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Computational genes: a tool for molecular diagnosis and therapy of aberrant mutational phenotype.

Martínez-Pérez IM, Zhang G, Ignatova Z, Zimmermann KH - BMC Bioinformatics (2007)

Bottom Line: The aberrant mutations trigger a cascade reaction: specific molecular markers as input are released and induce a spontaneous self-assembly of a wild type protein or peptide, while the mutational disease phenotype is silenced.We experimentally demostrated in in vitro translation system that a viable protein can be autonomously assembled.Our work demostrates the basic principles of computational genes and particularly, their potential to detect mutations, and as a response thereafter administer an output that suppresses the aberrant disease phenotype and/or restores the lost physiological function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Computer Technology, Hamburg University of Technology, Hamburg 21073, Germany. martinez-perez@tu-harburg.de

ABSTRACT

Background: A finite state machine manipulating information-carrying DNA strands can be used to perform autonomous molecular-scale computations at the cellular level.

Results: We propose a new finite state machine able to detect and correct aberrant molecular phenotype given by mutated genetic transcripts. The aberrant mutations trigger a cascade reaction: specific molecular markers as input are released and induce a spontaneous self-assembly of a wild type protein or peptide, while the mutational disease phenotype is silenced. We experimentally demostrated in in vitro translation system that a viable protein can be autonomously assembled.

Conclusion: Our work demostrates the basic principles of computational genes and particularly, their potential to detect mutations, and as a response thereafter administer an output that suppresses the aberrant disease phenotype and/or restores the lost physiological function.

Show MeSH
One-step assembly of the functional gene in eukaryotic in vitro translation system. The mutated DNA, the diagnostic complex and the non-assembled gene components were mixed in equimolar ratio and added to the in vitro translation reaction (lane: one step self-assembly). The translation product with a molecular mass of 17.3 kDa is marked by asterisk. Reaction containing only the intact hID1 gene served as positive control (lane: positive control); reaction lacking the mutated DNA was used to test the self-ligation of the diagnostic complex (lane: self-ligation control). The low concentration of the output product severely restricted the detection techniques that can be used to detect the translation product, i.e., in this case only by radioactivity. Note that the eukaryotic transcription/translation coupled system gives two high background signals at 10–13 and 28 kDa. The background that arises from the in vitro translation kit itself was tested in a negative control reaction containing pIVEX vector without any insert (lane: negative control). P and S denote pellet and supernatant, respectively.
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Figure 3: One-step assembly of the functional gene in eukaryotic in vitro translation system. The mutated DNA, the diagnostic complex and the non-assembled gene components were mixed in equimolar ratio and added to the in vitro translation reaction (lane: one step self-assembly). The translation product with a molecular mass of 17.3 kDa is marked by asterisk. Reaction containing only the intact hID1 gene served as positive control (lane: positive control); reaction lacking the mutated DNA was used to test the self-ligation of the diagnostic complex (lane: self-ligation control). The low concentration of the output product severely restricted the detection techniques that can be used to detect the translation product, i.e., in this case only by radioactivity. Note that the eukaryotic transcription/translation coupled system gives two high background signals at 10–13 and 28 kDa. The background that arises from the in vitro translation kit itself was tested in a negative control reaction containing pIVEX vector without any insert (lane: negative control). P and S denote pellet and supernatant, respectively.

Mentions: We tested the concept of computational gene in an in vitro translation system. As structural gene of the computational gene we took the human inhibitor of DNA binding 1 (hID1) gene [34] comprised of two exons (452 bp and 115 bp) separated by one intron (239 bp). Its original intron sequence was modified to construct the initial and accepting final sites: the intron was split into two parts (by HindIII digestion), releasing intact exons and the upstream promoter region. The conserved splicing signals, i.e., 5'-splice signal, AG/GTRAG (995), CTSAY branch point (1213), strong poly(Y) signal between branch point and 3'-splice signal consisting of 13/14 pyrimidines (1218), and an NYAG/G 3'-splice signal (ACAG/G, 1232) were used as constants in the computational gene and remained unchanged. Two 16 nt long ssDNAs (overhang A, 5'-GGCCGCAATTCCAAAC-3', and overhang B, 5'-CAATTCCAAATGAGCT-3') were attached to the released free overhangs after enzyme digestion, leaving 12 nt free overhangs, complementary to the 24 nt long activated diagnostic signal AB' (5'-CATTTGGAATTGGTTTGGAATTGC-3'). Note that the ligation point between overhangs A and B is located precisely at the mismatched positions in the diagnostic signal AB' (Figure 2B). Addition of the mutated DNA and diagnostic complexes, whose diagnostic signal is complementary to the single stranded overhangs in the initial and final accepting state, led to successful self-assembly of the double stranded long fragment, resembling the expected size of the gene of interest [see Additional file 5]. A viable functional gene was assembled in an in vitro translation system (Figure 3). All the components, the diagnostic complex (Am/AB'), the mutated DNA (Am'), and the non-assembled gene fragments (Figure 2), were mixed together and added simultaneously to an eukaryotic in vitro translation system and the reaction proceeded for 3.5 h at the optimal conditions (for details see Methods section). The addition of mutated DNA (Am' single strand) initiated the programmed cascade reaction specified by the path: (i) mutation detection, (ii) gene self-assembly and linking, and (iii) transcription and translation, yielding a 155 amino acid long protein (17.3 kDa) as output. The size of the output was verified by a control reaction, containing the intact hID1 gene (Figure 3, positive control lane). Even thought the yield of the output is very low (weak band, Figure 3), it points out the potential for feasibility of the model. Without a released diagnostic signal no self-ligation of the therapeutic gene could be detected (Figure 3, self-ligation control). The in vitro translation system is representative of intact transcription/translation machinery in the cell and the results from this experiment suggest that successful self-assembly of the computational gene and translation into a viable product (protein or anti-drug) might occur in living cells.


Computational genes: a tool for molecular diagnosis and therapy of aberrant mutational phenotype.

Martínez-Pérez IM, Zhang G, Ignatova Z, Zimmermann KH - BMC Bioinformatics (2007)

One-step assembly of the functional gene in eukaryotic in vitro translation system. The mutated DNA, the diagnostic complex and the non-assembled gene components were mixed in equimolar ratio and added to the in vitro translation reaction (lane: one step self-assembly). The translation product with a molecular mass of 17.3 kDa is marked by asterisk. Reaction containing only the intact hID1 gene served as positive control (lane: positive control); reaction lacking the mutated DNA was used to test the self-ligation of the diagnostic complex (lane: self-ligation control). The low concentration of the output product severely restricted the detection techniques that can be used to detect the translation product, i.e., in this case only by radioactivity. Note that the eukaryotic transcription/translation coupled system gives two high background signals at 10–13 and 28 kDa. The background that arises from the in vitro translation kit itself was tested in a negative control reaction containing pIVEX vector without any insert (lane: negative control). P and S denote pellet and supernatant, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175521&req=5

Figure 3: One-step assembly of the functional gene in eukaryotic in vitro translation system. The mutated DNA, the diagnostic complex and the non-assembled gene components were mixed in equimolar ratio and added to the in vitro translation reaction (lane: one step self-assembly). The translation product with a molecular mass of 17.3 kDa is marked by asterisk. Reaction containing only the intact hID1 gene served as positive control (lane: positive control); reaction lacking the mutated DNA was used to test the self-ligation of the diagnostic complex (lane: self-ligation control). The low concentration of the output product severely restricted the detection techniques that can be used to detect the translation product, i.e., in this case only by radioactivity. Note that the eukaryotic transcription/translation coupled system gives two high background signals at 10–13 and 28 kDa. The background that arises from the in vitro translation kit itself was tested in a negative control reaction containing pIVEX vector without any insert (lane: negative control). P and S denote pellet and supernatant, respectively.
Mentions: We tested the concept of computational gene in an in vitro translation system. As structural gene of the computational gene we took the human inhibitor of DNA binding 1 (hID1) gene [34] comprised of two exons (452 bp and 115 bp) separated by one intron (239 bp). Its original intron sequence was modified to construct the initial and accepting final sites: the intron was split into two parts (by HindIII digestion), releasing intact exons and the upstream promoter region. The conserved splicing signals, i.e., 5'-splice signal, AG/GTRAG (995), CTSAY branch point (1213), strong poly(Y) signal between branch point and 3'-splice signal consisting of 13/14 pyrimidines (1218), and an NYAG/G 3'-splice signal (ACAG/G, 1232) were used as constants in the computational gene and remained unchanged. Two 16 nt long ssDNAs (overhang A, 5'-GGCCGCAATTCCAAAC-3', and overhang B, 5'-CAATTCCAAATGAGCT-3') were attached to the released free overhangs after enzyme digestion, leaving 12 nt free overhangs, complementary to the 24 nt long activated diagnostic signal AB' (5'-CATTTGGAATTGGTTTGGAATTGC-3'). Note that the ligation point between overhangs A and B is located precisely at the mismatched positions in the diagnostic signal AB' (Figure 2B). Addition of the mutated DNA and diagnostic complexes, whose diagnostic signal is complementary to the single stranded overhangs in the initial and final accepting state, led to successful self-assembly of the double stranded long fragment, resembling the expected size of the gene of interest [see Additional file 5]. A viable functional gene was assembled in an in vitro translation system (Figure 3). All the components, the diagnostic complex (Am/AB'), the mutated DNA (Am'), and the non-assembled gene fragments (Figure 2), were mixed together and added simultaneously to an eukaryotic in vitro translation system and the reaction proceeded for 3.5 h at the optimal conditions (for details see Methods section). The addition of mutated DNA (Am' single strand) initiated the programmed cascade reaction specified by the path: (i) mutation detection, (ii) gene self-assembly and linking, and (iii) transcription and translation, yielding a 155 amino acid long protein (17.3 kDa) as output. The size of the output was verified by a control reaction, containing the intact hID1 gene (Figure 3, positive control lane). Even thought the yield of the output is very low (weak band, Figure 3), it points out the potential for feasibility of the model. Without a released diagnostic signal no self-ligation of the therapeutic gene could be detected (Figure 3, self-ligation control). The in vitro translation system is representative of intact transcription/translation machinery in the cell and the results from this experiment suggest that successful self-assembly of the computational gene and translation into a viable product (protein or anti-drug) might occur in living cells.

Bottom Line: The aberrant mutations trigger a cascade reaction: specific molecular markers as input are released and induce a spontaneous self-assembly of a wild type protein or peptide, while the mutational disease phenotype is silenced.We experimentally demostrated in in vitro translation system that a viable protein can be autonomously assembled.Our work demostrates the basic principles of computational genes and particularly, their potential to detect mutations, and as a response thereafter administer an output that suppresses the aberrant disease phenotype and/or restores the lost physiological function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Computer Technology, Hamburg University of Technology, Hamburg 21073, Germany. martinez-perez@tu-harburg.de

ABSTRACT

Background: A finite state machine manipulating information-carrying DNA strands can be used to perform autonomous molecular-scale computations at the cellular level.

Results: We propose a new finite state machine able to detect and correct aberrant molecular phenotype given by mutated genetic transcripts. The aberrant mutations trigger a cascade reaction: specific molecular markers as input are released and induce a spontaneous self-assembly of a wild type protein or peptide, while the mutational disease phenotype is silenced. We experimentally demostrated in in vitro translation system that a viable protein can be autonomously assembled.

Conclusion: Our work demostrates the basic principles of computational genes and particularly, their potential to detect mutations, and as a response thereafter administer an output that suppresses the aberrant disease phenotype and/or restores the lost physiological function.

Show MeSH