ProducefullyRNaseA-resistantRNAthatisperfectlysuitedforRNAaptamersynthesis,aswellasantisenseRNAandRNAinterference(RNAi)experiments.
ProtecttranscribedRNAfromRNAsedegradation- ProducehighRNAyields
- SelectandoptimizeRNAaptamersusingSELEXscreening
- SeeFAQ
TheDuraScribe®T7TranscriptionKit*isaninvitrotranscriptionkitthatproducesRNA-calledDuraScript®RNA-thatiscompletelyresistanttoRNaseAdigestion.
TheDuraScribeT7TranscriptionKitincludesDuraScribeT7RNAPolymerase,anenhancedformulationoftheY639Fmutant1ofT7RNAPolymerasethatefficientlyincorporates2´-Fluorine-CTP(2´-F-dCTP),2´-Fluorine-UTP(2´-F-dUTP),ATPandGTPintoRNase-resistantRNAtranscriptscalledDuraScriptRNA(Figure1).TheDuraScribeT7RNAPolymeraseusesthesameT7promotersasthewild-typeT7RNAPolymerase.
Applications
DuraScriptRNAhasdemonstratedadvantagesforRNAaptamersynthesis,andribozyme,RNAinterference(RNAi)andantisenseRNAstudies.
Features
- DuraScriptRNAisresistanttoRnaseAandDnase
- HighYieldsofDuraScriptRNA
- T7invitrotranscriptionreations.DuraScribeT7RNAPolymeraseusesthesameT7promotersasthewildtypeT7RNAPolymerase.
- IdealforRNAaptamerstudies.NumerouscitationsusingtheDuraScribeKitwithRNAaptamers
- SELEX-compatIBLe.DuraScriptRNAcanbeusedinSELEXselectionprocesses.
- MakelongorshortDuraScriptRNA.Double-strandoligos,linearizedplasmids,andPCRproductswithaT7polymerasepromotercanbetranscribed.
Table1.YieldofDuraScript®RNAfromaDuraScribe®Kitreaction.Onemicrogramofa3-KbDNAtemplatewaslinearizedatdifferentsitesandthentranscribedinaDuraScribeT7TranscriptionKitreactionfor4hours.TheyieldofDuraScriptRNAproducedfromeachtemplateisshowninmicrograms(µg)andinpicomoles(pmol).
| SizeofDuraScriptRNAproduced | DuraScriptRNAYield(µg) | DuraScriptRNAYield(pmol) |
| 2600nts | 100µg | 116pmol |
| 1400nts | 58µg | 124pmol |
| 330nts | 18µg | 164pmol |
| 88nts | 9µg | 307pmol |
Figure1(clicktoenlarge).TheDuraScribe®T7RNAPolymeraseefficientlyincorporates2´-F-dCTPand2´-F-dUTPintofull-lengthDuraScript®RNA.Thepresenceofthefluorineatthe2´-positionofthe2´-F-dCand2´-F-dUnucleotidespreventsdigestionbyRnaseA. |
Figure2.YieldofRNAfromaDuraScribe®T7Transcriptionreaction.Astandardreaction(4-6hours)produced40-60µgofa1.4-kbDuraScript®RNA. |
Figure3.DuraScript®RNAisresistanttoRnaseAdigestion.A1.4-kbstandardRNAtranscriptanda1.4-kbDuraScriptRNAtranscriptwereeachincubatedwith1UofhighlypurifiedRnaseAfor30minutes.ThestandardRNAtranscriptwascompletelydegradedwhiletheDuraScriptRNAtranscriptremainedintact.LaneM,sizeladder;lane1,1.4-kbstandardRNAtranscript;lane2,standardRNAafterRnaseAtreatment;lane3,1.4-kbDuraScriptRNA;lane4,DuraScriptRNAafterRnaseAtreatment. |
Figure4.DuraScript®RNAiscompletelyresistantto"finger"nucleases.A1.4-kbstandardRNAtranscriptanda1.4-kbDuraScriptRNAtranscriptwereproducedusingsterilewaterorwaterthathadbeencontaminatedbyexposuretothehandsofatestsubject.ThestandardRNAshowsextensivedegradationfrom"finger"nucleasesinthecontaminatedwaterwhiletheDuraScriptRNAremainsfullyintact.M,Sizeladder;Lane1,StandardRNAtranscript;Lane2,StandardRNAafter"finger"nucleaseexposure;Lane3,DuraScriptRNA;Lane4,DuraScriptRNAafter"finger"nucleaseexposure. |
*Coveredbyissuedand/orpendingpatents.
References
- Sousa,R.andPADIlla,R.(1995)EMBOJ.14:184609-4621.
