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Biosynthetic DNA-Protein Conjugation in Live Cells.
Biosynthetic DNA-Protein Conjugation in Live Cells.

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자료유형  
 학위논문
Control Number  
0017165068
International Standard Book Number  
9798346785286
Dewey Decimal Classification Number  
571.6
Main Entry-Personal Name  
Verma, Shivam.
Publication, Distribution, etc. (Imprint  
[S.l.] : Stanford University., 2024
Publication, Distribution, etc. (Imprint  
Ann Arbor : ProQuest Dissertations & Theses, 2024
Physical Description  
103 p.
General Note  
Source: Dissertations Abstracts International, Volume: 86-06, Section: B.
General Note  
Advisor: Bertozzi, Carolyn.
Dissertation Note  
Thesis (Ph.D.)--Stanford University, 2024.
Summary, Etc.  
요약Conjugating DNA to protein has accelerated biotechnologies including high-throughput protein screening, super-resolution microscopy, and ultrasensitive diagnostics. Current approaches to synthesize DNA-protein conjugates (DPCs) are, however, limited in throughput. Here, we present a new method to generate DPCs entirely with biosynthetic machinery in live cells. We synthesize DPCs via fusion to bacterial HUH endonucleases, tyrosine autoconjugases that react with single-stranded DNA (ssDNA). The reactant ssDNA is produced in cells by repurposing bacterial retrons, specialized reverse transcriptases paired with template RNA. We start by reacting HUH endonucleases with retron ssDNA in live E. coli to produce biosynthetic DPCs for the first time. By discovering key factors degrading designer DPCs, we also study how native DPCs are processed endogenously. Next, we show that HUH endonucleases and retrons are active in the mammalian cell cytoplasm. Combining these pathways in human cells presents exciting opportunities to generate well-folded and post-translationally modified human DPCs. We believe that, in the near future, biosynthetic DPC synthesis could be developed into a high-throughput, pooled conjugation platform for diverse impact in future biotechnologies.
Subject Added Entry-Topical Term  
Cell death.
Subject Added Entry-Topical Term  
Acids.
Subject Added Entry-Topical Term  
Antibodies.
Subject Added Entry-Topical Term  
Mutation.
Subject Added Entry-Topical Term  
Bacteria.
Subject Added Entry-Topical Term  
Scientific imaging.
Subject Added Entry-Topical Term  
E coli.
Subject Added Entry-Topical Term  
Genotype & phenotype.
Subject Added Entry-Topical Term  
Cell culture.
Subject Added Entry-Topical Term  
Annealing.
Subject Added Entry-Topical Term  
Protein synthesis.
Subject Added Entry-Topical Term  
Plasmids.
Subject Added Entry-Topical Term  
Mass spectrometry.
Subject Added Entry-Topical Term  
Information storage.
Subject Added Entry-Topical Term  
Empowerment.
Subject Added Entry-Topical Term  
Microscopy.
Subject Added Entry-Topical Term  
Genetic engineering.
Subject Added Entry-Topical Term  
DNA polymerase.
Subject Added Entry-Topical Term  
Toxins.
Subject Added Entry-Topical Term  
Analytical chemistry.
Subject Added Entry-Topical Term  
Biochemistry.
Subject Added Entry-Topical Term  
Bioengineering.
Subject Added Entry-Topical Term  
Cellular biology.
Subject Added Entry-Topical Term  
Genetics.
Added Entry-Corporate Name  
Stanford University.
Host Item Entry  
Dissertations Abstracts International. 86-06B.
Electronic Location and Access  
로그인을 한후 보실 수 있는 자료입니다.
Control Number  
joongbu:655758

MARC

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■035    ▼a(MiAaPQ)Stanfordyx012pw2225
■040    ▼aMiAaPQ▼cMiAaPQ
■0820  ▼a571.6
■1001  ▼aVerma,  Shivam.
■24510▼aBiosynthetic  DNA-Protein  Conjugation  in  Live  Cells.
■260    ▼a[S.l.]▼bStanford  University.  ▼c2024
■260  1▼aAnn  Arbor▼bProQuest  Dissertations  &  Theses▼c2024
■300    ▼a103  p.
■500    ▼aSource:  Dissertations  Abstracts  International,  Volume:  86-06,  Section:  B.
■500    ▼aAdvisor:  Bertozzi,  Carolyn.
■5021  ▼aThesis  (Ph.D.)--Stanford  University,  2024.
■520    ▼aConjugating  DNA  to  protein  has  accelerated  biotechnologies  including  high-throughput  protein  screening,  super-resolution  microscopy,  and  ultrasensitive  diagnostics.  Current  approaches  to  synthesize  DNA-protein  conjugates  (DPCs)  are,  however,  limited  in  throughput.  Here,  we  present  a  new  method  to  generate  DPCs  entirely  with  biosynthetic  machinery  in  live  cells.  We  synthesize  DPCs  via  fusion  to  bacterial  HUH  endonucleases,  tyrosine  autoconjugases  that  react  with  single-stranded  DNA  (ssDNA).  The  reactant  ssDNA  is  produced  in  cells  by  repurposing  bacterial  retrons,  specialized  reverse  transcriptases  paired  with  template  RNA.  We  start  by  reacting  HUH  endonucleases  with  retron  ssDNA  in  live  E.  coli  to  produce  biosynthetic  DPCs  for  the  first  time.  By  discovering  key  factors  degrading  designer  DPCs,  we  also  study  how  native  DPCs  are  processed  endogenously.  Next,  we  show  that  HUH  endonucleases  and  retrons  are  active  in  the  mammalian  cell  cytoplasm.  Combining  these  pathways  in  human  cells  presents  exciting  opportunities  to  generate  well-folded  and  post-translationally  modified  human  DPCs.  We  believe  that,  in  the  near  future,  biosynthetic  DPC  synthesis  could  be  developed  into  a  high-throughput,  pooled  conjugation  platform  for  diverse  impact  in  future  biotechnologies.
■590    ▼aSchool  code:  0212.
■650  4▼aCell  death.
■650  4▼aAcids.
■650  4▼aAntibodies.
■650  4▼aMutation.
■650  4▼aBacteria.
■650  4▼aScientific  imaging.
■650  4▼aE  coli.
■650  4▼aGenotype  &  phenotype.
■650  4▼aCell  culture.
■650  4▼aAnnealing.
■650  4▼aProtein  synthesis.
■650  4▼aPlasmids.
■650  4▼aMass  spectrometry.
■650  4▼aInformation  storage.
■650  4▼aEmpowerment.
■650  4▼aMicroscopy.
■650  4▼aGenetic  engineering.
■650  4▼aDNA  polymerase.
■650  4▼aToxins.
■650  4▼aAnalytical  chemistry.
■650  4▼aBiochemistry.
■650  4▼aBioengineering.
■650  4▼aCellular  biology.
■650  4▼aGenetics.
■690    ▼a0486
■690    ▼a0487
■690    ▼a0202
■690    ▼a0379
■690    ▼a0369
■71020▼aStanford  University.
■7730  ▼tDissertations  Abstracts  International▼g86-06B.
■790    ▼a0212
■791    ▼aPh.D.
■792    ▼a2024
■793    ▼aEnglish
■85640▼uhttp://www.riss.kr/pdu/ddodLink.do?id=T17165068▼nKERIS▼z이  자료의  원문은  한국교육학술정보원에서  제공합니다.

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