JMB—MS 348 Cust. Ref. No. GO 38/94 [SGML]

J. Mol. Biol. (1995) 247, 60–68

Comprehensive Chemical Modification Interferenceand Nucleotide Substitution Analysis of an RNAPseudoknot Inhibitor to HIV-1 Reverse Transcriptase

Louis Green 1, Sheela Waugh 1, Jonathan P. Binkley 2

Zuzana Hostomska 3, Zdenek Hostomsky 3 and Craig Tuerk 1,4*

1NeXagen, Inc., Suite 200 We had previously used in vitro RNA selection techniques to describe aconsensus RNA pseudoknot that binds and inhibits HIV-1 reverse2860 Wilderness Place

Boulder, CO 80301, U.S.A. transcriptase (HIV-RT). In this work we constructed variants of thisconsensus pseudoknot in order to evaluate the contributions of individual2Department of Molecular nucleotide identities and secondary structure to affinity for HIV-RT. We have

Cellular and Developmental also used chemical modification of ligand RNAs to corroborate the predictedBiology, University of structure of the pseudoknot, to discover which modifiable groups areColorado, Boulder, CO 80309 protected from chemical attack when bound to HIV-RT, and to find whichU.S.A. modifications interfere with binding to HIV-RT. A novel interference study

is presented which involves selection of ligands from a pool created by mixed3Agouron Pharmaceuticalsreagent oligonucleotide synthesis in order to rapidly determine allowedInc., 3565 General Atomicssubstitutions of 2'-OCH3 groups for the usual 2'-OH group in such RNACourt, San Diego, CA 92121ligands.U.S.A.

4Department of Biological andEnvironmental Sciences327E Lappin Hall, MoreheadState University, MoreheadKY 40351, U.S.A.

Keywords: HIV-1 reverse transcriptase; RNA; modification interference;2'-methoxylribose*Corresponding author

Introduction

We have used a procedure for isolating nucleicacid ligands from in vitro synthesized, randomizedpools (calledSELEX;seeTuerk&Gold,1990) to isolatenovel RNAs that bind and specifically inhibit HIV-1reverse transcriptase (HIV-RT: Tuerk et al., 1992).The collection of RNA sequences thus isolateddescribed a consensus RNA ligand, 5'-(U/A)CCGNXXXXXXNCGGGANAAX'X'X'X'X'X'-3' inwhich the N, X and X' sequences are unconserved forprimary sequence and the X nucleotides base-pair toX' nucleotides as indicated by underlining. Becausethe conserved sequence 5'-UCCG could base-pair tothe conserved sequence 5'-CGGG and because there

were 3 of the 18 members of the ligand sequencecollection in which the potential base-pair G4 · C13was replaced with C · G (twice) and A · U (once), weproposed that these HIV-RT ligands folded as anRNA pseudoknot (Pleij et al., 1985).

We synthesized a model ligand based on theconsensus which is shown in Figure 1. The two 5' Gnucleotides are essential for efficient transcription byT7 RNA polymerase. We number the nucleotides ofthe consensus beginning with the 5'-most U of thesequence UCCG. Those nucleotides that arebold-faced in Figure 1 were highly conserved in theoriginal consensus. U1 was preferred (existing at thisrelative position in 11 of the 18 sequences thatcontributed to the consensus), but A1 was also foundfrequently (in 6 out of the 18). The preferred numberof nucleotides connecting the two strands of stem 1was eight (in 8 out of 18). The number and pattern ofbase-paired nucleotides comprising stem 2 and apreference for A5 and A12 were derived from theconsensus of a secondary SELEX in which thesequences comprising loop 1 and stem 2 wererandomized, with stem 1 and loop 2 held constant. In

Abbreviations used: HIV-RT, HIV-1 reversetranscriptase; SELEX, systematic evolution of ligands byexponential enrichment; ENU, ethylnitrosourea; DMS,dimethylsulfate; CMCT, 1-cyclohexyl-3-(2-morpholino-ethyl) carbodiimide metho-p-toluene sulfonate; DEPC,diethylpyrocarbonate; RT, reverse transcriptase.

0022–2836/95/110060–09 $08.00/0 7 1995 Academic Press Limited

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Analysis of an RNA Pseudoknot Inhibitor to HIV-RT 61

Figure 1. Shown is the model RNA ligand of HIV-RT folded as a pseudoknot with 2 stems (1 and 2 as labeled) and2 loops (1 and 2 as labeled). The conserved nucleotides derived from the SELEX consensus (Tuerk et al., 1992) are bold-faced.The 3rd nucleotide of the ligand is numbered 1 as the 5'-most essential nucleotide of the ligand for optimal binding toHIV-RT. The results of nucleotide substitution experiments in which the calculated Ka values of each variant (calculatedfrom the binding data as described in Materials and Methods) is divided by the Ka value of the model RT ligand are shownin the boxes. Asterisks indicate results from 5' truncation experiments (see Figure 2). The value associated with theasterisked G · G which replaces U1 · G16 comes from ligand C of Figure 2; while that with the asterisked G · G replacingC2 · G15 comes from ligand D. The value of C · G substitutions for U1 · G16 is derived from the ligand E of Figure 2. Thearrows from the boxes labeled ‘‘truncation’’ indicate the positions of the 3' end of those particular truncations on the modelRT ligand sequence.

order to evaluate which nucleotides and atomicgroups are crucial to the interaction of this RNAligand with HIV-RT, we conducted a number ofnucleotide substitution and chemical modificationexperiments.

Results

Refinement of the information boundaries

The first two SELEX experiments in which 32nucleotide positions were randomized providedhigh affinity ligands in which there was variablelength for stem 1 at its 5' end; that is, some ligandshad the sequence: 5'-UUCCG which could base-pairto 5'-CGGGA, 5'-UCCG which could base-pair to5'-CGGG or 5'-CCG which could base-pair to 5'CGG(Tuerk et al., 1992). Determination of the boundariesof the sequences donating high-affinity to theinteraction with HIV-RT was accomplished byselection from partial alkaline hydrolysates of end-labeled clonal RNAs, a rapid but qualitative analysis.We conducted binding experiments with various 5'ends (A, 5'-GGUUCCG. . . ; B, 5'-GGUCCG. . . ; C,5'-GGCCG. . . ; D, 5'-GGCG. . .) to quantitativelyevaluate the boundary of highest affinity binding to

HIV-RT by the consensus ligand. The result shownin Figure 2 is that one 5'U is sufficient for thehighest-affinity binding to HIV-RT (ligands A andB), that with no U there is reduced binding (ligandC), and that any further removal of 5' sequencesreduces binding to that of non-specific sequences(ligand D). We used the design (hereafter referred toas the RT ligand) with only one 5'U (U1, see Figure 1)for the rest of the experiments reported here.

We also tested dependence on the length of stem2 by making various truncations at the 3' end of theRT ligand (see Figure 1). Deletion of as many as threenucleotides from the 3' end (A24 to U26) made nodifference in affinity of the molecule for HIV-RTwhich confirmed the boundary determined pre-viously (Tuerk et al., 1992). Deletion of the 3'-terminalfour nucleotides (C23 to U26) resulted in fivefoldreduced binding, deletion of five nucleotides (G22 toU26) resulted in an approximately 17-fold reductionand deletion of six nucleotides (U21 to U26, or no 3'helix) resulted in an approximately 50-fold reductionin affinity (data not shown). Save for the last deletion,such reductions were less drastic than reductionsfound for single-base substitutions reported below,suggesting (with other data reported below) that thishelix serves primarily a tethering role that aids thepositioning of crucial groups in loop 2.

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Analysis of an RNA Pseudoknot Inhibitor to HIV-RT62

Figure 2. Refinement of the 5' information boundary. Aset of model ligands were synthesized with T7 RNApolymerase from template oligonucleotides as describedby Milligan et al. (1987). Concentrations of HIV-RT aremolar. Illustrated are the various predicted base-pairingsthat would result from the 5' truncations for ligands Athrough E. The graph shows the individual binding curvesfor these model ligands, obtained as described by Tuerket al. (1990). The relative Ka values are reported in Figure 1for ligand B = 1.0 (RT ligand) and ligands C, D and E.

Chemical probing of the pseudoknot structure

We conducted a number of chemical modificationexperiments to probe the native structure of the RTligand (data not shown; However, see Figure 3,where the unselected lane shows the poormodification of A6, C10, A11, C23, A24 and C25 byDMS, which is indicative of base-pairing ascompared to the heavy modification of A5, A12, A17,A18 and A19, which is suggestive of single-strandedregions). In general the pseudoknot structure isconfirmed by these structure probing experimentswith the following exceptions. The base-pairA6 · U26 is susceptible to chemical attack, especiallywhen bound to HIV-RT. The potential base-pairU1 · G16 is also susceptible to chemical attack in thenative conformation.

Modification interference studies of the RTligand

We partially modified the RT ligand, bound thismodified population with varying concentrations ofthe HIV-RT, isolated the bound species and assayedthem for the modified positions. We thus haveknowledge about where modification interferes withbinding, and where there is no or little effect. Theresults from two such modification interferenceexperiments are shown in Figures 3 and 4. Notein Figure 3 the especially strong interference bymodifications of the 5' phosphate of G15. The indexvalue as calculated by the formula given in Materialsand Methods is 10.5 at this position normalized forA20 = 1.0. The next most prominent interference wasG14’s value of 4.1.

A schematic diagram summarizing all modifi-cation interference results (as calculated in Materialand Methods) is shown in Figure 5. As shown, mostof the significant interference with binding isclustered on the left-hand side of the pseudoknotwhich contains stem 1 and loop 2. This is also the partof the molecule that was highly conserved (primarysequence) in the collection of sequences isolated bySELEX and where substitution experiments pro-duced the most drastic reduction in binding affinityto HIV-RT.

Substitution of 2'-methoxyl for 2'-hydroxyl onribose groups of the RT ligand

‘‘RNA’’ molecules in which there is a 2'-methoxylgroup bonded to the 2' carbon atoms of the riboseinstead of the normal hydroxyl group are resistant toenzymatic and chemical degradation. In order to testhow extensively 2'-methoxyl groups can be substi-tuted for 2'-hydroxyl groups in the RT ligands, wesynthesized four oligonucleotides as shown inFigure 6. Because fully substituted 2'-methoxylligand binds poorly (curve D in Figure 6), andbecause we had found that most of the modificationinterference sites were clustered at one end of thepseudoknot, subsequent attempts to substitute wereconfined to the non-specific 3' helix as shown in

Testing the SELEX consensus for stem 1

We then tested various nucleotide substitutionsin the conserved stem 1. As shown in Figure 1,substitution of an A for U1 in model RNAsmade little difference in affinity for HIV-RT.As shown in Figure 2, curve E, substitution ofC at U1 (which would increase the stability ofstem 1) lowers the affinity about 20-fold. A Gat U1 (represented by curve C in Figure 2) alsoresulted in an approximately 20-fold lowering inaffinity.

Substitution of A for G16 (which would base-pair to U1) abolished specific binding. We sub-stituted a G · C pair for C2 · G15, which alsodrastically reduced binding, and for C3 · G14which reduced binding about tenfold. Thesetwo positions were highly conserved in thephylogeny of SELEX ligands. We substitutedvarious combinations for the G4 · C13 base-pair.The order of effect of these on affinity wereG4 · C13 = C · G > U · A > A · U��A · C whereA · U is about twofold reduced in affinity comparedto G4 · C13, and A · C is at least 100-fold reduced.These results suggest that the primary role of thisbase-pair is structural and that stronger base-pairsare preferred (see above and Tuerk et al., 1992).

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Analysis of an RNA Pseudoknot Inhibitor to HIV-RT 63

Figure 3. Ethylnitrosourea modification interferencewith binding of RT ligand to HIV-RT. Shown is anautoradiogram of a gel in which the assayed products ofethylnitrosourea modification were separated by electro-phoresis as described in Materials and Methods. Indicatedis a lane in which a partial digest of the 5' labeled RT ligandby T1 RNase (G-specific) was loaded. In the indicated lanes3 different concentrations of HIV-RT were used to selectfrom the partially modified pool RNAs that bind. Thosephosphate modifications that fail to appear in selectedlanes as compared to unselected lanes indicate modifi-cations that interfere with binding to HIV-RT. Theautoradiograms were scanned and the degree ofinterference determined (see Materials and Methods) andsignificant interferences are shown in Figure 5.

groups do not participate in alkaline hydrolysis as do2'-hydroxyl groups as is shown by the absence ofbands for positions A6 through A12 and U21through U26 in Figure 7). As shown in Figure 7, theligands selected by low HIV-RT from the mixedincorporation populations showed significantlyincreased hydrolysis at positions C13 and G14indicating enrichment for 2'-hydroxyl groups at thesetwo positions or, in other words, binding interferenceby 2'-methoxyl groups at these positions. To testthese results more quantitatively, we synthesizedoligonucleotides in which all nucleotides were2'-methoxyl except for 2'-hydroxyl at C13 and G14 inone, 2'-hydroxyl only at C13 in another, and one with2'-hydroxyl only at G14. As shown in Figure 8, thereis little reduction in affinity when both of thesepositions are 2'-hydroxyl, and partial reductionwhen one or the other is substituted with 2'-methoxylgroups, as compared to the dramatic reduction whenall positions have 2'-methoxyl groups.

Discussion

The results of substitution experiments, quantitat-ive boundary experiments and chemical probingexperiments are highly informative about the natureof the pseudoknot inhibitor of HIV-RT and highlightcrucial regions of contact on this RNA. Thepseudoknot structure is supported by structure-specific interference of chemical modification bybase-pairing. It is further supported by the previousSELEX consensus and the allowable base-pairsubstitutions for G4 · C13. We will discuss our resultsfurther within the context of this pseudoknotstructure on a nucleotide by nucleotide basis.

U1 can be replaced with A with little loss in affinitybut not by C or G. Although U1 may make transientbase-pairing to G16 this is not detectable by chemicalprobing of the structure. In addition, modification ofU1 N-3 with CMCT (which would preventbase-pairing) does not interfere with binding toHIV-RT. However, binding by HIV-RT protects theN-3 of U1 (data not shown) perhaps by steric orelectrostatic shielding of this position. Substitution ofU1 with C (which forms a more stable base-pair withG16) reduces affinity 20-fold (see Figure 1 and 2).Replacement of G16 with A (which forms a stableU1 · A16 pair) abolishes specific affinity for HIV-RTand modification of G16 N-1 strongly interferes withbinding to HIV-RT. Probably this modification ofG16 N-1 prevents a crucial contact with the protein.Why G substitutions for U1 reduce affinity and Asubstitutions do not, is not revealed by theseexperiments or by visual comparison of thestructures of the individual bases with their differentfunctional groups. Perhaps A at U1 replaces apotential U interaction with a similar or differentinteraction with HIV-RT, a replacement that cannotbe performed by C or G at this position. Alternatively,the G16 interaction could be the only crucialinteraction of this pair, and substitution of C or G forU1 interacts with G16 so as to interfere with G16’sactivity whereas U1 and A for U1 do not.

Figure 6B and C. Both of these ligands bind withhigh affinity to HIV-RT.

We next synthesized oligonucleotides in which theallowed substitutions at the ribose of stem 2 were all2'-methoxyl as in Figure 6C and at the remaining14 positions did mixed synthesis (50:50) with2'-methoxyl and 2'-hydroxyl phosphoramidite re-agents. These oligonucleotides were subjected toselection by HIV-RT followed by alkaline hydrolysisof selected RNAs and gel separation. (2'-methoxyl

JMB—MS 348

Analysis of an RNA Pseudoknot Inhibitor to HIV-RT64

Figure 4. DMS modification interference with binding of RT ligand to HIV-RT. Shown is an autoradiogram of a gelin which labeled reverse transcripts of modified RT ligand were separated by electrophoresis as described in Materialsand Methods. Also shown are 4 dideoxy sequencing lanes and an extension-only lane to indicate the individual bandidentities. Otherwise, selection and analysis is the same as that described in the legend to Figure 3 and in Materials andMethods.

The next base-pair of stem 1 (C2 · G15) cannot bereplaced by a G · C base-pair without complete lossof specific affinity for HIV-RT. Modification of thebase-pairing faces of either nucleotide stronglyinterferes with binding to HIV-RT and binding withHIV-RT protects susceptible bases from thesemodifications (data not shown). Modification of theN-7 of G15 interferes with binding and modificationof the 5' phosphate between this position and G14gives dramatic interference with binding. Substi-tution of the next base-pair, C3 · G14, with a G · Cpair shows less drastic reduction of affinity, butmodification is strongly interfering at this position,including modification of the ribose with a2'-methoxyl group.

Substitution of a C · G pair for the closingbase-pair G4 · C13 has no effect on binding, andsubstitution of the less stable A · U and U · A pairsslightly reduces affinity. Substitution of the non-pair-ing A-C for these positions abolishes specificbinding. This correlates with the appearance of C · G

substitutions and one A · U substitution in theoriginal SELEX phylogeny at this position, thenon-reactivity of this base-pair in the native state,and the high degree of modification interferencefound for these bases. The 2'-methoxyl modificationsof C13 here and the 3' phosphate between C13 andG14 strongly interfere with binding to HIV-RT.

The chemical modification data of loop 2corroborate well with the phylogenetic conservationseen in the original SELEX experiments. Strongmodification interference is seen at positions A17and A19. Weak modification interference occurs atA20, which correlates with the finding of some loop2s of the original SELEX that are deleted at thisrelative position (although the chemical interferenceexperiments we have conducted do not exhaustivelytest all potential contacts that a base may make withHIV-RT). A18 is unconserved in the original SELEXand modification at this position does not interfere,nor is this position protected from modification bybinding to HIV-RT (data not shown).

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Analysis of an RNA Pseudoknot Inhibitor to HIV-RT 65

Figure 5. Modification interference results for the RT ligand complexing with HIV-RT. Symbols for modification areas described in the boxed legend. The modifications indicated are those that are strongly (filled symbols) or partially (opensymbols) selected against, regarding binding to HIV-RT (reflected by decreased modification at those positions in theselected population). The asterisks indicate those predicted base-pairs whose hydrogen bonding groups are reactive undernative conditions. For types and methods of modification and assay see Materials and Methods. For the formula by whichthe densitometric scanning data was used to determine degree of modification interference see Materials and Methods.

Modification of phosphate does not differ betweennative and denatured forms of the RT ligand, so wepresume that the phosphate groups whose modifi-cation interferes with binding interfere with bindingdirectly and not because of perturbed conformationof the ligand. Modifications of phosphate groupsbetween C13-G14 and between G14-G15 stronglyinterfere with binding. Substitution of the corre-sponding 3'-hydroxyl groups (at C13 and G14) alsostrongly interferes with binding and thereforehighlight and corroborate these specific nucleotidesas regions of contact with HIV-RT.

Taken together the above data suggest that theessential components of stem 1 are a single-stranded5' nucleotide (U or A), which may make sequencespecific contact with the protein and a three base-pairhelix (C2 · G15, C3 · G14, G4 · C13) where there aresequence-specific interactions with the HIV-RT atthe first two base-pairs and a preference for a strongbase-pair (i.e. either C · G or G · C) at the thirdloop closing position of G4 · C13. The second strandof this helix (CGG) is especially sensitive tomodification-interference at phosphate and 2'-hy-droxyl groups. Loop 2 should be more broadlydescribed as GANAA (16 to 20) due to thesingle-stranded character of G16, which probablyinteracts with HIV-RT in a sequence-specific

manner, as likely do A17 and A19. As shown inprevious work, stem 2 varies considerably in thepattern and number of base-pairing nucleotides, butfrom 3' deletion experiments reported here one couldhypothesize that a minimum of three base-pairs instem 2 are required for maximum affinity. Within thestructural context of this model of the RT ligand, atleast two nucleotides are required in loop 1 of thebound ligand.

A revised model of the essential components ofthe RT ligand is illustrated in Figure 9. The majordifferences between this ligand description and thatperceived by the original SELEX consensus is thelength of stem 2, the more degenerate specification ofthe base-pair G4 · C13, the size of loop 1 (which isdirectly related to the size of stem 2) and thesingle-stranded character of U1 and G16. How canone reconcile these differences? The SELEX strategyrequires 5' and 3' fixed sequences for replication. Inany RNA sequence, such additional sequencesincrease the potential for other conformations thatcompete with that of the high-affinity ligand. As aresult, additional structural elements that do notdirectly contribute to affinity, such as lengthenedstem 2, may be selected. Assuming that the first twobase-pairs of stem 1 must be C · G because ofsequence-specific contacts, the most stable closing

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Analysis of an RNA Pseudoknot Inhibitor to HIV-RT66

Figure 6. The effect of 2'-methoxylribose substitutionson the RT ligand affinity for HIV-RT. Open circlesrepresent 2'-hydroxyl groups at indicated positions andfilled circles indicate 2'-methoxyl group substitution.Concentrations of HIV-RT are molar.

protecting groups. The deprotected RNAs were thenextracted with phenol, precipitated with ethanol andpurified by gel electrophoresis.

Affinity assays with labeled RNA and HIV-RT

Model RNAs for refinement of the 5' and 3' boundariesand for determination of the effects of substitutions werelabeled during transcription with bacteriophage T7 RNApolymerase as described by Tuerk et al. (1990) except that[a-32P]ATP was used, in reactions of 0.5 mM C, G and UTPwith 0.05 mM unlabeled ATP. Synthetic oligonucleotidesand phosphatased transcripts (as described by Tuerk et al.,1990) were kinased as described by Gauss et al. (1987). AllRNA–protein binding reactions were done in a ‘‘bindingbuffer’’ of 200 mM KOAC, 50 mM Tris-HCl (pH 7.7), 1 mMdithiothreitol. RNA and protein dilutions were mixed andstored on ice for 30 minutes, then transferred to 37°C forfive minutes. The reaction volume was 60 ml of which 50 mlwas assayed. Each reaction was suctioned through apre-wet (with binding buffer) nitrocellulose filter andrinsed with 3 ml binding buffer after which it was driedand counted. In comparisons of binding affinity, resultswere plotted and Ka values were determined by theleast-squares fit of the data points to a binding equationthat assumes a simple biomolecular ligand–HIV-RTinteraction (Jellinek et al., 1994).

Selection of modified RNAs by HIV-RT

Binding reactions were as above except that rather thanvary the amount of HIV-RT added to a reaction, the volumeof reaction was increased in order to lower HIV-RTconcentration. RNAs were modified under nativeconditions and were selected at concentrations of 20, 4 and0.8 nM HIV-RT (in respective volumes 1, 5 and 25 ml ofbinding buffer). The amount of RNA added to eachreaction was equivalent for each experiment (approxi-mately 1 to 5 pmol). RNA was eluted from filters asdescribed by Tuerk et al. (1990) and assayed for modifiedpositions. In each experiment a control was included inwhich unselected RNA was spotted on a filter, eluted andassayed for modified positions in parallel with the selectedRNAs. Determinations of variation in chemical modifi-cation for selected versus unselected RNAs were madefrom exposed films of electrophoresed assay products withthe following exceptions. The extent of modificationinterference by ENU was determined by densitometricscanning of films using an LKB laser densitomer. An indexof modification interference (M.I.) at each position wascalculated as follows:

M.I. = (O.D.unselected P/O.D.unselected N )

/(O.D.selected P/O.D.selected N ),

where the value at each position (P ) assayed for selectedmodified RNA (O.D.selected P ) is divided by that value forposition N (O.D.selected N ) and divided into likewisenormalized values for the unselected lane. N was A20 forENU, A5 for DMS, U26 for CMCT, and G(−1) for kethoxal.All values of M.I. greater than 2.0 are reported asinterfering (with an open symbol in Figure 5) and greaterthan 4.0 as strongly interfering (with a filled symbol inFigure 5).

Chemical modification of RNA

The chemicals used were ethylnitrosourea (ENU) whichmodifies phosphate groups, dimethylsulfate (DMS) which

base-pair would be G4 · C13 (Freier et al., 1986),selected due to a reduced tendency to formalternative, lower-affinity conformations. The se-quence-specific selection of U1 and G16 may becoincidental to their ability to base-pair; in othernucleic acid ligand–protein complexes such asKlenow fragment/primer–template junction andtRNA/tRNA synthetase there is significant localdenaturation of base-paired nucleotides (Freemontet al., 1988; Rould et al., 1989), which may occur to alimited extent in the RT ligand.

Materials and Methods

RNA synthesis

In vitro transcription with oligonucleotide templates wasconducted as described by Milligan et al. (1987). Allsynthetic nucleic acids were made on an AppliedBiosystems model 394-08 DNA/RNA synthesizer usingstandard protocols. Deoxyribonucleotide phosphoramid-ites and DNA synthesis solvents and reagents werepurchased from Applied Biosystems. Ribonucleotide and2'-methoxylribonucleotide phosphoramidites were pur-chased from Glen Research Corporation. For mixed basepositions, 0.1 M phosphoramidite solutions were mixed byvolume to the proportions indicated. Base deprotectionwas carried out at 55°C for six hours in 3:1 ammoniumhydroxide:ethanol. The synthetic RNAs (1 mmol synthesiseach) were dried and resuspended in 1 ml tetrabutylam-moniumfluoride to remove the t-butyl-dimethylsilyl

JMB—MS 348

Analysis of an RNA Pseudoknot Inhibitor to HIV-RT 67

Figure 7. Selection by HIV-RT from mixed populations of 2'-methoxylribose versus 2'-hydroxyl at positions U1 throughA5 and A12 through A20. An oligonucleotide was synthesized as described in Materials and Methods with the followingsequence:

5'-(AAAAA)d(UCCGA)x(AGUGCA)m(ACGGGAAAA)x(UGCACU)m-3'

where d indicates 2'-deoxy; x indicates that those nucleotides are mixed 50:50 for phosphoramidite reagents resulting in2'-methoxyl or 2'-hydroxyl groups on the ribose (indicated in the cartoon as half-filled circles); m indicates that thosenucleotides are all 2'-methoxyl groups on the ribose (full circles in the cartoon). The 2'-hydroxyl groups in the selectedligand are indicated in the cartoon as open circles. Note: the 5' 2-deoxyA sequences were added to enhance resolutionof the 5' positions on the gel.

modifies the base-pairing faces of C (at N-3) and A (at N-1),1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide (CMCT)which modifies the base-pairing face of U (at N-3) and tosome extent G (at N-1), diethylpyrocarbonate (DEPC)which modifies N-7 of A and to a lesser extent the N-7 ofG, and kethoxal which modifies the base-pairing N-1 andN-2 to G (for a review, see Ehresmann et al., 1987). Mostof the assays of chemical modification were done on the RTligand sequence which was transcribed as part of a longer3' sequence to which a 32P-labeled primer could beannealed and extended with AMV reverse transcriptase.Assays of ENU or DEPC modified positions were doneby respective modification-dependent hydrolysis, ormodified base removal followed by aniline scission of thebackbone at these sites. Modification of RNA under nativeconditions was done at 200 mM KOAC, 5 mM Tris-HCl(pH 7.7) at 37°C with ENU (1/5 dilution (v/v) of roomtemperature ENU-saturated ethanol) for one to three hoursDMS (1/750-fold dilution (v/v)) for eight minutes,kethoxal (0.5 mg/ml) for eight minutes, CMCT (8 mg/ml)for two minutes, and DEPC (1/10 dilution (v/v) for native

conditions or 1/100 dilution for denaturing conditions) for45 minutes, and under the same conditions bound toHIV-RT with the addition of 1 mM dithiothreitol. Theconcentrations of modifying chemical reagent wereidentical for denaturing conditions (except where noted forDEPC); those conditions were 7 M urea, 5 mM Tris-HCl(pH 7.7), 1 mM EDTA at 90°C for one to five minutes exceptduring modification with ENU which was done in theabsence of 7 M urea.

Assay of chemical modification

Positions of chemical modification were assayed byreverse transcription for DMS, kethoxal and CMCTon the lengthened ligand RNA, 5'-GGUCCGAAGUG-CAACGGGAAAAUGCACUAUGAAAGAAUUUUAUA-UCUCUAUUGAAAC-3' (the consensus HIV-RT bindingsequence is underlined), to which is annealed theoligonucleotide primer 5'-CCGGATCCGTTTCAATAGA-GATATAAAATTC-3'; reverse transcription products (ob-tained as described by Gauss et al., 1987) were separated

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Analysis of an RNA Pseudoknot Inhibitor to HIV-RT68

Figure 8. Affinity assays of 2'-methoxyl groupsubstituted oligonucleotide versions of ligand B. As shown,there are 4 ligands in which, unless otherwise indicated,the substitution at each ribose is 2'-methoxyl. Concen-trations of HIV-RT are molar.

Figure 9. Revised ligand description of the RT ligandessential features. S-S' indicates a G · C or C · G bondas per IUPAC nomenclature. N, indicates any allowablenucleotide and X base-pair to X'. The box labeledsingle-stranded indicates that at least 2 nucleotides of thisloop 1 are single-stranded.

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by electrophoresis on 10% (w/v) polyacrylamide gels.Positions of ENU and DEPC modification were assayed asdescribed by Vlassov et al. (1980) and Peattie & Gilbert(1980), respectively (separated by electrophoresis on 20%polyacrylamide gels). Assay of 2'-methoxyl ribose versusribose at various positions was assayed by alkalinehydrolysis for 45 minutes at 90°C in 50 mM sodiumcarbonate (pH 9.0).

Acknowledgements

This work was supported in part by NIH grant no. 1 UO1A133380. We wish to further thank all of those people whohave smoothed the transition from academia to industry andback again, especially Patrick J. Mahaffy, Larry Gold, KathyPiekarski, Geoff Gearner, and David Magrane. Thanksalso to Brian Reeder for critically reading the abstract.

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(Received 13 July 1994; accepted 22 November 1994)