Recording and Analyzing Nucleic Acid Distance Distributions with X‐Ray Scattering Interferometry (XSI)

Abstract

Most structural techniques provide averaged information or information about a single predominant conformational state. However, biological macromolecules typically function through series of conformations. Therefore, a complete understanding of macromolecular structures requires knowledge of the ensembles that represent probabilities on a conformational free energy landscape. Here we describe an emerging approach, X-ray scattering interferometry (XSI), a method that provides instantaneous distance distributions for molecules in solution. XSI uses gold nanocrystal labels site-specifically attached to a macromolecule and measures the scattering interference from pairs of heavy metal labels. The recorded signal can directly be transformed into a distance distribution between the two probes. We describe the underlying concepts, present a detailed protocol for preparing samples and recording XSI data, and provide a custom-written graphical user interface to facilitate XSI data analysis. © 2018 by John Wiley & Sons, Inc.

The structural ensemble of a Holliday junction determined by X-ray scattering interference

“…Our results underscore the ability of XSI to reveal precise ensemble properties across a wide range of ionic strength, even for rapidly interchanging conformations ( 28 , 29 , 32 , 36 ). XSI, therefore, will provide a valuable reference for emerging FRET approaches that provide calibrated distances ( 50 ) and ≤ms time resolution ( 45 , 51–53 ).…”

“…XSI measures the interference scattering pattern between two site-specifically attached electron-rich markers together with scattering terms from the molecule and the cross-scattering terms between the labels and the macromolecule. The interference between the two labels is extracted from measurements of the doubly-labeled, singly-labeled, and unlabeled samples and transformed into an absolute distance distribution using a weighted linear combination of basis scattering functions ( 23 , 24 , 26 , 28 , 29 , 31 , 32 ).…”

“…We assembled the DNA junction for XSI measurements from four synthetic oligonucleotides, with identical lengths and sequences as used in the smFRET experiments (see Materials and Methods). To provide the characteristic signal in XSI, we attached pairs of gold nanocrystal to generate four doubly labeled constructs (HR, BR, BH and RX, Figures 3 and 4 ) following published approaches ( 23 , 24 , 26 , 28 , 29 , 31 , 32 ) (see Materials and Methods). We also assembled, purified, and recorded data for the corresponding single-labeled and unlabeled constructs that are required to extract the gold-gold interference pattern.…”

“…Gold nanocrystals with particle radius of 7 Å were synthesized and purified as described previously ( 23 , 24 , 32 , 36 ). Unmodified single-stranded DNA (ssDNA) was purchased from Integrated DNA Technology (IDT) and C3-thiol-modified oligonucleotides were synthesized at the Protein and Nucleic Acids Facility at Stanford University on an automated ABI 394 DNA synthesizer.…”

“…One full set of data for further analysis ( Supplementary Figure S1 ) contains Holliday junction scattering profiles of a double labeled sample (AB), two single labeled samples (A label and B label), the unlabeled macromolecule (U), the gold nanocrystals (Au) and finally the buffer (Buf). The profiles are weighted, summed (AB and U) and subtracted (A and B) to calculate the gold–gold scattering interference pattern I Au–Au ( 32 ). The resulting interference patterns were fitted using a maximum entropy algorithm to obtain the final distance distributions as described previously ( 23 , 24 , 36 ).…”