David Tabatadze

Targeting the Mycobacterium tuberculosis 30/32-kDa mycolyl transferase complex as a therapeutic strategy against tuberculosis: Proof of principle by using antisense technology

We have investigated the effect of sequence-specific antisense phosphorothioate-modified oligodeoxyribonucleotides (PS-ODNs) targeting different regions of each of the 30/32-kDa protein complex (antigen 85 complex) encoding genes on the multiplication of Mycobacterium tuberculosis. Single PS-ODNs to one of the three mycolyl transferase transcripts, added either once or weekly over the 6-wk observation period, inhibited bacterial growth by up to 1 log unit. A combination of three PS-ODNs specifically targeting all three transcripts inhibited bacterial growth by ≈2 logs; the addition of these PS-ODNs weekly for 6 wk was somewhat more effective than a one-time addition. Targeting the 5′ end of the transcripts was more inhibitory than targeting internal sites; the most effective PS-ODNs and target sites had minimal or no secondary structure. The effect of the PS-ODNs was specific, as mismatched PS-ODNs had little or no inhibitory activity. The antisense PS-ODNs, which were highly stable in M. tuberculosis cultures, specifically blocked protein expression by their gene target. PS-ODNs targeting the transcript of a related 24-kDa protein (mpt51) had little inhibitory effect by themselves and did not increase the effect of PS-ODNs against the three members of the 30/32-kDa protein complex. The addition of PS-ODNs against the transcripts of glutamine synthetase I (glnA1) and alanine racemase (alr) modestly increased the inhibitory efficacy of the 30/32-kDa protein complex-specific PS-ODNs to ≈2.5 logs. This study shows that the three mycolyl transferases are highly promising targets for antituberculous therapy by using antisense or other antimicrobial technologies.

Fluorescence resonance energy transfer in near-infrared fluorescent oligonucleotide probes for detecting protein–DNA interactions

Optical imaging in the near-infrared (NIR) range enables detecting ligand-receptor interactions and enzymatic activity in vivo due to lower scattering and absorption of NIR photons in the tissue. We designed and tested prototype NIR fluorescent oligodeoxyribonucleotide (ODN) reporters that can sense transcription factor NF-κB p50 protein binding. The reporter duplexes included donor NIR Cy5.5 indodicarbocyanine fluorochrome linked to the 3′ end of the first ODN and NIR acceptor fluorochromes (indodicarbocyanine Cy7 or, alternatively, a heptamethine cyanine IRDye 800CW) that were linked at the positions +8 and +12 to the complementary ODN that encoded p50 binding sites. Both Cy7 and 800CW fluorochromes were linked by using hydrophilic internucleoside phosphate linkers that enable interaction between the donor and the acceptor with no base-pairing interference. We observed efficient fluorescence resonance energy transfer (FRET) both in the case of Cy5.5–Cy7 and Cy5.5–800CW pairs of fluorochromes, which was sensitive to the relative position of the dyes. Higher FRET efficiency observed in the case of Cy5.5–Cy7 pair was due to a larger overlap between the ODN-linked Cy5.5 emission and Cy7 excitation spectra. Fluorescent mobility shift assay showed that the addition of human recombinant p50 to ODN duplexes resulted in p50 binding and measurable increase of Cy5.5 emission. In addition, p50 binding provided a concomitant protection of FRET effect from exonuclease-mediated hydrolysis. We conclude that NIR FRET effect can be potentially used for detecting protein–DNA interactions and that the feasibility of detection depends on FRET efficacy and relative fluorochrome positions within ODN binding sites.

Hairpin extensions enhance the efficacy of mycolyl transferase-specific antisense oligonucleotides targeting Mycobacterium tuberculosis

We have investigated the efficacy of modifying gene-specific antisense phosphorothioate oligodeoxyribonucleotides (PS-ODNs) by the addition of 5′ and 3′ hairpin extensions. As a model system, we have targeted the Mycobacterium tuberculosis 30/32-kDa mycolyl transferase protein complex genes encoding three highly related enzymes (antigens 85 A, B, and C). Whereas the addition of a hairpin extension at only one end of the PS-ODNs did not improve their inhibitory capacity, the addition of hairpin extensions at both ends enhanced their capacity to inhibit M. tuberculosis multiplication in comparison with unmodified PS-ODNs. A combination of three 5′-, 3′-hairpin-modified PS-ODNs (HPS-ODNs) targeting each of the three mycolyl transferase transcripts inhibited bacterial growth in broth culture by ≈1.75 log units (P < 0.0001) and in human THP-1 macrophages by ≈0.4 log units (P < 0.0001), which to our knowledge has not previously been demonstrated for any PS-ODN; reduced target gene transcription by ≥90%; caused ≈90% reduction in mycolyl transferase expression; and increased bacterial sensitivity to isoniazid by 8-fold. The growth-inhibitory effect of the HPS-ODNs was gene-specific. Mismatched HPS-ODNs had no growth-inhibitory capacity. This study demonstrates that 5′- and 3′-HPS-ODNs are highly efficacious against M. tuberculosis and supports the further development of antisense technology as a therapeutic modality against tuberculosis.

A NOVEL THYMIDINE PHOSPHORAMIDITE SYNTHON FOR INCORPORATION OF INTERNUCLEOSIDE PHOSPHATE LINKERS DURING AUTOMATED OLIGODEOXYNUCLEOTIDE SYNTHESIS

A novel thymidine phosphoramidite synthon was synthesized and successfully used for incorporation of primary amino groups, attached through a triethylene glycol linker to the internucleoside phosphates, at desired locations during automated oligodeoxynucleotide synthesis. The synthesized amino-linker bearing oligonucleotides are stable under deprotection conditions and exhibit Watson-Crick base-pairing properties. Covalent labeling of oligonucleotides with carbocyanine near-infrared fluorochromes resulted in 2.5 times higher labeling yields when compared with oligonucleotides containing base-attached aminolinkers. We anticipate that the developed synthetic approach will be useful for nucleotide sequence-specific attachment of single or multiple ligands or reporter molecules.