Anticoagulant Oligonucleotide–Peptide Conjugates: Identification of Thrombin Aptamer Conjugates with Improved Characteristics

Abstract

Oligonucleotide–peptide conjugates (OPCs) are a promising class of biologically active compounds with proven potential for improving nucleic acid therapeutics. OPCs are commonly recognized as an efficient instrument to enhance the cellular delivery of therapeutic nucleic acids. In addition to this application field, OPCs have an as yet unexplored potential for the post-SELEX optimization of DNA aptamers. In this paper, we report the preparation of designer thrombin aptamer OPCs with peptide side chains anchored to a particular thymidine residue of the aptamer. The current conjugation strategy utilizes unmodified short peptides and support-bound protected oligonucleotides with activated carboxyl functionality at the T3 thymine nucleobase. The respective modification of the oligonucleotide strand was implemented using N3-derivatized thymidine phosphoramidite. Aptamer OPCs retained the G-quadruplex architecture of the parent DNA structure and showed minor to moderate stabilization. In a series of five OPCs, conjugates bearing T3–Ser–Phe–Asn (SFN) or T3–Tyr–Trp–Asn (YWN) side chains exhibited considerably improved anticoagulant characteristics. Molecular dynamics studies of the aptamer OPC complexes with thrombin revealed the roles of the amino acid nature and sequence in the peptide subunit in modulating the anticoagulant activity.

The Regioselective Conjugation of the 15-nt Thrombin Aptamer with an Optimized Tripeptide Sequence Greatly Increases the Anticoagulant Activity of the Aptamer

“…Chemical reagents and solvents were purchased from various commercial suppliers and used without further purification. Modified thymidine phosphoramidite ( Figure S1 ) was prepared as previously described [ 13 ]. Research-grade human thrombin from plasma for clotting studies was purchased from Renam (Moscow, Russia; #P33).…”

“…Molecular dynamics studies were performed as described previously [ 13 ]. Briefly, the crystal structure of the thrombin complex with T3-modified TBA (PDB id 6Z8X) was used as a starting model to build TBA–peptide conjugates.…”

“…After the two-stage optimization procedure [ 13 ], TBA–peptide complexes with thrombin were run in MD simulations using constant temperature (T = 300 K) and constant pressure ( p = 1 atm) for 80 ns. The plots of energy of interaction vs. time were smoothed using the moving average method (span = 5).…”

“…Depending on the functional roles of subunits in the oligonucleotide conjugate, this may include lipids, carbohydrates, peptides, or some other functional elements [ 9 , 10 ]. Particularly, aptamer–peptide conjugates may exhibit improved precision of targeting, enhanced affinity [ 11 , 12 , 13 ], or better delivery [ 10 , 11 ] as compared to unmodified aptamers or unconjugated peptides.…”

“…Recently, we reported the preparation of thrombin binding aptamer (TBA)—peptide conjugates with improved anticoagulant characteristics [ 13 ]. A TBA is a 15-nt G-rich DNA sequence 5′-GGTTGGTGTGGTTGG that forms a two-shelf anti-parallel G-quadruplex and binds with high affinity thrombin at its exosite I ( Figure 1 A).…”