Sequence-specific targeting of RNA with an oligonucleotide-neomycin conjugate.
The synthesis of neomycin covalently attached at the C5-position of 2'-deoxyuridine is reported. The synthesis outlined allows for incorporation of an aminoglycoside (neomycin) at any given site in an oligonucleotide (ODN) where a thymidine (or uridine) is present. Incorporation of this modified base into an oligonucleotide, which is complementary to a seven-bases-long alpha-sarcin loop RNA sequence, leads to enhanced duplex hybridization. The increase in Tm for this duplex (DeltaTm = 6 degrees C) suggests a favorable interaction of neomycin within the duplex groove. CD spectroscopy shows that the modified duplex adopts an A-type confirmation. ITC measurements indicate the additive effects of ODN and neomycin binding to the RNA target (Ka = 4.5 x 107 M-1). The enhanced stability of the hybrid duplex from this neomycin-ODN conjugate originates primarily from the enthalpic contribution of neomycin {DeltaDeltaHobs = -7.21 kcal/mol (DeltaHneomycin conjugated - DeltaH nonconjugated)} binding to the hybrid duplex. The short linker length allows for selective stabilization of the hybrid duplex over the hybrid triplex. The results described here open up new avenues in the design and synthesis of nucleo-aminoglycoside-conjugates (N-Ag-C) where the inclusion of any number of aminoglycoside (neomycin) molecules per oligonucleotide can be accomplished.
Charles
Irudayasamy
I
Laboratories of Medicinal Chemistry, Clemson University, Clemson, South Carolina 29634, USA.
Xi
Hongjuan
H
Arya
Dev P
DP
D001483:Base Sequence; D002151:Calorimetry; D002942:Circular Dichroism; D008958:Models, Molecular; D009355:Neomycin / Q000138:chemical synthesis / Q000737:chemistry*; D009584:Nitrogen / Q000737:chemistry; D009841:Oligonucleotides / Q000737:chemistry*; D012313:RNA / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013816:Thermodynamics; D017186:Titrimetry; D044366:Transition Temperature
D009841:Oligonucleotides; D012313:RNA; D009355:Neomycin; D009584:Nitrogen