Photoregulation of DNA polymerase I (Klenow) with caged fluorescent oligodeoxynucleotides

Tang, Xinjing; Richards, Julia L.; Peritz, Adam E.; Dmochowski, Ivan J.

doi:10.1016/j.bmcl.2005.08.058

Cited by 33 publications

(27 citation statements)

References 19 publications

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“…[1,2] Various oligonucleotides protected with photocleavable groups (caged oligonucleotides) have been reported to control events such as transcription, [3][4][5] translation, [6] RNA interference, [7] DNAzyme reactions, [8,9] DNA replication, [10,11] aptamer binding, [12] higher-order structure formation, [13][14][15] and PCR. [16] In this study we have A C H T U N G T R E N N U N G examined the use of a caged nucleotide as a site-selective terminator of the polymerase reaction in PCR.…”

Section: Introductionmentioning

confidence: 99%

“…These results show that the strategy proposed in Scheme 1 is fairly reasonable. According to Dmochowski et al, [10] DNA polymerization by the Klenow fragment is more efficiently blocked when photocleavable DABSYL and fluorescein are bound to dC residues in a template next to each other. However, the promoting effect of T NPP (3) , the second T NPP in 4, is not very explicit in the present LACE-PCR, since only one T NPP in the primer (and thus in the template for polymerase reaction) is able to stop elongation to provide the HindIII terminus, at least under the conditions employed here.…”

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confidence: 98%

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Site‐Selective Blocking of PCR by a Caged Nucleotide Leading to Direct Creation of Desired Sticky Ends in The Products

et al. 2008

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In order to terminate the polymerase reaction at a desired position, a caged thymine derivative--4-O-[2-(2-nitrophenyl)propyl]thymine--was incorporated into PCR primers. In the PCR cycles, the elongation of the nascent strand (5'-->3' direction) by polymerase was site-selectively terminated at the 3'-side of T(NPP). Accordingly, predetermined protruding ends were obtained after the removal of the protecting group by short UVA irradiation. Recombinant vectors coding the GFP gene were successfully prepared by direct ligation of these light-assisted cohesive-ending PCR (LACE-PCR) products with scission fragments obtained by use either of restriction enzymes or of artificial restriction DNA cutters and were used for transformation of E. coli.

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Section: Introductionmentioning

confidence: 99%

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confidence: 98%

Site‐Selective Blocking of PCR by a Caged Nucleotide Leading to Direct Creation of Desired Sticky Ends in The Products

et al. 2008

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“…Our work began a few years ago with the goal of photo-regulating DNA function while minimally perturbing DNA structure (40,41). This related to controlling DNA-mRNA and mRNA-rRNA interactions and thus was an important step toward regulating gene expression with light.…”

Section: Development Of Caged Fluorescent Oligonucleotidesmentioning

confidence: 99%

Taking control of gene expression with light-activated oligonucleotides

Dmochowski

Tang

2007

BioTechniques

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The recent development of caged oligonucletides that are efficiently activated by ultraviolet (UV) light creates opportunities for regulating gene expression with very high spatial and temporal resolution. By selectively modulating gene activity, these photochemical tools will facilitate efforts to elucidate gene function and may eventually serve therapeutic aims. We demonstrate how the incorporation of a photocleavable blocking group within a DNA duplex can transiently arrest DNA polymerase activity. Indeed, caged oligonucleotides make it possible to control many different protein-oligonucleotide interactions. In related experiments, hybridization of a reverse complementary (antisense) oligodeoxynucleotide to target mRNA can inhibit translation by recruiting endogenous RNases or sterically blocking the ribosome. Our laboratory recently synthesized caged antisense oligonucleotides composed of phosphorothioated DNA or peptide nucleic acid (PNA). The antisense oligonucleotide, which was attached to a complementary blocking oligonucleotide strand by a photocleavable linker, was blocked from binding target mRNA. This provided a useful method for photomodulating hybridization of the antisense strand to target mRNA. Caged DNA and PNA oligonucleotides have proven effective at photoregulating gene expression in cells and zebrafish embryos.

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“…RNA is stable for at least 6 months under these conditions. Synthesis of a full-length caged pre-mRNA: branch region oligonucleotide synthesis TIMING 10 days total 24| An oligonucleotide representing the branch region of the PIP85B pre-mRNA 28 , 5¢-GGGUGCUGA*C-3¢ (A* represents 2¢-caged adenosine) is synthesized, deprotected and purified as described above for the hammerhead substrate (Steps [16][17][18][19][20][21][22][23] giving an B20% (200 nmol) yield of caged oligonucleotide as determined by UV absorption at 260 nm (a molar extinction coefficient of 124,100 M À1 for the caged RNA, including a value of 3,700 M À1 for the nitrobenzyl group, is used in this calculation) 27 .…”

Section: |mentioning

confidence: 99%

“…Subsequent to our initial report 3 , the site-specific caging of nucleic acids has been widely applied to regulate a variety of activities or processes including transcription, aptamer and DNAzyme activity, DNA replication and the formation of higher order RNA structures [12][13][14][15][16][17][18][19] . These experiments have involved either direct modification of pyrimidine or purine functionalities or of groups appended to them.…”

Section: Introductionmentioning

confidence: 99%