POTENT 2'-AMINO-2'-DEOXYRIBONUCLEOTIDE, AND 2'-FLUORO-2'-DEOXYRIBONUCLEOTIDE RNA INHIBITORS OF KERATINOCYTE GROWTH-FACTOR

Reiterative in vitro selection-amplification from random oligonucleoti de libraries allows the identification of molecules with specific func tions such as binding to specific proteins. The therapeutic usefulness of such molecules depends on their high affinity and nuclease resista nce. Libraries of RNA molecules containing 2' amino-(2'NH2)- or 2'fluo ro-(2'F)-2'-deoxypyrimidines could yield ligands with similar nuclease resistance but not necessarily with similar affinities. This is becau se the intramolecular helices containing 2'NH2 have lower melting temp eratures (Tm) compared with helices containing 2'F, giving them thermo dynamically less stable structures and possibly weaker affinities. We tested these ideas by isolating high-affinity ligands to human keratin ocyte growth factor from libraries containing modified RNA molecules w ith either 2'NH2 or 2'F pyrimidines. We demonstrated that 2'F RNA liga nds have affinities (K-d approximately 0.3-3 pM) and bioactivities (K- i approximately 34 pM) superior to 2'NH2 ligands (K-d approximately 40 0 pM and K-i approximately 10 nM). In addition, 2'F ligands have extre me thermostabilities(Tm approximately 78 degrees C in low salt, and sp ecificities).