction. The need to have such structural properties in the selected nucleic acids essentially reduces the proportion of compounds in the population that are suitable candidates as winners . The end result is that much of the value of a library ' s r;ty is lost and the number of cycles required to narrow down the population of nucleic acids to those that have appropriate scaffolding is unnecessArily great. The use of libraries in which the member nucleic acids are ~lP~i~nPd to possess such 3LLU~LUL~n in fact optimizes the s~-lec~inn procedure.
Among structured nucleic acids that are obseLv- d, stem-loop, bubbled, psel~nknnt ~nd tetraplex structures are known. Since base sequence is for the most part irrelevant in the structured regions 80 long as base pairs are formed, for the instant invention nucleic acid libraries are ~lD~jqnDd with fixed-sequence base-paired regions . If it is prP~lPtPnmi nP~ that a library in which all nucleic acids possess A particular structure contains one or more winners, such a structured library would present the following advantages in determination of the winner sequence ( 8 ): ( 1 ) SUB~T!TUTE St~E~T (~ULE 26l -~ Wo 95l27072 2 1 8 ~ 9 1 8 r~ c ;os selection is completed with fewer selection cycle6; (2) because structure is already integrated into the ', more complexity i6 introduced into variable region6 of the molecule without ~.Ycef~3in~ the sequence complexity that can be s~1ect-~1 for; and (3) if the structure contain6 a random region of the m;n;--l ly permitted size for a winner sequence, this prevents a winning sequence from occupying multiple registers of the random region in different winner molecules, i.e., this reduces the number of dif f erent winner compounds .
Structured nucleic acid libraries are generated with automated nucleic acid synthesizers by procedures well known in the art. Libraries can be generated to /-ssume structures that include, but are not =limited to, stem-loops, bubbles, rC~ nknnts or tetraplexes. Libraries can contain one or more of these structures. Structured libraries can contnin, RNA, DNA, a mixture of the two, and/or ~ _ _..ds comprised of '; f; ed nucleotides .
Stem-loop structures are generated by constructing a molecule with complimentary ends that base-pair with adequate a~finity to maintain a stem-loop rt~nfo~r-tion. A non-limiting example of a simple stem-loop structure might have the sequence GINyCI wherein x is at least 2 up to lO or more and Ny L~L~:S.~11LS the r~ndom loop sequence in which y can be at le~lst 3 up to 20 or more.
In such a library, each G in the Gl region would be expected to pair with a corresponding C in the Cl region to form the following structure (x=5, y=6 is shown as an example):
NN~
5 '--GGGGG N
3 ' -CCCCC N
\ NN ~
T'TIJ~ Ct~'~T ~ 26) Wogs/27072 ;~ ~ 8~ B,,_!Olo~

The loop can contain natural nucleotides tribo A,C,G,U or deoxyribo A,C,G,T) or nucleotide6 -;f;F--l in the base, sugar andJor harlrhnnP moiety. ~ rles o~ such ';f;e~ base6 include, but are not limited to, 5' methyl U, 2,6 diaminopurine, inosine.
r leR of such ';fi~l 6ugars include, but are not limited to, 2~ alkylated, 2'-0-alkyl, 2' h~ln7~n~ted, 2' amino 6ubstituted sugrrs. r le8 of such -';f;e~3 barkhnn~ groups include, but are not limited to, rhnsphorothioates, pho~,yllu-udithioates, methyl-rhnsrlhnnAtes, and amino alkyl hACI~hnn~6. The loop can also contain abasic-nucleotides or non-nucleotides, ;nn1~ ;n~, but not limited to, so-called PNAs, glycols or ~mino acids, or other monomeric units described hereinabove.
In the stem, the bases can be varied 80 long as they are cnnfigllred to base-pair appropriateIy. In f~ct, it is preferable to vary the bases in the stem region to avoid slippage in base pairing: e.g., A can be substituted for G and T (or U) for C in the aful Lioned structure or the stem can consist of a mixture of A-T (or A-U) and G-C base pairs. In addition, ribo G-U or ';f;~A
ribo G-U base pairs cnn be used in the stem. Any `; f; ~-i bases are ^llowed in the stem 80 long as they ~orm suitable base pairs.
The stem can also contain ocnA~;nnAl non-base-paired regions. The 3 ~ or 5 ' end of the molecules ( or both ends ) can be attached to moieties that can be used for ~UUL,UUBE5 of quantit~tion or identi~ication. ISxamples of such moieties include, but are not limited to, unpaired nucleotides, biotin, fluorescein, amino acids, 32P. One or both ends o~ the stem can be covalently attached to an ol i~nn~lcleotide "coding sequence" via an orthns~onAl ulld _s described by Beutel et al. ~US Patent ~rplicAtinn 08/079,677) or to a tag sequence, as described above.
Bubbled structures are similar to stem loops except that stem regions surround the loop. A non-limiting way to generate a bubble library is by synthesis the following ol i,~ , r. N ~ M '1' N r SUBSTITUTE SHE~I ~RUI 2In Wossl27o72 ~ g ~ .,,51'C ~ _ In such structures, G~ would base-pair with C~ to form a stem on one side of the bubble and A~ would base-pair with T~ to form a stem on the other side of the bubble. The compounds in the libraries would then h_ve the following structure (v~4, w=4, x=4, y-4, and z~4 is shown as an example ):
~M~
5 '--GGGG M
3 '--CCCC TTTT~ J
NNNN M
As envisaged, v can represent at least 2 up to 10 or more nucleotides and x can represent at least 2 up to 10 or more nucleotides. N,,and Nztogether form the random loop structure, although N~, or N~ can have a ~ixed sequence . w and z can each be as 6mall as one nucleotide up to 20 or more nucleotides. w and z can be equal or unequal.
The bubble librariea can contain naturally-occurring nucleotides or can contain ';fied nucleotides or non-nucleosidic Ls as described above for the stem-loop libraries. The molecules in the bubble libraries can contain adducts attached to the 3 ' or 5 ' end as described above for the stem-loop libraries .
c;m;lAr1y~ librariea can contain pseudoknot or tetraplex structures .
These and other structures used in libraries can be envisaged based on existing knowledge of such structures in the art (e.g., Puglisi et al., Acc. Chem. Res. 24, 152, 1991).
In addition to the foregoing, structural libraries can be constructed with I..ds other than ol; ~rn~lrl eotides, such SllB~TlTUT~ SIIEET (~ULE 261 W095/27072 ~ 9 ~ P~IIIJ ~0l03s including, but not limited to, peptides and rigid org~nic , , u,-d~ .
As one: _ i L of the present invention, the pre6creen is used to determine both the nature and size of the structured nucleic ~cid that might best be tested for a given target. The procedure includes contacting the target with a series of libraries containing structured ~_ c that differ in the size of their random portion. As a non-limiting example, the target is contacted with stem-loop librarie6 containing progressively larger loops, such loops containing random nucleotide sequences. Thus, the different libraries can contain 4-nucleotide loops, 5-nucleotide loops, 6-nucleotide loops, etc., up to 20-nucleotide, or greater, loops. Following reiterative contact of the target with each of the stem-loop libraries, it is det~rm; n-~d whether each of the libraries is a winner or loser library. If it is A~t~rmin~d that winner libraries mu6t contain a loop of at least a given size, then only structured libraries containing a loop size equal to, or larger than, that minimum size are used to identify the winner sequence~ s ) .
It is a f urther aspect of the instant invention to test different structures for affinity to target. For example, a prescreen in accordance with the invention is carried out with st~m-loop, bubble, psellrirknnt~ tetraplex and linear libraries, such th~lt the molecular size of the ~ _ ~~ ir. each library is approximately the same. kibraries scored as winners are subsequently used for s~lert;~)n of optimal binding sequences as described below.
In yet another aspect of this invention, libraries are constructed that dif f er in net charge . For example, libraries can be constructed containing phosphorothioate b~rkh- nec ( Stein et al., ~ucl. Acids Res. 16, 3209, 1988), methyl phosphonate h~rkh~nes SURSTITUTE SHEET (RULE 26) wo 95/2~0 J2 ~ ~ 8 5 9 ~ 8 P~ o (Niller et al., U.S. Patent 4,469,863~ or ATnin~ Lhyl phosphonate b~rkhonPs (Fathi et al., Bioconjugate Chem. 5, 47, 1994). In these libraries, the net charges will be negative, neutral or positive, respectively. Alternatively, the library ^ntS can contain nucleotides with a mixture of barkh~ ne~ so that the charge can be varied. By using the yLeS~ L~:~II of the instant invention with these nucleic acid libraries, it can be detPrm; nP~7 which libraries contain winners. Prescreening can also be carried out with libraries varied with regard to both structure and charge.
In a very general ' i L, the pre~ent invention can be used with any polymeric or nonpolymeric library that can be synthesized in a stepwise manner (this is important for the winner n~if;ration described below) as long as the library members can be detected and quantitated by any mea~s. There is no a priori reason f or the libraries to be biopolymers such as nucleic acids, peptides, or derivatives and i f j rar; ~ thereof, though there are practical reasons for using these such as the convenient and widely practiced methods of synthesis known in the art.
In accordance with yet another aspect of the present invention there is provided a process for ~ term;n;n~ the identity or sequence of a compound that binds to a target molecule with at least the sperif;ed activity, wherein such sequence is IPtPnm;nPd indirectly, i.e., without actually having to sequence the ulld.
The present invention, in this aspect, is also directed to a wide variety of ~1~ of the type hereinabove described. Thus, for example, the ~ d8 that may be employed in accordance with this ~spect of the invention may be nucleic acids, or o~ nurleotides~
peptides, proteins, polymeric materials that are not naturally occurring, or organic u--ds. The present invention, in this ~spect, also does not require I l;f;rAtion~ although it is poss;h~P when using nucleic acids to employ GUch ~l;fi--Ation although it is not required in most aspect6.
5UB~lTurE SHEE~ 26 W0 95/27072 i~ r~ c ~
In accordance with this aspect of the present invention, thore is prepared a plurAlity of individual sublibraries of the type of u--d which is to be screened against a target molecule to ascertain the sequence of the compound or _ u..ds that bind6 to the target molecule with at least the Sper; f i ~1 activity . In each of the sublibraries, the ullds therein have a known entity at a known position in the u--d. Thereafter, e~-ch of the sublibraries is cont~cted with the target molecule to determine whether or not each of the sublibraries has a winner therein, i.e., a ' or, u--ds that bind to the target molecule with at least the sper; f; ed activity. Based on the fact that for each of the sublibraries containing a winner compound or _ u--ds, an entity of the compound is known and the position of the entity in the ~ ' is known. s~sed on a determination of winner libraries there can be indirectly det~rmin~; a u--d(s) with a sequence(s) or structure(s) that will bind to the target molecule with at least the ~per; f i ~d activity.
Thus, if for example, a prescreen of oli~nnllnleotide libraries in a m/mner a8 herein~bove described rl~t~m;ne~ that a 10 nucleotide random ol; ~nn~C 3 eotide library contains a winner sequence, then in accordance with this aspect of the present invention, 40 gublibrarie6 would be synthegized, each contA;n;n~
one of the four nucleotides defined at one of the 10 positions.
Each of the libraries would then be evaluated to determine, for example, in the manner hereinabove described, which of the librAries rnntAin~d a winner sequence. Then the nucleotide se~uence for a 10-nucleotide long ol; ~nn~ eotide would be indirectly deduced by knowing the defined nucleotide at the defined poS;tinn for each of the 6ublibraries that rnnt~in~d a winner.
For example, if the winner sequence wa6 5 '-AGGCTATACG-3 ', the sublibraries AN9, NGN8, N2GN~, N3CN6, etc. would all have a plateau in their respective semi-log plots and be scored as E SHE~ 26~

Wo 95127072 2 ~ 8 ~ 9 ~ 8 ~ c positive, or as ~winner librarie6", whereas CNg, NAN~ N2TN7, N3GN6, etc. would be scored as negative, or "loser libraries". In total, 10 sublibraries corrp~prnri;n~ to the 10 nucleotides in the winner sequence would be scored as positive whereas 30 libraries would be scored as negative.
Thus, in accordance with the present invention there is a combinatorial approach f or identifying a sequence that binds to a target molecule with at least a sperifiecl activity. As hereinabove described, such activity may be a de6ired affinity, or may be a desired level of inhibition of activity of a target molecule, etc.
Similarly, the _u-.ds that are screened in a plurality of sublibraries may be any one of a wide variety of ~ '~ such as, for example, ol i~rn~lrleotide6, peptides, polymers, organic ul.ds, etc. Although the invention is further described with respect to a nucleic acid that binds to a target with at least the sperifi~d activity, 6uch description i5 equally ArplirAhle to other u.lds, such as peptides and other org~nic u-.ds, ~nd is equally Arrl;rAhlP to ~lPf;n;t;~n~ of activity other than affinity.
Thus, as a representative example, after detprm;n;n~7 that a singlc sLL~Il.led six-nucleotide ol ;rJnnllrleotide will bind to the t_rget molecule with at least the sper; f; Pd ilctivity, there are then ~ c~ d 24 sublibrAries, eAch one fixed at one position with a known nucleotide. For extlmple, if the sublibr~ries are constituted of natural r;hr)nllrlp~tides~ the sublibrary set iB the following, where N is a mixture of the four ribonucleotides:
ANNNNN NANNNN NNANNN NNNANN NNNNAN NNNNNA
CNNNNN NCNNNN NNCNNN NNNCNN NNNNCN NNNNNC
GNNNNN NGNNNN NNGNNN NNNGNN NNNNGN NNNNNG
UNNNNN NUNNNN NNUNNN NNNUNN NNNNUN NNNNNU
SUBSTITUTE SHEET (RU~E 261 W0 95/27072 ;2 ~ i 9 1 ~3 PCr/US95/04098 -3~
Each of the 6Ublibraries is contacted with target and submitted to iteration6 of exposure to target, isolation and rc ~u~lu~e to target, as described above, to determine whether the sublibrary contains one or more winners. By ~ tf~rminin~ whether each sublibrary is a winner or loser library, a winner sequence can be deduced. For example, suppose that the reiterative described procedure of the invention is used to dist;n~l; ~h between winner and lo~er sublibraries within this set, and only the underlined sublibraries contain winners:
NANNNN NNANNN NNNANN ~a~I NNNNNA
CNNNNN NCNNNN NNCNNN NNNCNN NNNNCN
GNNNNN NGNNNN ~Ç2~[ NNNGNN NNNNGN NNNNNG
~lNNNNN NUNNNN NNUNNN NNNUNN NNNNUN NNNNNU
In this instance, it~ cr~n be deduced that the winner sequence is ACGCAC. For re~sons mentioned Above, a compound having as much as one-third the affinity of the winner compound acts, in effect, as a loser. Nevertheless, it is possible that a library contains ~ small number of, ' that have similar optimal affinities for a target. For example, there might be ~ consensus sequence in which some percentage of a winning sequence is critical in the inter~ction with t~rget whereas other parts of the seql~nce are essentially irrelevant, serving as sr~ffn~l;ng In such instances, there might be more than one winner sublibrary at a given f ixed-base position, e . g .:
ANNNNN NANNNN NNANNN NNNANN ~ ~a CNNNNN NCNNNN NNCNNN NNNCNN NNNNCN NNNNNC
GNNNNN NGNNNN NNGNNN NNNGNN NNNNGN NNNNNG
UNNNNN ~ NN~INNN NNNUNN NNNNUN NNNNNU
In such an instance, it can be inf erred that there is either consensu8 sequence AYGYAR (i.e, ACGCAA, ACGCAG, ACGUAA, SUB~TUTE ~1EE~ (RULE 26) Wo ssl27072 2 ~ 8 5 q 1 8 r~l,.,. C~,!, .~

ACGUAG, AUGCAA, AUGCAG, AUGUAA and AUGUAG are all winners ~
(hereinafter Alternative 1), or a limited number of in~lPrPn~Pnt winner se~u~ncP~, for example, ACGCAA and AUGUAG (hereafter Alternative 2 ) .
It is possible to deduce which of the alternatives is likely to be correct through the quantitative analyses with the sublibraries. For example, if Alternative 1 is correct, the ANNNNN, NNGNNN and NNNNAN sublibraries all contain eight winner sequences, whereas if AltPrnAtive 2 is correct, the same sublibraries contain two winner geqUpn~ p~. Similarly, if Alternative 1 is correct, the NCNNNN, NUNNNN, NNNCNN, NNNUNN, NNNNNA and NNNNNG 6ublibraries each contain four winners whereas if Alternative 2 is correct, they contain one winner sequence. These kinds of differences in numbers of winners in the sublibraries are measurable as shown in ~igure ~, and could be used to deduce the correct alternative. In any event, since it is unlikely that a library will contain a large number of winners of unrelated sequence with very similar affinities for a target, it is a simple matter to synthesize the candidate winner sequences and test them individual ly f or af f inity .
Whereas the above example of the invention is for i~lPnt;firAt;nn of winner ol;gnnucleotidesr a broader: ` 'i t of this invention is generalized to virtually any type of polymeric or non-polymeric _ . lld library. For example, a large number of different ~--n7o~ 7epine derivatives c~n be systematically synthesized such that they differ oIle from the other by substitution of rhpmic~l moieties present at various positions of the basic bpn7o~ 7erinf~ structure, much as members of an nl i ~nnllCleotide library differ in the nucleotide at~Arh~d to each position of the polymer. By syn~hP~i~in~ sublibraries of bPn7~riiA7Pr;nP~, each of which is char~cterized by having a fixed t-h~ Al moiety, at a p~rticular position but a v~riety of ~ JB~TlTL!TE ~ T (,q'~E 2fi~

Wo 9S/2~072 ~ 3 PCT/US95/04098 different moieties at other positions (see, e.g., Figure 5), one can test these libraries for winners and indirectly indentify the most active b~n7O~ 7Rpin~ derivative in a large library. The screening procedure with such libraries is analogous to the aforementioned analysis of nl;gnnll--leotide libraries wherein each sublibrary has a particular nucleotide def ined at a particular position. In general, nny library that can be systematically divided or synthesized as no.. ~,vellapping sublibraries, each of which has one distinct rhF-~n;c;~l characteristic at a particular site on the molecule, is hlR to the analysis of the invention.
Although the ~ u~ u .: of the inst~nt invention does not require direct identif;ri~t;n~ of a winner _..d, the selection ~lùc~lulê can be coupled with direct i~l~nt;f;cation. If the amount of material left after a given number of rounds is sufficient to allow ir~nt;ftcation u8ing procedures well ~cnown in the art, such as DNA sequencing or clo~ing and seq~nc;ng for nucleic acid libraries, then such dircct ;~l~.n~;f;~ ~t;nn may be useful. This would only be the case if the winners are nbundAnt enough and/or the library size is large enough to allow such direct ident;f;r~t;nll without amplific~tion of the material. In these cases, the method of the invention e~sentiaLly serves as an indicntor that a library cont~ins winners nnd that the winners are abundant enough to identify directly. The winners can then either be identified via the indirect method or by direct procedures known in the art.
The invention will be further described with respect to the following les; however, it is to be understood that the examples do not limit the scope of the invention:
SUBSTITUTE SHEET (RULE 26) Wo 95127072 2 ~ ~ ~ `9 ~ ~ P~l/u~ o ,~.
Example 1. Identification of Eligh A~finity 01 ;sonucleotide Sequences .
Six ol i ~nnllrl entide libraries ~re compared to determine which, if any, of the libraries contains ol;~onllcleotides that have a dissociation equilibrium constant of Kd = 10 nM for binding to basic Fibroblast Growth Factor (bFGF). The libraries are:
1. Three " stem-loop N libraries, each with the sequence 5'-GGCCG(N579)CGGCC-3' such that one library co~tains 5 positions of L ' i 7ed sequence, another contains 7 L ' ; 7ed positions, and another contains 9 L ' ; 7~cl positions . Each of the three libraries rnntAinc the 10 nonrandom bases shown flanking the rAn~' i 7ed region, five complementary bases on each side, so that the random region is located within a looped region o~ a stem-loop structure .
2. Three "bubble" libraries, each with the sequence 5 l -GGCCG ( N4 5 6 ) GAcuc~ r~r-uc ( N~ ) cGGcc- 3 l such that one l ibrary contains 4, another library 5, and another library 6 L ' 7~1 positions across a bubble region from four (in all three libraries) additional L~ ' ' 7~cl positions . Each of the three libraries contains 24 nul-l ' bases as shown so that the random regions are located on each strand o~ a bubble in the center of a 10 base stem region with the loop sequence 'AAAA ' at one end of the stem .
All six libraries are composed of 2'-ûMe-RNA nucleotides at all nv..L ' positions and contain 8 different nucleotides mixed in equal proportions in the rAnl' i 7~rl po8itions . The 8 nucleotides in the random positions are dA, dC, dG, dU, 2 '-OMe-A
( "A" ), 2 '-OMe-C ( "C" ), 2 '-OMe-G( "G" ), and 2 '-015e-U ( "U" ) . Each library, therefore, nnnt~in~ 8N unique members, 80 that, for example, the smallest library (the 5N loop library) contains 85 =
32,768 ol i~onll~leotides and the largest library (the 6+4N bubble SUBSTITUTE SIIEET (RULE 26~

WO gs/27072 ' ~ 8 ~ q ~ ~3 r~ ,,' o library) contains 81-1.07 x 109 oliql~n~cleotides. In addition, all six libraries are synthe6ized 0 that a single fluorescein tag is at the 5 ' end of every molecule.
A total of 10l5 molecules of each library are incubated for 30 minute with bFGF in a 1 ml re~ction of standard buffer (Tris-C1 pH 7.5, 150 mM NaCl, 3 mM MgCl2) with 100 nM bFGF. Note that the 1015 molecules of library conist of approximately 3 x 10l copies of each of the 32,768 unique members of the 5N loop library, or approximately 10~ copies of each of the 109 unique members of the 6+4N bubble library, and L.ULL' F.~ ;ngly different numbers of copies of unique members of each of the other libraries such that the total number is equal to 1015 in each case.
The six reactions are separately filtered through nitrocellulose filters (M; 11 ;rore type HA) under vacuum and washed with lO ml reaction buffer. The wet filters are then extracted with 200 ul 7M urea and 400 ul phenol, followed by a chloroform extraction, and the aqueous phae that cont~ins the extracted nl;~rnllrl~tides is roll~ct~. The ol;~nn-lrl~tides are then ethanol precipitated by standard methods and an aliquot of 19~ of each sample is removed and measured by capillary ele~ Llu~hvLcsis with a fl~lul~ E_.~C~ detector to quantitAte the total amount of fluorescent material in the sample from which the 1~ was taken for meaD UL ~
At thi~ point, all six library samples have roughly 1013 molecules (observed as a ~luorescent signal from the 19~ aliquots equal to that of a lOIl molecule fluorescent standard), indicating that 9995 of the I ~ _ ` in each of the libraries has passed through the nitrocellulose filter and therefore that no more than 1~5 of each libr~ry is bound to the bFGF and thereby retained on the filter. This i consistent with none o~ the six libraries having SUBSEITUTE SHEET (~ULE 26~

wo ssn7072 2 1 8 5 9 1 8 1~ 'O i~5 an average library af f inity f or bFGF high enough to bind bFGF at a high percentage under these conditions.
The six samples collected after the first exposure to bFGF
are then incubated once again under the same conditions with bFGF
in new reactions with the addition of 101s molecules of an unrelated non-fluorescent ol;~nnl-cl~otide sequence, 5'-AGTAGCTTGACGATCCG-3' that is added simply to be a "carrier" molecule to protect the library s~mples from being nnn~pe--;f;c~lly lost to test-tube surfaces. As before, the renctions ~re filtered and w~shed; the library samples are each extracted, precipitated, and quantitated by measurement of a 1% aliquot.
At this point (after two e~oDuLes to bFGF) approximately 101l molecules rem~Lin in e~ch of the six samples, indicating that once ~gain the library samples have very little measurable binding to bFGF.
Two additional iterations of this procedure (;nCllh~;nn with bFGF, separation of bound molecules by f ilter, extraction, and mea~uL~ ) result in ever decreasing numbers of molecules in each of the six samples, with apprn~;r~t~ly 9996 of each libr~ry sample lost during each iteration. At this point (after a total o~ 4 iterations ), each of the six s~mples is found to contain approximately 107 molecules. After one more iteration, the 5+4~
bubble library is found to contain nearly 107 molecules, indicating that these L~ ;nin~ molecules, representing a rare subset of about one in 10~ molecules of this library, a level indicative of a single unique sequence in this library, has an affinity for bFGF high enough (i.e., a E~d low enough) to bind to the 100 nM bFGF and be retained by the filter. The other five libraries all cnnt;n~ to decre~se ~8 in previous iter~tions 80 thAt e~ch rnnt~in~ only about 105 molecules after this fifth round. After yet another iteration, this result is conf; ~ because these other 5 libraries SUBSTITUTE SHEET ~RULE 26) WO9S/27072 ~ 859 ~ 8 r~l~u~ o~
4o-all decrease to about 103 molecules and cannot even be measured by _luorescence detection while the 5+4N bubble library still has between 106 ~nd 107 molecules.
From these results, the cnnr]l~cinn is reached that there i6 a bubble sequence in the 5+4N bubble library, as yet lln; ~.nt i f; ed, that has very high affi~ity for bFGF; this sequence has higher affinity than any SN loop, 7N loop, 9N loop, 4+4N bubble, or 6+4N
bubble seauences in the other five libraries, And higher affinity than ~ny of the other se~uences in the 5+4N bubble librAry.
Based on this conclusion, 72 sublibraries are Synt~rci 7~
Each sublibrary has the same 5+4N bubble f;Yorl s~rUctllr~l seauence.
In the 9 bubble positions, however, only 8 of the positions are L ' 7~rl while a single ~ ;n;nrJ position is specified AS one of the 8 nucleotides (dA, dC, dG, dU, 2 'O~e-P., etc. ) . There are 8 sublibraries, eelch with a different sp~r;f;r~l nucleotide, for each of 9 single sper;f;~d positions, making a total of 8 x 9 = 72 sublibraries .
All 72 sublibraries and the original 5+4N bubble library ~with all 9 positions ;7F~rl) are u8ed exactly as described above in ~ reiterative procedure of incubAtion with bFGF, separation by filter, extraction, and measurement by aliauot. By the fifth and sixth iter~tions, only 9 sublibrary saTnples and the original library sample have greater than 105 molecules. The 9 sublibrary samples each have 5-10 times more molecules than the reiterated nri~;nFIl libr~ry sample. The 9 sublibraries that have ~amples with more molecules after the reiterations are:
5~-GGC~rr~ ~Nr~rTJr.~ r~-u~ NNNN~ rc-5 '--GGCCbNI Irur7~ hr~iu~ NNNr L~GC~--3 5~-GGC~rr.NN~NNr.~rTJr.~A~r~ u~ - -3, 5 '--GrC~ ~ ~N~TT~r~rut:~h~r~u~:NNNL~ _ --T;TI.~TE S~IEEt ~ r 26 W095127072 ~ ~ ~591~ r~,l/rJ~ J~J
5 '--~jr:r~ NNNNArArurAAP~rAl-u~ NNr ~ ,CC--3 5 ~ 'liNI'''''' ';Arur-AAAArAl-lJL~.~ G iCC--3 ~
5 '--li~ ic~ rlr:r~r:l iAr~ Ur.AAAArArUrNr~l INN~ IjGCC--3 ' 5 --r;iriCcl:NNNNNl-~rur-AAAArAl-Jl Nrl~ - ' ib~--3 ' 5 '--GGCcl;NNr- -.-~..CUrAAAArArTTrNNNrlrrrr.CC--3 ' From this ; nfnrr-t i ~n, it is deduced that the sequence of the high af f inity 5+4 bubble sequence must be 5~-riGcrr~ATTrlAduAr-AruriAAAArAriucçs~r~GGr-r--3~ wherein the 6equences from the experi 'Ally rA ' i7r~rl pogitions are underlined.
Thi6 sequence is synthesized nnd shown to have a Rd = 10 nM
for bFGF, which is consistent with its binding nearly quantitatively in the reiterative procedure that led to its identification when bFGF was 100 nM.
Example 2. Identification of ~iigh Affinity Peptide Sequences.
ThrSe peptide libraries are synth~ci7~rl with lengths 4, 5, And 6 amino acids, respectively. Each ha6 a single fluorescein moiety covalenty linked to the amino-terminus. Each of these libraries is incubated under standard buf f er conditions with a particular antibody o~ interest. ~he incubations are in 1 ml of reaction bu~fer with 101S molecules of peptide. After an initial 30 minute in~rllhatinn~ the reactions are tran8ferred into a dialysis apparatus in which the 1 ml samples are separated from 10 ml o~
reactio~ buffer by a 5,000 molecular weight cut-off dialysis membrane. Unbound peptides ~with molecular weights well below the dialysis cut-off ~ can freely diffuse through the membr~ne whereas ilntibody and antibo~ b.~l-d peptides c~mnot. Because the volume of the dialysate is ten times larger than the sample volume, 909~ of all unbound peptides (losers) migrate across the membrane, thereby SUBSTITUTE SHEET (RULE 26) -WO gs/27072 7 ~ o ~

preferentially decreasing the number of losers in the sample relative to the number of winner5 that are retained in the sample because they are bound to antibody. After di~lysis for 15 minutes, 10 microliters of the reaction are sampled and me~sured for fluorescent intensity to d~rmin~ the number of peptide molecules in the 1 ml reaction chamber. The dialysate i8 then removed and replaced with 10 ml buffer. Thi8 procedure of dialysis, removal of sample, and buffer ~Yrh~n~e is repe~ted ten times.
The meaDuL~ Ls ;nrl;cAte that the amount of 4 amino acid library in the reaction chamber decreases by a factor of 10 each time the 10 ml dialysate is ~YrhAnged.
The 5 and 6 amino acid libraries, however, decrease by a factor of 10 each time the dialysate is ~YrhAn~e~ until the seventh round. Thereafter, more th~n half of the L~ ;ninr peptides are retained in the reaction chamber during each buffer ~-Yrh~n~e. This indicates that very rare subsets of these libraries bind antibody with a high affinity such that they are unavailable for diffusion across the ~llt. For the 5 amino acid library, this u ULL~UOnd5 to only one unique peptide sequence. The 6 amino acid library contains 20 times more sequences with 20 time8 fewer copies of e~ch, -~d to the 5 amino acid library. ~lI.eLeLore, the ~act that it shows the same dialy~is behavior as the 5 amino acid library indic~tes th~t there Are 20 peptides in it that bind with high af f inity to the antibody .
Based on these data, the 5 amino acid library is used as the basis for synthesis and testing of 100 peptide sublibraries, using the same di~lysis procedure. From the results of these experiments, the identity of the high affinity 5 amino acid peptide is deduced.
SUBSTiTUTE SHEET IRU~E 21i~

WO95/27072 2 ~ ~5 ~ .l/U.,,_,'0I~

Numerous -i f i cations and variations of the present invention are possible in light of the above t~'h i n~ and, therefore, within the scope of the Arr~nded cl~ims, the invention may be practiced otherwise than as particularly described.

~ jP~ L gH~ '~E ~6~

Claims (16)

WHAT IS CLAIMED IS:

1. A process for identifying chemical entities at defined positions in a compound which binds to a target molecule with at least a predetermined binding affinity, comprising:
(a) contacting a plurality of individual libraries with a target molecule, wherein each individual library has a plurality of compounds having defined positions, with each compound of a library having the same defined chemical entity at the same one of the defined positions, and wherein the compounds of a library differ from the compounds other libraries by at least one of the defined chemical entity or the defined position for a defined chemical entity, said compound being selected from the group consisting of polymers and nonpolymers, and said chemical entities at the defined positions for polymers being monomeric units and said chemical entities at the defined positions for nonpolymers being chemical substituents;
(b) determining the libraries in which there has been an increase in the percentage of compounds that bind to the target molecule with at least the predetermined binding affinity; and (c) based on the defined chemical entity at the defined position for each of the libraries determined in step (b), identifying chemical entities at defined positions for a compound which binds to the target molecule with at least the predetermined binding affinity.

2. The process of Claim 1 wherein the contacting is a reiterative procedure involving a plurality of rounds and the determining of the libraries comprises determining a change in the recovery of compounds between at least two of the rounds.

3. The process of Claim 1 wherein the contacting of the libraries with the target molecule is under condition to eliminate compounds that bind to the target molecule with at least the predetermined binding affinity at a rate slower than the compounds that bind to the target molecule with less than the predetermined binding affinity.

4. The process of Claim 3 wherein the contacting and eliminating is effected by dialysis.

5. The process of Claim 3 wherein the determining comprises measuring a change in rate of recovery of compounds to determine said increase in percentage.

6. The process of Claim 1 wherein said target molecule is a protein.

7. The process of Claim 1 wherein said contacting comprises recovering compounds which bind to the target molecule and recontacting the recovered compounds with the target molecule for eliminating compounds from the library which bind to the target molecule at less than the predetermined binding affinity.

8. The process of Claim 2 wherein the compound is a polymer.

9. The process of Claim 8 wherein the monomeric units are amino acids and the polymer is a peptide.

10. The process of Claim 8 wherein the monomeric units are nucleotides and the polymer is an oligonucleotide.

11. The process of Claim 10 wherein the oligonucleotide is a modified oligonucleotide.

12. The process of Claim 1 wherein the compounds are labeled.

13. The process of Claim 3 wherein the determining comprises measuring a change in the rate of elimination of compounds to determine said increase in percentage.

14. The process of Claim 1 wherein the compounds are nucleic acids.

15. The process of Claim 1 wherein the compounds are peptides.

16. The process of Claim 1 wherein the compounds are organic compounds.