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Summary The ability to visualize endogenous proteins in living neurons provides a powerful means to interrogate neuronal structure and function. Here we generate recombinant antibody-like proteins, termed FingRs (Fibronectin intrabodies generated with mRNA display), that bind endogenous neuronal proteins PSD-95 and Gephyrin with high affinity and which, when fused to GFP, allow excitatory and inhibitory synapses to be visualized in living neurons. Design of the FingR incorporates a novel transcriptional regulation system that ties FingR expression to the level of the target and reduces background fluorescence. In dissociated neurons and brain slices FingRs generated against PSD-95 and Gephyrin did not affect the expression patterns of their endogenous target proteins or the number or strength of synapses. Together, our data indicate that PSD-95 and Gephyrin FingRs can report the localization and amount of endogenous synaptic proteins in living neurons and thus may be used to study changes in synaptic strength in vivo.

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Synthesis of Aminoacylated tRNAThe pdCpA dinucleotide (tetrabutylammonium salt) and a sample of αOH-Phenylalanyl-dCA were obtained as a gift from Neurion Pharmaceuticals (Pasadena, CA). Subsequent preparation of αOH-Phenylalanyl-dCA was carried out according to the protocols in references 17-18. An example synthesis is described below. Synthesis of αOH-Phenylalanine cyanomethyl ester (1)L-phenylactic acid (266 mg, 1.6 mmol) was dissolved in 3 mL DMF. To this was added chloroacetonitrile (3 mL, 47.4 mmol) and TEA (651 μL, 4.6 mmol). The reaction was allowed to proceed under nitrogen at room temperature overnight. The desired product was purified by flash chromatography (silica gel, 3:7 EtOAc:Hexanes). The final yield (amber oil) was 18.9 mg (68% Synthesis of αOH-Phenylalanine-dCA (2)αOH-Phenylalanine cyanomethyl ester (11 mg, 54 μmol) was dissolved in 400 μL DMF and added to the tetrabutyl ammonium salt of pdCpA (9 μmol) in the presence of a catalytic amount of TBA-acetate. The reaction was allowed to proceed under nitrogen for 4 hr. at room temperature. The aminoacylated dinucleotide was purified by RP-HPLC using a gradient from 25 mM NH 4 OAc (pH=4.5) to CH 3 CN. Following lyophilization of the pooled fractions, the product was dissolved in 10 mM HOAc and lyophilized again. The final yield was determined by absorbance at 260 nm. and found to be 97 nmol (1%). The product was analyzed by ESI- In vitro Transcription of THG73 tRNAThe plasmid harboring the THG73 gene was linearized with FokI and transcribed with T7 RNA polymerase. The transcription product was gel-purified by Urea-PAGE, dissolved in dH 2 O, and quantitated by absorbance at 260 nm.Ligation to THG73 tRNA 20 μg THG73 tRNA (8 μL in dH 2 O) in HEPES (22 μL, 10 mM, pH=7.5) was heated to 94 ºC for 3 min and allowed to cool slowly at room temperature. 8 μL αOH-Phenylalanine-dCA (3 mM in DMSO), 32 μL 2.5 X Reaction Buffer (25 μL 400 mM HEPES pH=7.5, 10 μL 100 mM DTT, 25 μL 200 mM MgCl 2 , 3.75 μL 10 mM ATP, 10 μL S1 5 mg/mL BSA, 26.25 μL dH 2 O), 7 μL water, and 4 μL T4 RNA Ligase (N.E.B.). After incubation at 37 ºC for 1 hr, the reaction was extracted once with an equal volume of phenol (saturated with 300 mM NaOAc, pH=5.0:CHCl 3 :isoamyl alcohol (25:24:1)) and ethanol precipitated. The pellet was washed with 70% EtOH, dried under vacuum, and dissolved in 1 mM NaOAc to a concentration of 2 μg/μL. Construction of Fusion Templates (Phe(K), UAG(K), UAG(V))These dsDNA templates were created by PCR using overlapping primers: (Forward primer = Gen-FP: 5'-TAATACGACTCACTATAGGGACAATTACTATTTACAATTACA-3') and a unique reverse primer (Phe(K)- hr. The mRNA-DNA template was gel purified by Urea-PAGE, dissolved in water, and quantitated by absorbance at 260 nm. Construction of MK(n) Library Templates (MK0, MK2, MK4, MK6, MK8, MK10)Antisense ssDNA templates were synthesized at the Keck Oligonucleotide Synthesis Facility (Yale). The sequences for the MK Library ssDNA templates are as follows: MK library dsDNA was amplified by PCR using the forward primer Gen-FP (5'-TAATACG...

Peptides were generated on an Applied Biosystems 432A peptide synthesizer using solid phase, F-Moc chemistry. Crude peptides were deprotected by TFA/ethanedithiol/thioanisole treatment and purified on a C-18 reverse phase HPLC column to a final purity greater than 95% (MALDI-TOF, Analytical C-18 HPLC). Peptides without a naturally occurring tryptophan or tyrosine residue were synthesized with a carboxy-terminal Gly-Tyr tag for quantification purposes.Unlabeled RNA hairpins (λboxB R15 and P22boxB L15 ) were synthesized by in vitro transcription using T7 RNA polymerase. 1 The RNA was purified by 20% urea-PAGE, desalted on a NAP column (Amersham Pharmacia), and freeze-dried. RNA was quantified by UV absorption at a wavelength of 260 nm.Labeled RNA hairpins containing 2-amino purine (2AP) at loop position 2 (2AP-2), 3 (2AP-3), or 4 (2AP-4) were constructed by automated RNA synthesis using either 2-aminopurine-TOM-CE phosphoramidite or 2'-O-methyl 2-aminopurine phosphoramidite (Glen Research, Sterling, VA). Steady-State Fluorescence MeasurementsMeasurements were conducted following the procedures of Barrick et. al. 2 Titrations were performed on a ShimadzuSpectrofluorophotometer at 20º C with Excitation/Emission wavelengths at 310/370 nm . Peptides were titrated iteratively into a constantly stirred solution of 2AP labeled RNA hairpin (20-200 nM RNA). Binding buffer contained 20 mM Tris-OAc, with a variable concentration of KOAc (15 mM-500 mM) at pH 7.5. Binding Constants were calculated for a one step binding mechanism by nonlinear least squares regression using the computer program DynaFit. 3 All isotherms were fit with < 10% uncertainty. Stopped-Flow Fluorescence MeasurementsExperiments were conducted following the procedures of Lacourciere et al. 4 Measurements were performed at 20º C under standard buffer conditions (20 mM Tris-OAc, 50 mM KOAc, pH 7.5) using a stop-flow device from Applied Photophysics (Surray, U.K.) in twosyringe mode. Fluorescence excitation was performed at 310 nm and emmision was measured with a filter cutoff > 360 nm. The

RNA loops that adopt a characteristic GNRA ''tetraloop'' fold are common in natural RNAs. Here, we have used in vitro selection by means of mRNA-peptide fusions to select peptides that bind an example of this RNA loop motif. Starting with the RNA recognition domain from the N protein, we have constructed libraries containing 150, 1,600, and 9 trillion different peptide sequences as mRNA-peptide fusions and isolated those capable of high-affinity RNA binding. These selections have resulted in more than 80 different peptides that bind the same RNA loop. The highest affinity peptides exhibit low nanomolar dissociation constants as well as the ability to discriminate RNA hairpins differing by a single loop nucleotide. Thus, our work demonstrates that numerous, chemically distinct solutions exist for a particular RNA recognition problem.

Peptides constructed with the 20 natural amino acids are generally considered to have little therapeutic potential because they are unstable in the presence of proteases and peptidases. However, proteolysis cleavage can be idiosyncratic, and it is possible that natural analogues of functional sequences exist that are highly resistant to cleavage. Here, we explored this idea in the context of peptides that bind to the signaling protein Gαi1. To do this, we used a two-step in vitro selection process to simultaneously select for protease resistance while retaining function–first by degrading the starting library with protease (chymotrypsin), followed by positive selection for binding via mRNA display. Starting from a pool of functional sequences, these experiments revealed peptides with 100–400 fold increases in protease resistance compared to the parental library. Surprisingly, selection for chymotrypsin resistance also resulted in similarly improved stability in human serum (~100 fold). Mechanistically, the decreases in cleavage results from both a lower rate of cleavage (kcat) and a weaker interaction with the protease (Km). Overall, our results demonstrate that the hydrolytic stability of functional, natural peptide sequences can be improved by two orders of magnitude simply by optimizing the primary sequence.