Method of SElection with Modified Aptamers (SELMA) Enabling Incorporation of Fluorinated or Methoxylated RNA

A method for selecting fully modified RNA aptamers that bind to a target molecule without reverse transcription, comprising:

• providing a first pool of oligonucleotide complexes that each comprise a ds-DNA molecule tethered to a fully modified RNA molecule, wherein the ds-DNA molecule comprises a coding strand and a complementary non-coding strand which encodes the RNA molecule;
• exposing the first pool of oligonucleotide complexes to the target molecule and allowing the fully modified RNA molecules to bind the target molecule to provide a pool of selected oligonucleotide complexes comprising selected fully modified RNA aptamers which bind the target molecule;
• amplifying the tethered ds-DNA molecules in the pool of selected oligonucleotide complexes using an affinity labeled forward primer and an unlabeled reverse primer to provide amplified affinity labeled ds-DNA molecules, wherein the affinity labeled forward primer binds a primer binding site near the 3′ end of the coding strand and the unlabeled reverse primer binds a primer binding site near the 3′ end of the non-coding strand, and wherein the affinity labeled ds-DNA molecules comprise an affinity labeled strand;
• contacting the amplified affinity labeled ds-DNA molecules with an affinity label-binding molecule and selecting amplified affinity labeled ds-DNA molecules which bind the affinity labeled-binding molecule to provide affinity label-binding ds-DNA molecules;
• and
• isolating the non-affinity labeled template strand from the affinity label-binding ds-DNA molecules to provide a first amplified single-stranded DNA template for the selected fully modified RNA aptamers,
• wherein a fully modified RNA molecule comprises 2′-fluoro-nucleotides, 2′-methoxy-nucleotides, or a combination of 2′-fluoro-nucleotides and 2′-methoxy-nucleotides, and no un-modified nucleotides.