fluorescent proteins with ties that bind

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NEWS AND VIEWS Harnessing the immune system’s ability to generate high-affinity binding pockets has proven to be an enormously important tech- nology for biomedical science. The ability to generate protein-specific affinity reagents has formed the basis of thousands of types of assays for purifying, detecting and probing the molecular components encoded by genomes—assays that have touched virtually every facet of modern molecular biology. In this issue, Bradbury and colleagues 1 demonstrate a powerful new hybrid reagent: intrinsically fluorescent proteins that sport the complementarity-determining regions (CDRs) normally found in antibody variable domains. This new class of reagent, termed a ‘fluorobody’, thus marries one of the greatest recent boons to cell biology and physiology research, fluorescent proteins (such as green fluorescent proteins or GFPs), with the ability to select for high-affinity binding to a target of choice. Although antibodies have proven pow- erful and versatile, they require a secondary detection scheme to be useful, which typically requires the use of at least two other reagents. With fluorobodies the detection scheme is built in. The structure of an intact antibody is com- plex, and high-affinity binding to antigens arises from the detailed crevices, surfaces and pockets formed by the apposition of six CDRs, three from each of two different gene prod- ucts, the heavy- and light-chain variable dom- ains, respectively. Considerable efforts have gone into creating reagents that maintain high- affinity binding and large diversity but are more amenable to high-throughput expres- sion systems. Single-chain antibodies (scFvs) maintain the diversity and high-affinity capacity of intact antibodies in a simpler pack- age, whereby the variable domains of the light and heavy chains are expressed as a single gene product connected by a flexible linker 2 . Recently, this technology was simplified even further with the creation of single-domain antibody (IDab) libraries, which, although combinatorially less complex than scFvs, maintain excellent antigen-binding proper- ties 3 . These schemes have allowed the creation of high-diversity libraries that can be com- bined with efficient expression and selection schemes to identify high-affinity reagents 4,5 that work well both inside and outside cells. Although in principle the gene for GFP could be fused directly into either scFv or IDab libraries to provide a built-in detection scheme, this would significantly increase the size of the reagent and would be likely to decrease the efficiency of production because GFPs normally fold in the intracellular space, whereas antibodies normally fold within lumenal domains of intracellular organelles. Instead, Bradbury and colleagues take advan- tage of the fact that GFPs arise from a novel three-dimensional fold called a β-can formed by 11 β-strands wrapping into a cylinder (Fig. 1a). In intact antibodies, the CDRs are brought together at the top of a cylindrical surface defined by two β-sheets, one from each of the heavy- and light-chain variable domains (Fig. 1b). The similarity in these structures suggested that GFPs themselves might provide an appropriate scaffold to allow CDRs to define a high-affinity binding arr- angement. As the various β-strands in GFP also have connecting loops at the top of the cylinder, Bradbury and colleagues replaced four of the GFP loops (Fig. 1c) with a library of the CDR with the greatest diversity (CDR3); and thus fluorobodies were born. Expression of only a modestly diverse library (10 7 elements) with a phage-display system led to the isolation of numerous fluo- robodies against various antigens with affini- ties as high as the nanomolar range. The intrinsic fluorescence itself is very useful dur- ing the selection and purification procedures, as the binding properties are inherently linked to the stability of the protein and therefore its Timothy A. Ryan is in the Department of Biochemistry, Joan and Sanford I. Weill Medical College, Cornell University, 525 E. 68th Street, New York, New York 10021, USA. e-mail: [email protected] Fluorescent proteins with ties that bind Timothy A Ryan Antigen-binding fluorescent proteins provide a powerful tool for immunodetection-based assays. NATURE BIOTECHNOLOGY VOLUME 21 NUMBER 12 DECEMBER 2003 1447 HCDR1 HCDR3 HCDR2 LCDR1 LCDR3 LCDR2 a c b Figure 1 The making of a fluorobody. (a) β-can structure of GFP made up of 11 β-strands into a cylinder. At the top of the can, loop regions join the strands. (b) The antigen-binding site of antibodies is made by two sets of β-sheets from both heavy and light chains organized in a cylindrical surface. The hypervariable regions, the loops at the top of this cylindrical arrangement (HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3), define the antigen-binding site. (c) In a fluorobody, four of the loops of GFP are replaced by a library of HDCR3. These loops define a new binding site using the β-can as a scaffold rather than the light chain– and heavy chain–defined β-cylinder. Bob Crimi © 2003 Nature Publishing Group http://www.nature.com/naturebiotechnology

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N E W S A N D V I E W S

Harnessing the immune system’s ability togenerate high-affinity binding pockets hasproven to be an enormously important tech-nology for biomedical science. The ability to generate protein-specific affinity reagentshas formed the basis of thousands of types of assays for purifying, detecting and probingthe molecular components encoded bygenomes—assays that have touched virtuallyevery facet of modern molecular biology.In this issue, Bradbury and colleagues1

demonstrate a powerful new hybrid reagent:intrinsically fluorescent proteins that sport the complementarity-determining regions(CDRs) normally found in antibody variabledomains. This new class of reagent, termed a‘fluorobody’, thus marries one of the greatestrecent boons to cell biology and physiologyresearch, fluorescent proteins (such as greenfluorescent proteins or GFPs), with the abilityto select for high-affinity binding to a target ofchoice. Although antibodies have proven pow-erful and versatile, they require a secondarydetection scheme to be useful, which typicallyrequires the use of at least two other reagents.With fluorobodies the detection scheme isbuilt in.

The structure of an intact antibody is com-plex, and high-affinity binding to antigensarises from the detailed crevices, surfaces andpockets formed by the apposition of six CDRs,three from each of two different gene prod-ucts, the heavy- and light-chain variable dom-ains, respectively. Considerable efforts havegone into creating reagents that maintain high-affinity binding and large diversity but aremore amenable to high-throughput expres-sion systems. Single-chain antibodies (scFvs)

maintain the diversity and high-affinitycapacity of intact antibodies in a simpler pack-age, whereby the variable domains of the lightand heavy chains are expressed as a single geneproduct connected by a flexible linker2.Recently, this technology was simplified evenfurther with the creation of single-domainantibody (IDab) libraries, which, althoughcombinatorially less complex than scFvs,maintain excellent antigen-binding proper-ties3. These schemes have allowed the creationof high-diversity libraries that can be com-bined with efficient expression and selectionschemes to identify high-affinity reagents4,5

that work well both inside and outside cells.Although in principle the gene for GFP

could be fused directly into either scFv orIDab libraries to provide a built-in detectionscheme, this would significantly increase thesize of the reagent and would be likely todecrease the efficiency of production because

GFPs normally fold in the intracellular space,whereas antibodies normally fold withinlumenal domains of intracellular organelles.Instead, Bradbury and colleagues take advan-tage of the fact that GFPs arise from a novelthree-dimensional fold called a β-can formedby 11 β-strands wrapping into a cylinder (Fig. 1a). In intact antibodies, the CDRs arebrought together at the top of a cylindricalsurface defined by two β-sheets, one fromeach of the heavy- and light-chain variabledomains (Fig. 1b). The similarity in thesestructures suggested that GFPs themselvesmight provide an appropriate scaffold to allowCDRs to define a high-affinity binding arr-angement. As the various β-strands in GFPalso have connecting loops at the top of thecylinder, Bradbury and colleagues replacedfour of the GFP loops (Fig. 1c) with a libraryof the CDR with the greatest diversity(CDR3); and thus fluorobodies were born.

Expression of only a modestly diverselibrary (107 elements) with a phage-displaysystem led to the isolation of numerous fluo-robodies against various antigens with affini-ties as high as the nanomolar range. Theintrinsic fluorescence itself is very useful dur-ing the selection and purification procedures,as the binding properties are inherently linkedto the stability of the protein and therefore its

Timothy A. Ryan is in the Department ofBiochemistry, Joan and Sanford I. Weill MedicalCollege, Cornell University, 525 E. 68th Street,New York, New York 10021, USA.e-mail: [email protected]

Fluorescent proteins with ties that bindTimothy A Ryan

Antigen-binding fluorescent proteins provide a powerful tool for immunodetection-based assays.

NATURE BIOTECHNOLOGY VOLUME 21 NUMBER 12 DECEMBER 2003 1447

HCDR1

HCDR3

HCDR2LCDR1

LCDR3

LCDR2

a

c

bFigure 1 The making of a fluorobody. (a) β-canstructure of GFP made up of 11 β-strands into acylinder. At the top of the can, loop regions jointhe strands. (b) The antigen-binding site ofantibodies is made by two sets of β-sheets fromboth heavy and light chains organized in acylindrical surface. The hypervariable regions, theloops at the top of this cylindrical arrangement(HCDR1, HCDR2 and HCDR3 and LCDR1,LCDR2 and LCDR3), define the antigen-bindingsite. (c) In a fluorobody, four of the loops of GFPare replaced by a library of HDCR3. These loopsdefine a new binding site using the β-can as ascaffold rather than the light chain– and heavychain–defined β-cylinder.

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N E W S A N D V I E W S

fluorescent properties. Fluorobodies haveproven useful for detection of proteins in gels,enzyme-linked immunosorbent assays, flowcytometry and immunocytochemistry oncells, indicating that they are essentially capa-ble of replacing conventional antibodies formost uses with the benefit that secondaryreagents are not needed to detect the antibody.

In cell biological applications, the ability tolabel proteins in their cellular context withexogenous reagents is very valuable. As intactantibodies are bivalent, labeling often res-ults in crosslinking of the target molecule.Although less of a concern in fixed tissue, thiscan be a significant perturbation in live cells.Furthermore, although the production of use-ful Fab fragments that have been covalentlycoupled to a fluorophore is possible, they frequently lose antigen-binding capacity atthis step. Fluorobodies will almost certainlybind monovalently and are immediately use-ful for fluorescence detection without theneed for secondary antibodies or fluorophorecoupling.

One exciting avenue for development is thepotential to use the ever-expanding repertoireof GFPs in conjunction with this technology.Since their original discovery, GFPs have bec-ome an invaluable tool in many aspects of bio-medical research and have been coaxed intorevealing not only the dynamics and subcellu-lar locations of given proteins but also infor-mation such as small-molecule-messenger

dynamics, enzyme activation, protein-proteininteractions and intracellular as well asintralumenal pH6. The possibility of couplingthese types of changes in fluorescence proper-ties with antigen binding would almost cer-tainly expand the number of useful app-lications of intrinsically fluorescent proteins.Finally, this technology may be a boon simplyin the production of protein-specific affinityreagents. In contrast to antibody productionfrom immune cells, which is expensive in largequantities, fluorobodies should be easily gen-erated reproducibly and in large quantities inbacterial expression systems.

Although it remains to be seen whether fluorobodies can replace antibodies for alluses, particularly because secondary reagentsused for detection often increase sensitivity,this technological breakthrough will likelycompel many researchers to change how theynormally use protein-specific affinityreagents, for the simple reason that where onepreviously needed three reagents one nowonly needs one.

1. Zeytun, A., et al. Nat. Biotechnol. 21, 1473–1479(2003).

2. Huston, J.S. et al. Proc. Natl. Acad. Sci. USA 85,5879–5883 (1988).

3. Tanaka, T., Natividad, L. & Rabbitts, T.H. J. Mol. Biol.331, 1109–1120 (2003).

4. Sheets, M.D. et al. Proc. Natl. Acad. Sci. USA 95,6157–6162 (1998).

5. Feldhaus et al. Nat. Biotechnol. 21, 163–170 (2003).6. Zhang, J. Campbell, R.E., Ting, A.Y. & Tsien, R.Y. Nat.

Rev. Mol. Cell Biol. 3, 906–918 (2002).

repair is the nascent primary transcript, whichon the average spans ∼ 27,000 nucleotides andis interrupted by seven introns (see ref. 2 andreferences therein). These primary transcriptsare frequently alternatively spliced to producemany different mRNA isoforms, and thisprocess can be redirected to preferentiallyexpress some isoforms over others in a type ofreprogramming where no new information is provided to the revised transcript4. The nascent transcript is also the target for spliceosome-mediated RNA trans-splicing2,5.Endogenous spliceosomes catalyze splicing of an exon in the target transcript (exon 2 inFig. 1) with an exon in an engineered pre-trans-splicing molecule (PTM)2,5. The PTMexon replaces the mutant exon 3 (m) for awild-type exon (Fig. 1, reaction 1). PTM tar-geting is accomplished through base-pairinginteractions, which in the figure are schemati-cally drawn within the second intron of thetranscript. After successful trans-splicing, thereprogrammed mRNA is transported to thecytoplasm and translated to produce func-tional protein.

Spliceosome-mediated trans-splicing hasbeen used to repair endogenous mRNAs andrestore function not only in cell culture butalso in animals5–7. Although these studiesmerit cautious optimism, several issues limitthe potential of this methodology—mostimportantly, this type of trans-splicing re-quires the presence of nuclear introns and tar-gets trans-splicing only to available splice sites.The specificity of spliceosome-mediatedtrans-splicing has not yet been completelyascertained, and competing reactions, such asnonspecific trans-splicing, may lead to expres-sion of undesirable products.

After nuclear RNA processing and nucleo-cytoplasmic transport, mutant mRNAs havebeen reprogrammed by trans-splicing medi-ated by group I ribozymes (Fig. 1, reaction 2).This reaction, which provided the first exam-ple of RNA reprogramming8, requires theexpression of a targeting transcript thatencodes for both the trans-splicing group Ienzyme and the coding sequence to beincluded in the revised RNA. Group Iribozyme–mediated trans-splicing has beenused to effect mRNA repair in cells in cul-ture9,10, but has yet to restore function in ani-mals. An advantage of this technique is that itis not limited to naturally occurring splicesites, thus extending the potential range ofRNA reprogramming to mRNAs fromintronless genes and also to noncoding RNAs.As with spliceosome-mediated trans-splicing,targeting by group I introns is achieved bybase pairing of the ribozyme to the targetmRNA, and therefore similar questions about

1448 VOLUME 21 NUMBER 12 DECEMBER 2003 NATURE BIOTECHNOLOGY

Gene therapy, just like the current economicrecovery, is buoyed by hopeful long-term fore-casts but encumbered by important everydayhurdles. To overcome these problems, we relyon the evolution of novel approaches andtechniques. One such general approach isknown as mRNA reprogramming—a form ofgene therapy that modifies transcripts directly.Messenger RNA reprogramming in mam-

malian cells has been achieved primarily byrecombining mRNA molecules via trans-splicing using introduced group I ribozymes1

or endogenous spliceosomes2. In this issue,Deidda et al.3 report on a third and intriguingvariation of this reprogramming theme. Theauthors use a combination of a splicing endo-nuclease from an archaeal bacterium and atargeting RNA to alter mRNAs in cells in cul-ture. Because the splicing endonuclease medi-ates trans-splicing only if it encounters a veryspecific structure, this reprogramming meth-od could offer significant advantages in casesrequiring exquisite specificity.

RNA reprogramming can be achieved atmultiple sites during the process of geneexpression (Fig. 1). The first target for RNA

Mending the messageMariano A Garcia-Blanco

An ancient, archaeal splicing endonuclease provides a promising alternativefor gene therapy.

Mariano A. Garcia-Blanco is in the Departmentof Molecular Genetics and Microbiology,Department of Medicine, Box 3053 (424 CARL),Duke University Medical Center, ResearchDrive, Durham, North Carolina 27710, USA.e-mail: [email protected]

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