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DNA translocation through reconstituted planar bilayers- results and perspectives
Gyoergy Bathori1, Ildiko Szabo2, Francesco Tombola2 and Mario Zoratti2
1) Dept.of Physiology, Semmelweis Medical University, H-1088, Puskin u. 9, Budapest, Hungary
2) CNR Center for Biomembranes, Dept. Biomedical Sciences, University of Padova,Viale G. Colombo 3, 35121 Padova, Italy
Transport of nucleic acids through biological membranes is of great practical importance. Cellular uptake of naked DNA may transfect bacteria and mammalian cells. Antisense RNA offers potential impact in the anticancer therapy. Moreover, the nucleic acid transporters also participate in several naturally occurring processes as bacterial conjugation, horizontal gene transfer, viral infection, differentiation by RNA acquired from neighboring cells and t-RNA or sr-RNA uptake by mitochondria. Despite of the great interest, our knowledge about the mechanism of these processes is still surprisingly insufficient.
Reconstitution of the transporters into artificial membranes (liposomes or planar bilayers) has been a very effective method to explore the properties of small molecule transporters and ion channels. At the same time, it has to be emphasized, that reconstitution of the transport activity is an absolute requirement in the process of isolation of any transporter protein. In the case of macromolecule transporters, however, the lack of an appropriate reconstitution method considerably hindered the progress. Observations with reconstituted planar bilayers have suggested that proteins might be translocated through high conductance
channels. The interaction has been indicated by the altered electrophysiological activity of the channel. The translocated material however could not be detected due to the low transport rate. (Typically, one or a few channels are incorporated into the planar bilayer; the transport rate is therefore exceptionally low.) Theoretically, the nucleic acids too, should use large pores for the translocation. Recently, a method allowing direct observation of translocation of genetic material through reconstituted planar bilayers has been introduced [1,2,3]. This method utilizes the advantage of the high sensitivity of PCR. Amplification by PCR multiplies the number of DNA molecules resulting in detectability of the translocated material. The unique advantage of the method is that simultaneously with the detection of translocation, the electrophysiological events related to the transport may also be monitored.
We tested this method on membrane fractions from Bacillus subtilis and isolated mitochondrial porin. In planar bilayers reconstituted with B. subtilis membrane fractions we observed a large, stretch activated (SA) channel (conductance .100 pS in 100 mM KCl). The translocation of double stranded DNA appeared only in the presence of SA channels. In control experiments there was no translocation through "empty" (nonreconstituted) bilayers, membranes reconstituted with gramicidin, maltoporin (LamB) or with mammalian plasma membrane fractions containing small channels (conductance 30 pS in 150 mM KCl). An unfavorable electrophoretic driving force (positivity on the side of the addition of DNA) also prevented the translocation.
In the membrane fractions we have a multitude of various proteins. Our observation that the translocation appeared in the presence of large bacterial channels made likely, but not proved the penetration of the nucleic acid through the aqueous pore formed by the channel. Direct evidences for translocation through large channels could be obtained by means of isolated pores, where the interference of other proteins has been excluded. In our experiments we used isolated mitochondrial porin for that purpose. The mitochondrial outer membrane contains only two large channels, the protein import channel and porin. There are some sporadic data on translocation of DNA through mitochondrial membranes. Porin seems
more suitable for the mediation of the translocation of polyanionic DNA, because of its large pore diameter (approximately 4 nm) and its anion selective characteristic. (The diameter of the protein translocating channel is only 2 nm and it is cation selective.)
Indeed we found that in presence of favorable electrophoretic driving force DNA was translocated. In control experiments the translocation was prevented by unfavorable electroforetic driving force and blocked - in all likelihood sterically - by antibodies developed against porin.
Theoretically our method might be extended to the investigation of RNA and ribonucleoprotein translocation. Investigation of t-RNA and sr-RNA transport into the mitochondria is an emerging topic in the current literature. Reconstitution of the suspected components of the transport machinery may help their identification. Accumulating evidences show that porin is also present in the plasma membrane. Another intriguing possibility is that porin would mediate the cellular uptake of DNA. Reconstitution of porin containing plasma membrane fractions into planar bilayers may provide direct evidence of this hypothesis. The present paper also discusses some other perspectives of the application of the method.
References
- Kasianowicz, J.J., E. Brandin, D. Branton, and D.W. Deamer. 1996. Characterization of individual polynucleotide molecules using a membrane channel. Proc. Natl. Acad. Sci. (USA) 93: 13770-13773
- Szabo, I., Gy. Bathori, F. Tombola, M. Brini, A. Coppola, and M. Zoratti. 1997. DNA translocation across planar bilayers containing Bacillus subtilis ion channels, J. Biol. Chem. 272: 25275-25282
- Szabo, I., Gy. Bathori, F. Tombola, A. Coppola, M. Brini, I. Schmehl, A. Ghazi, V. De Pinto, and M. Zoratti. 1998. DNA can be translocated across planar membrane containing purified mitochondrial porin, FASEB J. 12: 495-502
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