Nanocircles - Revision history

Dajordan at 19:25, 8 December 2007

2007-12-08T19:25:35Z

Dajordan: /* Importance of Ribozyme Secondary Structure */

2007-12-08T19:24:18Z

‎Importance of Ribozyme Secondary Structure

Dajordan: /* Importance of Ribozyme Secondary Structure */

2007-12-06T21:32:40Z

‎Importance of Ribozyme Secondary Structure

Dajordan: /* Importance of Ribozyme Secondary Structure */

2007-12-06T21:32:15Z

‎Importance of Ribozyme Secondary Structure

Dajordan at 21:30, 6 December 2007

2007-12-06T21:30:47Z

Dajordan: /* Activity of Nanocircle Vector in ''E. coli'' */

2007-12-06T21:27:54Z

‎Activity of Nanocircle Vector in ''E. coli''

Dajordan at 20:03, 6 December 2007

2007-12-06T20:03:40Z

Dajordan: /* = */

2007-12-06T19:51:33Z

‎=

Dajordan at 19:51, 6 December 2007

2007-12-06T19:51:11Z

Dajordan: /* Results */

2007-12-06T19:38:30Z

‎Results

@@ Line 44: / Line 44: @@
 === Activity of Nanocircle Vector in ''E. coli'' ===
-To test whether "mar"A can be used in another system, the ribozyme was encoded in the upstream end of a CAT gene. When "mar"A RNA is cleaved "in trans," down-regulation of CAT activity would occur. Thus, not only was CAT-activity downregulated by the marA nanocircle vector, but it also showed a concentration dependence (Figure 5A and 5B). ''Image Permission Pending.''
+To test whether ''mar''A can be used in another system, the ribozyme was encoded in the upstream end of a CAT gene. When ''mar''A RNA is cleaved ''in trans,'' down-regulation of CAT activity would occur. Thus, not only was CAT-activity downregulated by the marA nanocircle vector, but it also showed a concentration dependence (Figure 5A and 5B). ''Image Permission Pending.''
 http://www.pnas.org/content/vol99/issue1/images/medium/pq0125890007.gif

@@ Line 56: / Line 56: @@
 http://www.pnas.org/content/vol99/issue1/images/medium/pq0125890008.gif
-Figure 6. (A) Sequences and predicted secondary structures of the monomer ribozymes: active and inactive marA, and short marA. The inactivating A  C mutation is boxed in the first ribozyme. (B) Effect of 10 µM various nanocircle vectors on the inhibition of CAT activity. The plotted data were averaged from three independent experiments (Olmichi et al., 2002). ''Image Permission Pending.''
+Figure 6. "(A) Sequences and predicted secondary structures of the monomer ribozymes: active and inactive marA, and short marA. The inactivating A  C mutation is boxed in the first ribozyme. (B) Effect of 10 µM various nanocircle vectors on the inhibition of CAT activity. The plotted data were averaged from three independent experiments" (Olmichi et al., 2002). ''Image Permission Pending.''
 == Continuing Research ==

@@ Line 52: / Line 52: @@
 === Importance of Ribozyme Secondary Structure ===
-The investigators used variations of ''mar''A that either lacked a ''trans'' cleavage point in the inactive ''mar''A or was missing a significant part of the ''mar''A in the short ''mar''A. This simply shows the only ''mar''A has a drastic decrease in % CAT activity, underscoring the importance of secondary structures in ribozymes. On the other hand, the inactive ''mar''A exhibited a signifant repression in the gene expression, which suggests that some of the ability that ''mar''A has in down-regulation of CAT may be attributed to http://en.wikipedia.org/wiki/Antisense_mRNA anti-sense] activity.
+The investigators used variations of ''mar''A that either lacked a ''trans'' cleavage point in the inactive ''mar''A or was missing a significant part of the ''mar''A in the short ''mar''A. This simply shows the only ''mar''A has a drastic decrease in % CAT activity, underscoring the importance of secondary structures in ribozymes. On the other hand, the inactive ''mar''A exhibited a signifant repression in the gene expression, which suggests that some of the ability that ''mar''A has in down-regulation of CAT may be attributed to [http://en.wikipedia.org/wiki/Antisense_mRNA anti-sense] activity.
 http://www.pnas.org/content/vol99/issue1/images/medium/pq0125890008.gif

@@ Line 52: / Line 52: @@
 === Importance of Ribozyme Secondary Structure ===
-The investigators used variations of ''mar''A that either lacked a ''trans'' cleavage point in the inactive ''mar" A or was missing a significant part of the ''mar''A in the short ''mar''A. This simply shows the only ''mar''A has a drastic decrease in % CAT activity, underscoring the importance of secondary structures in ribozymes. On the other hand, the inactive ''mar''A exhibited a signifant repression in the gene expression, which suggests that some of the ability that ''mar''A has in down-regulation of CAT may be attributed to http://en.wikipedia.org/wiki/Antisense_mRNA anti-sense] activity.
+The investigators used variations of ''mar''A that either lacked a ''trans'' cleavage point in the inactive ''mar''A or was missing a significant part of the ''mar''A in the short ''mar''A. This simply shows the only ''mar''A has a drastic decrease in % CAT activity, underscoring the importance of secondary structures in ribozymes. On the other hand, the inactive ''mar''A exhibited a signifant repression in the gene expression, which suggests that some of the ability that ''mar''A has in down-regulation of CAT may be attributed to http://en.wikipedia.org/wiki/Antisense_mRNA anti-sense] activity.
 http://www.pnas.org/content/vol99/issue1/images/medium/pq0125890008.gif

@@ Line 16: / Line 16: @@
 http://www.pnas.org/content/vol0/issue2001/images/data/012589099/DC1/5890Fig9.gif
-Figure 1A. Structrure of single-stranded DNA nanocircle composed of 63 nucleotides encoding a hammerhead ribozyme and 41 nucleotides of randomized sequences.  The randomized sequence acts as a promoter that allow initiation with RNA polymerase to transcribe the nanocircle.
+Figure 1. (A) Structrure of single-stranded DNA nanocircle composed of 63 nucleotides encoding a hammerhead ribozyme and 41 nucleotides of randomized sequences.  The randomized sequence acts as a promoter that allow initiation with RNA polymerase to transcribe the nanocircle. (B) Schematic of artificial ribozymes using error prone reverse transcripase PCR.
+''Image Permission Pending.''
 == Results ==
@@ Line 26: / Line 25: @@
   http://www.pnas.org/content/vol99/issue1/images/medium/pq0125890002.gif
-Figure 2.   "Improvement of transcription activity over successive rounds of in vitro selection. RNA amount was measured for each successive population at 37°C after 1.5 h. Dark and light bars correspond to the relative RNA amounts (>80-nt product) for the successive population with and without ligation, respectively" (Olmichi et al. 2002).
+Figure 2.   "Improvement of transcription activity over successive rounds of in vitro selection. RNA amount was measured for each successive population at 37°C after 1.5 h. Dark and light bars correspond to the relative RNA amounts (>80-nt product) for the successive population with and without ligation, respectively" (Olmichi et al., 2002). ''Image Permission Pending.''
 === Production of Monomeric Ribozymes ===
@@ Line 33: / Line 32: @@
 http://www.pnas.org/content/vol99/issue1/images/medium/pq0125890004.gif
-Figure 3. "Selected circular DNA motifs engender RNA synthesis in vitro with E. coli RNAP. (A) Autoradiogram of denaturing 10% polyacrylamide gel showing in vitro transcription of the 103-nt initial library, a control 63-nt nanocircle lacking the randomized domain, and selected individual nanocircles E1, E15, and E38 (after 1.5 h). (B) The relative total RNA amounts (all lengths >80 nt) for the 103-nt initial library, 63-nt nanocircle lacking the randomized domain, and E1, E15, and E38. (C) Time course of the production of monomeric ribozyme for the 103-nt initial library, 63-nt nanocircle lacking the randomized domain, E1, and E15" (Ohmichi et al. 2002).
+Figure 3. "Selected circular DNA motifs engender RNA synthesis in vitro with E. coli RNAP. (A) Autoradiogram of denaturing 10% polyacrylamide gel showing in vitro transcription of the 103-nt initial library, a control 63-nt nanocircle lacking the randomized domain, and selected individual nanocircles E1, E15, and E38 (after 1.5 h). (B) The relative total RNA amounts (all lengths >80 nt) for the 103-nt initial library, 63-nt nanocircle lacking the randomized domain, and E1, E15, and E38. (C) Time course of the production of monomeric ribozyme for the 103-nt initial library, 63-nt nanocircle lacking the randomized domain, E1, and E15" (Ohmichi et al., 2002). ''Image Permission Pending.''
 === Specificity and Modularity of Ribozyme ===
@@ Line 41: / Line 40: @@
 http://www.pnas.org/content/vol99/issue1/images/medium/pq0125890006.gif
-Figure 4.  "Assessment of transplantability of E15 selected motif to a new nanocircle encoding ''mar''A ribozyme. Autoradiogram of denaturing 10% polyacrylamide gel showing in vitro transcription of the 103-nt initial library, nanocircle E15, the new marA nanocircle, marA nanocircle with inactivated ribozyme, and two 63-nt nanocircle controls" (Ohmichi et al. 2002).
+Figure 4.  "Assessment of transplantability of E15 selected motif to a new nanocircle encoding ''mar''A ribozyme. Autoradiogram of denaturing 10% polyacrylamide gel showing in vitro transcription of the 103-nt initial library, nanocircle E15, the new marA nanocircle, marA nanocircle with inactivated ribozyme, and two 63-nt nanocircle controls" (Ohmichi et al., 2002). ''Image Permission Pending.''
 === Activity of Nanocircle Vector in ''E. coli'' ===
-To test whether "mar"A can be used in another system, the ribozyme was encoded in the upstream end of a CAT gene. When "mar"A RNA is cleaved "in trans," down-regulation of CAT activity would occur. Thus, not only was CAT-activity downregulated by the marA nanocircle vector, but it also showed a concentration dependence (Figur 5A and 5B)
+To test whether "mar"A can be used in another system, the ribozyme was encoded in the upstream end of a CAT gene. When "mar"A RNA is cleaved "in trans," down-regulation of CAT activity would occur. Thus, not only was CAT-activity downregulated by the marA nanocircle vector, but it also showed a concentration dependence (Figure 5A and 5B). ''Image Permission Pending.''
 http://www.pnas.org/content/vol99/issue1/images/medium/pq0125890007.gif
-Figure 5. "Effect of nanocircle vectors on the inhibition of CAT activity. (A) Thin-layer chromatogram showing levels of CAT expressed in the presence of 10 µM marA vector and E15 vector. The control lane is with no nanocircle vector. (B) Concentration dependence of down-regulation of CAT activity with ''mar''A vector" (Olmichi et al. 2002).
+Figure 5. "Effect of nanocircle vectors on the inhibition of CAT activity. (A) Thin-layer chromatogram showing levels of CAT expressed in the presence of 10 µM marA vector and E15 vector. The control lane is with no nanocircle vector. (B) Concentration dependence of down-regulation of CAT activity with ''mar''A vector" (Olmichi et al., 2002). ''Image Permission Pending.''
 === Importance of Ribozyme Secondary Structure ===
@@ Line 57: / Line 56: @@
 http://www.pnas.org/content/vol99/issue1/images/medium/pq0125890008.gif
-Figure 6. (A) Sequences and predicted secondary structures of the monomer ribozymes: active and inactive marA, and short marA. The inactivating A  C mutation is boxed in the first ribozyme. (B) Effect of 10 µM various nanocircle vectors on the inhibition of CAT activity. The plotted data were averaged from three independent experiments (Olmichi et al. 2002)
+Figure 6. (A) Sequences and predicted secondary structures of the monomer ribozymes: active and inactive marA, and short marA. The inactivating A  C mutation is boxed in the first ribozyme. (B) Effect of 10 µM various nanocircle vectors on the inhibition of CAT activity. The plotted data were averaged from three independent experiments (Olmichi et al., 2002). ''Image Permission Pending.''
 == Continuing Research ==

← Older revision		Revision as of 21:27, 6 December 2007
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	=== Activity of Nanocircle Vector in ''E. coli'' ===		=== Activity of Nanocircle Vector in ''E. coli'' ===
		+
		+	To test whether "mar"A can be used in another system, the ribozyme was encoded in the upstream end of a CAT gene. When "mar"A RNA is cleaved "in trans," down-regulation of CAT activity would occur. Thus, not only was CAT-activity downregulated by the marA nanocircle vector, but it also showed a concentration dependence (Figur 5A and 5B)

	http://www.pnas.org/content/vol99/issue1/images/medium/pq0125890007.gif		http://www.pnas.org/content/vol99/issue1/images/medium/pq0125890007.gif

@@ Line 1: / Line 1: @@
 Nanocircles are small circular single-stranded DNA that can be transcribed by phage and bacterial RNA polymerases. These plasmid-like structures were originally developed by [http://www.stanford.edu/group/kool/kool.htm Eric T. Kool]'s lab. The new technology uses a method called rolling circle transcription (RCT) to encode hammerhead, hairpin and hepatitis delta ribozymes.
-[http://www.stanford.edu/group/kool/research.htm Rolling Circle Animation] Click on Rolling Circles & Artificial Telomeres
+[http://www.stanford.edu/group/kool/research/rollingcircle.swf Rolling Circle Animation]
 == Goals ==