DNA sequencing using reversible terminators, as one sequencing by synthesis strategy, has garnered a great deal of interest due to its popular application in the second-generation high-throughput DNA sequencing techno...DNA sequencing using reversible terminators, as one sequencing by synthesis strategy, has garnered a great deal of interest due to its popular application in the second-generation high-throughput DNA sequencing technology. In this review, we provided its history of develop- ment, classification, and working mechanism of this technology. We also outlined the screening strategies for DNA polymerases to accommodate the reversible terminators as substrates during polymerization; particularly, we introduced the ＂REAP＂ method developed by us. At the end of this review, we discussed current limitations of this approach and provided potential solutions to extend its application.展开更多
According to the information on the hammerhead structure of ribozyme known so far,the cleavage occurs at the 3′-side of the specific position.The 3′-half molecule of yeast alanyl tRNA with GTΨ sequence is chosen as...According to the information on the hammerhead structure of ribozyme known so far,the cleavage occurs at the 3′-side of the specific position.The 3′-half molecule of yeast alanyl tRNA with GTΨ sequence is chosen as substrate.Consequently,a 38-mer ribozyme is designed by Haseloff and Gerlach mod- el.In the presence of Mg^(2+),the expected cleavage really occurs at the 3′-side of GTΨ in vitro.The Michaelis-Menten kinetic parameters for the reaction are:K_m,6-7 μmol/L,k_(cat),3.4×10^(-3)/min.At the same time,a 17-mer substrate analogue has been synthesized,with a GUU-sequence instead of GTΨ men- tioned above.The steady-state kinetic analyses reveal that K_m values are nearly the same for both sub- strates.But k_(cat) values decrease by 294 fold.These observations demonstrate that the modified nucleotide Ψ can be the cleavage site of hammerhead structure of ribozyme with a much lower k_(cat).展开更多
The role of base modification in yeast tRNAAl(?) function in protein synthesis was examined by the use of unmodified tRNA analogues. Unmodified full length tRNAs, 5'-half tRNAs (nucleotides 1-35) and 3'-half t...The role of base modification in yeast tRNAAl(?) function in protein synthesis was examined by the use of unmodified tRNA analogues. Unmodified full length tRNAs, 5'-half tRNAs (nucleotides 1-35) and 3'-half tRNAs (nucleotides 37-75) were transcribed in vitro using T7-RNA polymerase. Unmodified tRNA half molecules were joined to normally modified half molecules (5'-half, nucleotides 1-36; 3'-half, nucleotides 36-75) by T4-RNA ligase. Using this method, we synthesized three analogues of yeast tRNAAl(?): (i) tRNAAl(?) which lacks base modifications in the 5'-half of the molecule; (ii) tRNAAl(?) which lacks base modifications in the 3'-half of the molecule; and (iii) tRNAAla completely lacking base modifications. We determined the biological activities of these analogues. In rat aminoacyl-tRNA synthetase reactions, the alanine acceptance activity decreased by 52%, 79% and 85% when modified bases were absent from the 5'-half molecule, the 3'-half molecule or the total molecule, respectively. In rabbit reticulocyte lysates, alanine incorporation into proteins decreased by 3%, 57% and 47% for tRNA analogues lacking modified bases in the 5'-half, and 3'-half or the entire tRNA, respectively. These results suggest that the modified nucleotides, especially in the 3'-half molecule, plays an important role in yeast tRNAAla activity.展开更多
A thirty-nine nucleotide fragment (nucleotides 38—76) of yeast alanyl tRNA was prepared by using calf spleen phosphodiesterase to delete C_(36)m^1I_(37) from the 3′-half molecule of this tRNA under controlled condit...A thirty-nine nucleotide fragment (nucleotides 38—76) of yeast alanyl tRNA was prepared by using calf spleen phosphodiesterase to delete C_(36)m^1I_(37) from the 3′-half molecule of this tRNA under controlled conditions. Analogs of yeast alanyl tRNA with A, G or C instead of m^1I_(37) were synthesized and their biological activities determined. The results indicated that the aminoacylation activity of these analogs was not affected in the rat liver aminoacyl-tRNA synthetase system, compared with natural yeast alanyl tRNA. However, the incorporation activity (i. e. the activity of transferring alanine into proteins) of these analogs was significantly reduced to 20—30% of that of natural yeast alanyl tRNA in the rabbit reticulocyte lysate cell-free protein synthesizing system.展开更多
基金the National Natural Science Foundation of China (Grant No. 31270846)the Chinese Academy of Sciences "100-Talent Program" for the support of this work
文摘DNA sequencing using reversible terminators, as one sequencing by synthesis strategy, has garnered a great deal of interest due to its popular application in the second-generation high-throughput DNA sequencing technology. In this review, we provided its history of develop- ment, classification, and working mechanism of this technology. We also outlined the screening strategies for DNA polymerases to accommodate the reversible terminators as substrates during polymerization; particularly, we introduced the ＂REAP＂ method developed by us. At the end of this review, we discussed current limitations of this approach and provided potential solutions to extend its application.
基金the National Natural Science Foundation of China National High Technology Program
文摘According to the information on the hammerhead structure of ribozyme known so far,the cleavage occurs at the 3′-side of the specific position.The 3′-half molecule of yeast alanyl tRNA with GTΨ sequence is chosen as substrate.Consequently,a 38-mer ribozyme is designed by Haseloff and Gerlach mod- el.In the presence of Mg^(2+),the expected cleavage really occurs at the 3′-side of GTΨ in vitro.The Michaelis-Menten kinetic parameters for the reaction are:K_m,6-7 μmol/L,k_(cat),3.4×10^(-3)/min.At the same time,a 17-mer substrate analogue has been synthesized,with a GUU-sequence instead of GTΨ men- tioned above.The steady-state kinetic analyses reveal that K_m values are nearly the same for both sub- strates.But k_(cat) values decrease by 294 fold.These observations demonstrate that the modified nucleotide Ψ can be the cleavage site of hammerhead structure of ribozyme with a much lower k_(cat).
基金Project supported by the National Natural Science Foundation of China.
文摘The role of base modification in yeast tRNAAl(?) function in protein synthesis was examined by the use of unmodified tRNA analogues. Unmodified full length tRNAs, 5'-half tRNAs (nucleotides 1-35) and 3'-half tRNAs (nucleotides 37-75) were transcribed in vitro using T7-RNA polymerase. Unmodified tRNA half molecules were joined to normally modified half molecules (5'-half, nucleotides 1-36; 3'-half, nucleotides 36-75) by T4-RNA ligase. Using this method, we synthesized three analogues of yeast tRNAAl(?): (i) tRNAAl(?) which lacks base modifications in the 5'-half of the molecule; (ii) tRNAAl(?) which lacks base modifications in the 3'-half of the molecule; and (iii) tRNAAla completely lacking base modifications. We determined the biological activities of these analogues. In rat aminoacyl-tRNA synthetase reactions, the alanine acceptance activity decreased by 52%, 79% and 85% when modified bases were absent from the 5'-half molecule, the 3'-half molecule or the total molecule, respectively. In rabbit reticulocyte lysates, alanine incorporation into proteins decreased by 3%, 57% and 47% for tRNA analogues lacking modified bases in the 5'-half, and 3'-half or the entire tRNA, respectively. These results suggest that the modified nucleotides, especially in the 3'-half molecule, plays an important role in yeast tRNAAla activity.
基金Project supported by the National Natural Science Foundation of China. Part Ⅱ of this series is Ref. [3].
文摘A thirty-nine nucleotide fragment (nucleotides 38—76) of yeast alanyl tRNA was prepared by using calf spleen phosphodiesterase to delete C_(36)m^1I_(37) from the 3′-half molecule of this tRNA under controlled conditions. Analogs of yeast alanyl tRNA with A, G or C instead of m^1I_(37) were synthesized and their biological activities determined. The results indicated that the aminoacylation activity of these analogs was not affected in the rat liver aminoacyl-tRNA synthetase system, compared with natural yeast alanyl tRNA. However, the incorporation activity (i. e. the activity of transferring alanine into proteins) of these analogs was significantly reduced to 20—30% of that of natural yeast alanyl tRNA in the rabbit reticulocyte lysate cell-free protein synthesizing system.
