Solid-Phase Synthesis and Combinatorial Technologies

10,000,000 library members contain a specific 10-mer insert (410 = 107, 1014/107 = 107). Even if the shorter sequence is a weaker binder than the former, the relative abundance of the shorter motif will drive the selection and amplification cycles toward suboptimal, more abundant, shorter ONs. To overcome this limitation, extremely long randomized sequences are considered for ON libraries to allow the selection of long binders (with a high price to pay in terms of synthetic complexity); usually, the cutoff level is raised during the final iterative cycles in order to isolate a few long molecules with extremely high affinity for the target and to discard the weak, shorter binders.

The in vitro selection/amplification strategy has also been applied to modified ONs, especially where the 2′-ribose position has been changed and where phosphorothioates or other phosphate replacements have been used (Fig. 10.25, top). Several structures of modified ON chains that have been synthetically produced to obtain constrained sequences or sequences with higher stability to nucleases have also been reported (Fig. 10.25, bottom). Examples of biosynthetic modified ON libraries are covered in the next section.

Figure 10.25 Structures of modified building blocks, linkages, and constructs for biosynthetic ON libraries.

534 BIOSYNTHETIC COMBINATORIAL LIBRARIES

10.2.3 ON Libraries: Binding to Small Molecules, Drugs, and Proteins

In vitro selection of ON sequences from random libraries as binding motifs for a series of molecular targets ranging from small molecules up to macromolecules is a wellstudied area that has been extensively covered in several recent reviews (147–155).

Small molecules have often been the targets for aptamer selection. Haller and Sarnow (156) have reported the screening of a 1013-member, ON 90-mer library L15 that incorporated randomization of 40 inner residues to select for specific binders of 7-methyl guanosine 10.22 (Fig. 10.26) and of 10.22-containing oligonucleotides (capped oligonucleotides). An iterative cycle of the screening strategy is also depicted in Fig. 10.26. Library L15 was incubated with sepharose-bound m7-GTP (7-methyl guanosine tri phosphate) (step a) for 1 h at 4°C; then the unbound library fraction was washed with fresh assay buffer (step b), and the bound library members were eluted with an excess of free m7-GTP (step c). The selected RNAs were reverse transcribed to the corresponding cDNA, and the ODN sequences were amplified by PCR (steps d and e). Transcription using RNA polymerase (step f) produced an enriched ON library that was submitted to a second iterative cycle that was identical to the first, except for a preliminary elution of the bound library fraction with GTP to discard RNA binders acting on features of m7-GTP different from the methylated site (step g, Fig. 10.26). The amount of bound RNA was low after the first cycle (less than 1% of the library population) but steadily increased in the following cycles. After the eighth cycle, the PCR-amplified cDNA pool was cloned and several of these clones were characterized. Among them, the aptamer 10.23 showed significant specificity for 10.22 containing

a: incubation with sepharose-supported 10.22; b: washing of the unbound RNAs; c: elution of the bound RNAs with an excess of free 10.22; d: reverse transcription into DNA; e: PCR amplification;

f:transcription into RNA; g: a-f, seven cycles with preliminary elution with free GTP after step b;

h:cloning and sequencing of selected clones.

BEST BINDER: 10.23 IC50 = 500 nM

Figure 10.26 Screening of the biosynthetic ON library L15 for aptamers binding to m7-GTP (10.22): the selection/amplification process and the structure of the most active aptamer10.23.

nucleotides versus their desmethyl analogues (Table 10.1) and proved to be a useful tool in inhibiting capping-dependent translation in eukaryotic cells in vivo (157). Many other reports of in vitro selection of aptamers binding to small molecules have been reported, including coenzyme A (158), vitamin B12 (159), flavin adenine dinucleotide (FAD) (160), theophylline (161), and adenosine triphosphate (ADP) (162).

Burke et al. (163) reported two libraries of 1014–1015-member ON pools L16 and L17 containing 118-mers and 134-mers with inner randomization of 70 and 80 nucleotides, respectively. Both of these libraries were screened using agarose-bound chloramphenicol 10.25 (Cam, Fig. 10.27) to find families of aptamers able to shed light on the mode of binding of Cam (10.24, Fig. 10.27) to the bacterial ribosome. Library synthesis, transcription and reverse transcription from and to DNA sequences, selection, and amplification of selected sequences were performed using standard procedures (Fig. 10.26). Elution of resin-bound RNAs was performed with 10.24 with increased stringency for 12 cycles. The selected library population was low for the first seven cycles (<0.2%) but increased to 2–6% in the eighth round, eventually reaching 50% in the final cycle. Seventy-four aptamers were isolated after cloning of the individual sequences with various frequencies from both L16 and L17 (Fig. 10.27). It was found that, while several individuals shared common sequence elements, there were no sequences common to all of the selected aptamers. The most active aptamers showed a low micromolar dissociation constant, representing a 1000-fold increase of the original affinity of the random L16 and L17 sequences. The structural information acquired was then used to build a shorter 50-mer 10.26, retaining full Cam affinity, which was used to elucidate the characteristics of the ribosome–aptamer complex

TABLE 10.1 Specificity of Aptamer 10.23 Toward

m7-GTP-Containing Nucleosides and Nucleotides

536 BIOSYNTHETIC COMBINATORIAL LIBRARIES

a:incubation with agarose-supported 10.25; b: washing of the unbound RNAs;

c:elution of the bound RNAs with an excess of free 10.24; d: reverse transcription into DNA;

e:PCR amplification; f: transcription into RNA; g: a-f, 11 cycles; h: cloning and sequencing

of selected clones; i: truncation of ON sequences.

Figure 10.27 Screening of the biosynthetic ON libraries L16 and L17 for aptamers binding to chloramphenicol 10.24: the selection/amplification process using the supported selection substrate 10.25.

(163). Other reports regarding the selection of antibiotic-binding aptamers, including aminoglycosides, have also recently appeared (164–167).

The preparation of aptamers binding to RNA, DNA, or proteins has been the subject of many reports in the literature. Lebruska and Maher (168) has described the synthesis and screening of a library of 1014 ON 100-mers, L18, in which each member was made of 60 randomized bases flanked by two constant sequences. L18 was used to select aptamers for the proteic transcription factor NF-κB that binds to duplex DNA strands. The search for RNA strands with comparable affinity for the transcription factor was designed to produce potential tools, or even antisense therapeutics for antiviral and anticancer therapy. The library was incubated with the protein homodimer (p502, Fig. 10.28) for 2 h at 37°C (step a), then filtered through nitrocellulose membranes and washed (steps b and c) to remove the unbound members of the library (>99%). Elution with a mild denaturating buffer recovered the bound library fraction (step d), which was then reverse transcribed and amplified using standard procedures (steps e–g). The new RNA pool was submitted to several iterative cycles (step h), and the population obtained after 14 cycles was cloned to give two related sequences (step i), 10.27 (21 recurrences) and 10.28 (3 recurrences, Fig. 10.28). The two sequences displayed a low nanomolar affinity for the receptor, similar to the natural ligand. The sequences of

L18

a: incubation with p502; b: filtration on nitrocellulose; c: washing of the unbound RNAs; d: elution of

the bound RNAs; e: reverse transcription into DNA; f: PCR amplification; g: transcription

into RNA; h: a-g, 13 cycles; i: cloning and sequencing of selected clones; j: truncation of ON sequences.

Figure 10.28 Screening of the biosynthetic ON library L18 for aptamers binding to the transcription factor NF-κB: the selection/amplification process and the structures of the most active aptamers 10.29 and 10.30.

these two aptamers were used to identify the essential regions for binding to NF-κB, and two reduced ON sequences 10.29 (26-mer) and 10.30 (31-mer) were prepared (Fig. 10.28). An excess of 10.27 and 10.29 displaced the duplex DNA strand from NF-κB thus demonstrating the potential usefulness of these aptamers in modifying or influencing the process of transcription in a disease state. Other examples of aptamer selection for proteins (169–173), for receptors (174), or for nucleic acids (175, 176) have appeared in the literature recently.

Several reports have described the use of an in vivo selection method for the detection of biologically relevant interactions with macromolecules. In this work, the difficulties encountered in the in vivo selection/amplification cycle were compensated by the effectiveness of the selected sequences on the target in vivo. Ferber and Maher (177) screened ON libraries in E. coli cells and found positive effectors of amplification of an expressed plasmid. The selection of high-affinity RNA aptamers that could be used as markers or even as drugs targeted against the living cells of Trypanosoma protozoan parasites have been described by Homann and Goringer (178). Bruno and Kiel has reported the selection of DNA aptamers using anthrax spores (179) as biosensors in biological warfare detection. Davis et al. (180), Smith and co-workers (181, 182) have used aptamer sequences as staining agents and markers to track disease-related targets in vivo.

Therapeutic applications of selected RNA aptamers are usually prevented by the considerable drawbacks of such molecules, including their poor stability in biological media. Modified biosynthetic ON libraries, which are still accepted by the biological machinery of the cell during transcription, translation, and amplification but are considered to be more “druglike,” have been the subject of several reports. For example, Bridonneau et al. (183) have reported the synthesis and selection in vitro of a 3 × 1014, ≈100-mer modified ON library L19 for the detection of high-affinity aptamers for human nonpancreatic secretory phospholipase A2 (hnps-PLA2). The chemical synthesis of L19 employed the purine nucleotides A and G, and the 2′-NH2 pyrimidine nucleobases 10.31 and 10.32 (Fig. 10.29), a modification that is known to

538 BIOSYNTHETIC COMBINATORIAL LIBRARIES

a:incubation with hnps-PLA2 immobilized onto agarose with pAbs; b: washing of the unbound RNAs;

c:elution of the bound RNAs; d: reverse transcription into DNA; e: PCR amplification; f: transcription into RNA; g: a-f, 13 cycles, cloning and sequencing of selected clones; h: truncation of ON sequence.

Figure 10.29 Screening of the biosynthetic ON library L19 for aptamers binding to hnpsPLA2: the selection/amplification process and the structure of the most active aptamer10.33.

increase the stability of the ONs (184) without affecting the iterative cycles of selection/ amplification. The library was selected and amplified through 11 cycles following standard protocols (steps a–g, Fig. 10.29). The affinity binding was performed with hnps-PLA2 polyclonal antibodies supported on agarose beads (cycles 1–5, 8, and 9, step a, Fig. 10.29) or with nitrocellulose filtering using free hnps-PLA2 (cycles 6, 7, 10, and 11, step b, Fig. 10.29). With both procedures, incubation of the RNA pools with bound or free enzymes lasted 5 min at 37°C, and the final clones isolated and individually amplified to generate several low-affinity binders (which were discarded) together with a family of high-affinity binders that contained a number of aptamers with picomolar affinity (183). Refinement of the aptamer structure and detailed mechanistic studies resulted in a 51-mer sequence 10.33 (step h, Fig. 10.29) with specific and significant in vitro and in vivo affinity for hnps-PLA2 and with similar potency to some recently described small organic molecule inhibitors (185). Further examples of modified amplifiable ON libraries have also been presented recently (186, 187).

An intriguing expansion of this concept is represented by the so-called selection– reflection strategy. If a chiral molecule is the binding target, its enantiomer is prepared and in vitro selection is used to select a high-affinity natural D-RNA or D-DNA aptamer (188). The mirror image of this aptamer/ligand, that is, an unnatural L-RNA, L-DNA, or a D-peptide, is then prepared that has by definition the same binding/inhibitory activity on the mirror image of the chiral selector (i.e., the target). However, the L-ON or D-peptide nature of the selected oligomer makes it resistant to degradation by nucleases or proteases. This concept has been exploited by several groups to select and

540 BIOSYNTHETIC COMBINATORIAL LIBRARIES

interactions to compensate for the 2′-OH-based RNA interactions and possibly to switch the cleaving preference toward DNA strands.

The selection mechanism is shown schematically in Fig. 10.30. The members of the library that catalyzed the cleavage of the DNA substrate 10.34 ligated its 3′-portion onto the mutated ribozyme molecule (steps a and b). This 3′-portion was carefully selected to be recognized by a primer (step c) that initiated cDNA synthesis in the presence of reverse transcriptase leading to the amplification of the DNA-cleaving ribozymes through synthesis of dsDNA (step d) and transcription/amplification of the corresponding ribozymes (step e). The inactive library individuals were not recognized by the primer, thus preventing their amplification (step f, Fig. 10.30). Incubation of the library with 10.34 (10 µM) was done at 37°C for 1 h, and nine selection/amplification cycles were performed. Selected representatives of round 9 such as 10.35 showed a 100-fold increased DNA cleavage activity, which became appreciable at 37 °C; however, their efficiency as RNA-cleaving enzymes remained largely prevalent (Fig. 10.31). In a further report Tsang and Joyce (201) applied more stringent selection parameters for 18 additional selection/amplification cycles on the final population from the first paper (round 9, L21, Fig. 10.31); the concentration of substrate 10.34 was reduced to 0.2 µM and from round 19 the incubation period was reduced to 5 min. This stringency caused an increase in both the catalytic rate and the binding affinity for the substrate, producing selected representatives from round 27 with comparable DNAand RNA-cleaving performances (Fig. 10.32, top). The high degree of selection for DNA cleavage (10,000-fold increase from L20), though, was obtained together with a slight improvement in the RNA cleavage, which led to ribozymes such as 10.36 with broader specificity including DNA strands, rather than DNA-specific ribozymes (Fig. 10.32). In a following paper Tsang and Joyce (202) conceived a modified selection procedure (Fig. 10.32, bottom) in which an RNA inhibitor was added together

inactive RNA

a: DNA substrate recognition; b: cleavage and ligation of the 3' portion; c: treatment with a primer for cDNA synthesis; d: DNA amplification; e: transcription/amplification into RNA; f: no reaction.

Figure 10.30 Evolution of a natural, RNA-cleaving ribozyme into a DNA-cleaving enzyme: the selection/amplification process using the selection/amplification substrate10.34.

randomization of 35 positions in the catalytic domain

a: nine selection/amplification cycles as in Fig. 10.30; incubation with 10.34 (10 M) for 1 hr at 37°C.

SELECTED INDIVIDUAL FROM L21:

10.35

Kcat/Km for the cleavage of 10.34 = 3,600 M-1 min-1 at 37° with 10 M Mg++ Kcat/Km for the cleavage of natural RNA substrate = 107 M-1 min-1 at 37° with 10 M Mg++

WILD-TYPE Tetrahymena RIBOZYME:

Kcat/Km for the cleavage of 10.34 = 36 M-1 min-1 at 50° with 10 M Mg++

Kcat/Km for the cleavage of natural RNA substrate = 107 M-1 min-1 at 37° with 10 M Mg++

RESULTS:

>100-fold increase of DNA-cleavage activity; still 3x103 higher RNA-cleavage activity.

RNA-SPECIFIC CLEAVING RIBOZYME

Figure 10.31 Evolution of a natural, RNA-cleaving ribozyme into a DNA-cleaving enzyme: screening of the biosynthetic ON ribozyme library L20 and selection of the biosynthetic ON ribozyme library L21.

with the DNA substrate. In this case, selection of DNA-cleaving ribozymes was not changed (step b), but if a comparable or even higher activity on RNA was embedded into the ribozyme, it bound to the RNA inhibitor and was sequestered from the amplifiable library pool, which was then processed as described above. Thirty-six additional cycles were performed starting from the population of round 27 (L22, Fig. 10.32, top). Selected representatives from round 63 such as 10.37 showed a fivefold increase in DNA cleavage and a twofold decrease in RNA cleavage activity (Fig. 10.32). For the first time higher DNA cleavage activity was detected even though the specificity was not yet significant. Analysis of the mutation history from round 0 (L20) to round 63 allowed an understanding of the major interactions driving toward DNA specificity (200, 202). Other examples of focused ON libraries derived from natural sequences have been reported by Ekland and Bartel (204), Schmitt and Lehman (205) and Ordoukhanian and Joyce (206) who described the optimization of natural RNA ligases; by Tusoul et al. (207), reporting the synthesis of small nuclear RNA-inspired libraries; by Zarrinkar and Sullenger (208) and Pierce and Ruffner (209), who reported the synthesis of libraries inspired by the so-called hammerhead ribozymes; by Putlitz et al., who elaborated by combinatorial methods the structure of the so-called hairpin ribozymes (210); by Cole and Dorit (211) who evolved the M1 RNA natural ribozyme into a DNA-cleaving enzyme.

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L21 L22

413-mers

a: 18 selection/amplification cycles as in Fig. 10.30; incubation with 10.34 (0.2 M) for 1 hr at 37°C in rounds 10-18, for 5' in rounds 19-27.

SELECTED INDIVIDUAL FROM L22:

10.36

Kcat/Km for the cleavage of 10.34 = 2.9x106 M-1 min-1 at 37° with 10 M Mg++

Kcat/Km for the cleavage of natural RNA substrate = 2.1x107 M-1 min-1 at 37° with 10 M Mg++

RESULTS:

around 1000-fold increase of DNA-cleavage activity with respect to 10.35; around 10 times higher RNA-cleavage activity.

BROAD (RNA > DNA) SPECIFICITY RIBOZYME

a: 36 selection/amplification cycles as in Fig. 10.30; incubation with 10.34 (0.2 M) for 5' at 37°C in presence of an RNA inhibitor of the natural RNA cleavage reaction.

SELECTED INDIVIDUAL: 10.37

Kcat/Km for the cleavage of 10.34 = 1.5x107 M-1 min-1 at 37° with 10 M Mg++

Kcat/Km for the cleavage of natural RNA substrate = 6.9x106 M-1 min-1 at 37° with 10 M Mg++

RESULTS:

5-fold increase of DNA-cleavage activity with respect to 10.36; 2 times lower RNA-cleavage activity.

BROAD (DNA > RNA) SPECIFICITY RIBOZYME

Figure 10.32 Evolution of a natural, RNA-cleaving ribozyme into a DNA-cleaving enzyme: screening of the biosynthetic ON ribozyme libraries L21 and L22 and selection of the DNA-selective ribozyme 10.37.

Ribozymes acting on nucleotide sequences have often been selected from random libraries, and many artificial sequences have been obtained. Landweber and Porovskaya (212) recently reported the selection of a family of small ribozymes from a 1.6 × 1015-member, 132-mer library L23 able to ligate multiple RNA substrates. In vitro selection was performed with three different RNA substrates for six cycles in the presence of Mg2+ (step a, Fig. 10.33), and multiple substrates were used to select for