Insights Into The Biomedical Effects Of Carboxylated Single-wall Carbon Nanotubes On Telomerase And Telomeres

Telomeres play important roles in chromosome structural integrity to cap and protecttheir extremities from illegitimate recombination, degradation and end-to-end fusion.In humans, the telomere is composed of G-rich and C-rich duplex with asingle-stranded3′-overhang, the G-rich strand can form a four-stranded G-quadruplexstructure, and its complementary C-rich strand may adopt intercalated i-motifstructures. Both human telomeric G-rich and C-rich DNA have been considered asspecific drug targets for cancer therapy. G-quadruplex formation can inhibittelomerase activity. Many compounds that can stabilize G-quadruplex structure andinhibit telomerase activity have been reported. However, due to i-motif structureunstable and lack of i-motif specific binding agents, it remains unclear whetherstabilization of i-motif structure can inhibit telomerase activity.Single-walled carbon nanotubes (SWNTs) have been considered as the leadingnanodevice candidate, whose interactions with nucleic acids has attracted muchattention. A series of DNA interactions with SWNTs have been studied by our group.We found that SWNTs can destabilize duplex DNA and induce a sequence-dependentDNA B-A transition, and can facilitate self-structuring of single-stranded RNApoly(rA) to form A·A~+duplex structure. Intriguingly, SWNTs, not multi-walledcarbon nanotubes (MWNTs), can selectively stabilize human telomeric i-motif DNAand induce i-motif structure formation by binding to the5′-end major groove underphysiological conditions, and even under molecular crowding conditions. Furtherstudies indicate that SWNTs do not stabilize G-quadruplex DNA, and can causehuman telomeric duplex to disassociate by forming i-motif and G-quadruplexDNA. This makes SWNTs as the first selective i-motif DNA binding agent andsuitable probe to study the biological function of i-motif both in vitro and in livingcells.In this report, we applied TRAP-G4assay, conventional TRAP assay,immunofluorescence, immunoblot, immunoprecipitation, ChIP and telomere-FISH toanalyze the effects of SWNTs on telomerase activity, telomere structure and cellproliferation. The main results are as follows:1. SWNTs can enter the nucleus and localize at telomere when modified with PEG and labeled with FITC, which is clarified by the co-localization with interphasetelomere marker TRF1protein. This result may provide the possible biologicalactivities of SWNTs in living cells, especially at telomere.2. Based on TRAP-G4assay, SWNTs can inhibit telomerase activity in vitro throughthe indirect G-quadruplex stabilization on the telomeric G-rich strand induced by theformation of i-motif on the complementary C-rich strand. This has been confirmed inliving systems. Furthermore, the decreased telomerase activity in living cells was notassociated with the alteration of its localization or expression.3. Although SWNTs did not induce acute cytotoxicity, after a long time exposure,SWNTs can inhibit cell growth, which may be related to the telomere dysfunction, butnot merely to the telomerase activity inhibition. Furthermore, growth arrest inducedby SWNTs was associated with the production of DNA damage response, which wasfurther clarified to occur at telomere.4. SWNTs can induce telomere dysfunction characterized by the formation ofanaphase bridges, micronuclei and telomere fusion. SWNTs also induced the directunprotection of telomere visualized by the telomere-TdT assay.5. SWNTs can induce the reveal of telomere binding protein TRF2, POT1and PCBP1from telomere, which may provide the indirect evidence for G-quadruplex and i-motifformation in living cells. Moreover, SWNTs treatment caused the rapid degradationof telomeric3′-overhang from telomere without affecting the total telomere length.6. SWNTs induced the cell cycle arrest, apoptosis and senescence as consequence oftelomere dysfunction. This growth suppression maybe associated with theupregulation of p16and p21proteins.This is the first example that stabilization of i-motif structure can inhibit telomeraseactivity and interfere with the telomere functions in cancer cells. Our work mayprovide new insights into understanding the biomedical effects of SWNTs in cancercells and the biological importance of i-motif structure in vivo.