Mechanism of RNA Cleavage Catalyzed by Sequence Specific Polyamide Nucleic Acid-Neamine Conjugate

Abstract

In earlier studies, we found that a conjugate of neamine-polyamide nucleic acid targeting transactivation response element of HIV-1 RNA genome (HIV-1 TAR) displayed anti-HIV-1 activity and sequence-specific cleavage of the target RNA in vitro. Here we show that both the position of conjugation of polyamide nucleic acid (PNA) on neamine and the length of the spacer are critical parameters for conferring cleavage activity to the conjugate. The conjugation of PNA via a spacer incorporating 11 atoms to the 5-position of ring I of the neamine core conferred sequence-specific RNA cleavage activity on the conjugate, while conjugation to the 4'-position of ring II abolished this activity. Similarly, 5-neamine PNA complementary to TAR sequence of HIV-1 genome (PNA(TAR)) conjugates having either a 23-atom spacer or a bulky dansyl group between PNA and the neamine core also resulted in complete loss of cleavage activity. Based on these observations, we propose a mechanism for the observed RNA cleavage catalyzed by the conjugate involving unprotonated and protonated amino groups at the 3-position of ring I and the 6'-position of ring II of the neamine core, respectively.

Synthesis of Aminoglycoside Conjugates of 2′-O-Methyl Oligoribonucleotides

“…The residue was purified by silica gel chromatography (5% MeOH in CH 2 Cl 2 ) to yield 0.71 g (71%) of the product (26) Compound 27 was synthesized from 26 using the three-step procedure, as described for 23, but 10 molar equiv levulinic anhydride was used. After silica gel chromatography (2% MeOH in CH 2 Cl 2 ), product (27) was obtained as white foam in a 79% yield (0.45 g). 1 6,3′,4′, 2″, 3″-Penta-O-levulinoyl-N 1 ,N 3 ,N 2 ′,N 6 ′-tetra(trifluoroacetyl)ribostamycin (28).…”

“…Consistent with the foregoing, they noted that small structural differences perturbed the activity of the neamine-PNA conjugates; only one of the conjugates resulted in a remarkable cleavage. Chaubey et al proposed the following mechanism for the neamine catalyzed hydrolysis of RNA (27). The deprotonated amino group (pK a ) 6.9) at the 3-position of ring I extracts a hydrogen atom from the 2′-OH group of the ribose moiety of the RNA, while the protonated amino group (pK a ) 9.2) at the 6′-position of ring II polarizes the phosphate group by electrostatic interaction.…”

Metal-containing peptide nucleic acid conjugates

“…66 For the "cleaving unit", a metal moiety is usually required although purely organic alternative agents have been used such as neamine or diethylenetriamine. [68][69][70][71] Indeed, the cleavage with artificial metallo-nucleases is following either an oxidative or a hydrolytic mechanism. 67,[72][73][74] The former is observed with redox-active metal complexes while the latter one is based on the Lewis-acidity of metal ions.…”

Pharmacokinetic Analysis of Polyamide Nucleic-Acid-Cell Penetrating Peptide Conjugates Targeted against HIV-1 Transactivation Response Element

“…The PNA bound to the column was eluted with 60% acetonitrile in 0.1% TFA at 50 o C. An aliquot of each sample was analyzed by mass spectrometry using Applied Biosystems 4700 Proteomics Analyzer 7000. The remaining samples were lyophilized, dissolved in PBS, and subjected to gel-retardation assay as described in Chaubey et al (2007).…”