Aptamers against extracellular targets for in vivo applications.

Oligonucleotides are multifunctional molecules which can interfere with gene expression by different mechanism such as antisense, RNA interference, ribozymes, etc. For most in vivo diagnostic and therapeutic applications, oligonucleotides need to be delivered to the intracellular compartment of a specific organ, a difficult task which limits considerably their use. However, aptamer oligonucleotides which target extracellular markers obviate this problem. Aptamers are short oligonucleotides (<100 bases) selected from large combinatorial pools of sequences for their capacity to bind to many types of different targets, ranging from small molecules (amino acids, antibiotics...) to proteins or nucleic acid structures. Aptamers present the same high specificity and affinity for their targets as antibodies. In addition to efficient binding, aptamers have been shown in many cases to display an inhibitory activity on their targets. Moreover, they seem to lack immunogenicity and can be chemically modified in order to improve their stability against nucleases or extend their blood circulation time, two properties which are particularly useful for in vivo applications. Recently, aptamers have been selected against whole living cells, opening a new avenue which presents three major advantages 1) direct selection without prior purification of the targets; 2) conservation of membrane proteins in their native conformation similar to the in vivo conditions and 3) identification of (new) targets for a specific phenotype. Many aptamers are now being developed against biomedical relevant extracellular targets: membrane receptor proteins, hormones, neuropeptides, coagulation factors... Among them, one aptamer that inhibits the human VEGF165 has recently been approved by FDA for the treatment of age-related macular degeneration. Here we discuss the recent developments of aptamers against extracellular targets for in vivo therapy and as tools for diagnosis using molecular imaging.

RNA and N3'-->P5' kissing aptamers targeted to the trans-activation responsive (TAR) RNA of the human immunodeficiency virus-1.

We used in vitro selection to identify RNA aptamers able to selectively bind to the TAR RNA motif of HIV-1, an unperfect RNA hairpin involved in the transcription of the retroviral genome. We selected aptameric RNA hairpins giving rise to kissing complexes with TAR. The N3'-->P5' phosphoramidate variant of the aptamer bind to TAR with a Kd in the low nanomolar range. However, only the RNA-RNA loop-loop complex is recognized by the Rop protein of E. coli which is specific for kissing complexes.

Is a closing "GA pair" a rule for stable loop-loop RNA complexes?

RNA hairpin aptamers specific for the trans-activation-responsive (TAR) RNA element of human immunodeficiency virus type 1 were identified by in vitro selection (Ducongé, F., and Toulmé, J. J. (1999) RNA 5, 1605-1614). The high affinity sequences selected at physiological magnesium concentration (3 mm) were shown to form a loop-loop complex with the targeted TAR RNA. The stability of this complex depends on the aptamer loop closing "GA pair" as characterized by preliminary electrophoretic mobility shift assays. Thermal denaturation monitored by UV-absorption spectroscopy and binding kinetics determined by surface plasmon resonance show that the GA pair is crucial for the formation of the TAR-RNA aptamer complex. Both thermal denaturation and surface plasmon resonance experiments show that any other "pairs" leads to complexes whose stability decreases in the order AG > GG > GU > AA > GC > UA >> CA, CU. The binding kinetics indicate that stability is controlled by the off-rate rather than by the on-rate. Comparison with the complex formed with the TAR* hairpin, a rationally designed TAR RNA ligand (Chang, K. Y., and Tinoco, I. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8705-8709), demonstrates that the GA pair is a key determinant which accounts for the 50-fold increased stability of the TAR-aptamer complex (K(d) = 2.0 nm) over the TAR-TAR* one (K(d) = 92. 5 nm) at physiological concentration of magnesium. Replacement of the wild-type GC pair next to the loop of RNA I' by a GA pair stabilizes the RNA I'-RNA II' loop-loop complex derived from the one involved in the control of the ColE1 plasmid replication. Thus, the GA pair might be the preferred one for stable loop-loop interactions.

In vitro selection identifies key determinants for loop-loop interactions: RNA aptamers selective for the TAR RNA element of HIV-1.

We selected RNA aptamers specific for the trans-activation responsive (TAR) RNA, a stem-loop structure crucial for the transcription of the integrated genome of the human immunodeficiency virus. Most of the selected sequences could be folded as imperfect hairpins and displayed a 5'-GUCCCAGA-3' consensus motif constituting the apical loop. The six central bases of this consensus sequence are complementary to the entire TAR loop, leading to the formation of TAR RNA-aptamer "kissing" complexes. The consensus G and A residues closing the aptamer loop contributed to the high affinity (Kd = 30 nM at 23 degrees C) of the aptamers for the TAR RNA. This G A pair was shown to be crucial for binding to TAR at a low magnesium concentration. The selection also identified 5'-PuPy and 5'-PyPu base pairs at alpha and beta positions of the stem, next to the loop, respectively. This strategy offered a way to identify key determinants of loop-loop interactions and to generate high affinity ligands of TAR RNA structure.

Identification of aptamers against the DNA template for in vitro transcription of the HIV-1 TAR element.

We have extracted from a random population of about 10(9) oligodeoxynucleotides a series of 21-mers that are able to bind to a folded DNA 76-mer used as a template for in vitro transcription of the TAR element of the retrovirus HIV-1, by the T7 RNA polymerase. Five aptastrucs, that is, aptamers able to bind to the structure, out of 15 analyzed sequences, share the consensus motif 5'-PyGGG(TG)PyC, complementary in part to a weak double-stranded region of the target. (The parentheses indicate that either T or G is missing in one of these aptastrucs.) A dissociation constant of about 3 microM was evaluated by electrophoretic mobility shift assay for the winner sequence. Interactions between the aptastruc and the target sequences involve more than Watson-Crick base pairing of the consensus octamer. The binding is chemistry dependent. Phosphorothioate oligodeoxyribonucleotides and 2'-O-methyl oligoribonucleotides derived from the selected aptastrucs exhibit a weak if any affinity for the target.