Overstretching molecular duplexes made of DNA and RNA 

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Preparation of dsDNA

The sequence of interest is amplified by a polymerase chain reaction (PCR) from the plasmid pT7rrnB (Fig. I.9). The PCR primers (primer 1 and primer 2) were designed to introduce the sequence of a T7 polymerase promotor and an Afl2 restriction site (primer 1) at one extremity of the double-stranded PCR product, and the sequence of an Fse1 restriction site (primer 2) at the other extremity. The PCR product was then digested by the Fse1 restriction enzyme. The restricted piece was purified and replaced by a similar sequence carrying biotin via ligation. The resultant product was then digested by an Afl2 restriction enzyme generating a 4-nucleotide overhang at the 5’ end of the leading strand. The complementary nucleotides were filled in by Klenow DNA polymerase (Klenow exo-fragment). Some of them carry biotin groups for attachment.

Preparation of the RNA-DNA hybrid

As for dsDNA, the sequence of interest was first amplified by a polymerase chain reaction (PCR, step1 Fig. I.10). The PCR primers (primer 1 and primer 2) were designed to introduce the sequence of a T7 polymerase promotor and an Afl2 restriction site (primer 1) at one extremity of the double-stranded PCR product, and the sequence of an Fse1 restriction site (primer 2) at the other extremity (Fig. I.10, step 2). A part of the PCR product (2μg) was conserved at ≠20¶C for in vitro RNA transcription and the rest was used to prepare dsDNA with biotin modifications at three of its extremities following the same steps (Figures N5, N6, steps 3-6) as described in section I.3.1. Once
the dsDNA with biotin modifications was ready (Fig. I.10, step 6), the conserved PCR product was used to obtain in vitro transcribed RNA containing the sequences of 23S and 5S rRNAs (Fig. I.10, step 7). It is important to prepare fresh RNA for this step, since its stability is low and it degrades fast as compared to DNA. After preparation of ssRNA (Fig. I.10, step 7), the RNA and the dsDNA (Fig. I.10, step 6) were combined to do a strand exchange. This way, many copies of the RNA-DNA hybrid with biotin modifications at the two extremities of the DNA strand were obtained (Fig. I.10, step 8). The last biotin modification at the 3rd extremity of the RNA-DNA construct (3’ end of RNA strand) was introduced by a small RNA oligo of 20bp carrying two biotin-dT at its extremity. The remaining dsDNAs were degraded by an appropriate restriction enzyme to make sure that all the measurements were done on RNA-DNA hybrids only.

Preparation of dsRNA

Each strand of dsRNA was prepared separately. The leading strand was prepared as described in the previous section I.3.2. (Fig. I.10, steps 1, 2, 7, Fig. I.11, steps 1, 2, 3). The preparation of the lagging strand was done as follows. The sequence of interest (23S and 5S rRNA) was amplified by a polymerase chain reaction (PCR). The PCR primers 3 and 4 (Fig. I.11, step 1) were designed to introduce a random sequence at one extremity of the PCR product and a sequence of T7 polymerase promoter and a restriction site for FseI restriction enzyme at the other extremity of the PCR product (Fig. I.11, step 5). As one can see from Fig. I.11, to prepare the lagging strand the T7 polymerase promotor sequence is introduced at the opposite extremity of the PCR product (Fig. I.11, step 5) as compared to the preparation of the leading strand (Fig. I.11, step 2). After the PCR reaction, part of it was used to perform in vitro transcription of the lagging single stranded RNA (Fig. I.11, step 8). The latter was biotin-modified at both extremities via splint ligation. The idea of our particular splint ligation is the following. T4 RNA ligase 2 can ligate pieces of ssRNA (biotin-modified in our case) to DNA-RNA hybrids. This means that in order to ligate biotin-modified RNA oligonucleotides at two extremities of the ssRNA_lagg, it is necessary to create small regions of a DNA-RNA hybrid at both extremities. To do this, two DNA oligonucleotides (DNA1 and DNA2, Fig. I.11, step 7).

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Preparation of DNA-RNA hybrid

In this case, the leading strand is DNA and the lagging strand is RNA. The prepa- ration of this construct is done in three steps: a preparation of dsDNA with three biotin-modified extremities (Fig. I.12, steps 1-5), preparation of the lagging RNA sin- gle strand with biotin modifications at its 5’ and 3’ ends (Fig. I.12, steps 6-9) and an exchange of the DNA-lagging strand by a RNA-lagging strand via a hybridization reaction (Fig. I.12, step 10). The preparation of dsDNA is the same as in section I.3.1 (Fig. I.12, steps 1-6) and the preparation of the lagging ssRNA is the same as described in section I.3.3 (Fig. I.11, steps 4-7).

Further treatment of the constructs

When the molecular constructs are prepared, part of them are conserved at ≠20¶C for later use, while part of them are mixed with streptavidin-coated polystyrene beads. The mixture is first centrifuged 6 min at 30G and then incubated at room temperature (25¶C) for one hour. Centrifugation allows the beads and the molecules to come close together, thus increasing their binding efficiency and reducing the incubation time from 3 hrs to 1 h. After incubation, the sample is loaded into a fluidics chamber composed of two glass coverslips sealed together by 2 parallel parafilm layers. After loading with the molecular construct the two open edges of the chamber are sealed with wax (Fig. I.13). Finally, the sample is installed on the microscope stage between two objectives (100x oil and 60x water, described above), a bead couple with a molecule attached in between is searched for, found, trapped and a force measurement is performed (Fig. I.14). The results of these experiments are given in papers 1 and 2.

Table of contents :

Remerciements
Introduction
I Overstretching molecular duplexes made of DNA and RNA 
I.1 DNA, RNA, DNA-RNA hybrid
I.1.1 Chemical structure
I.1.2 Physical structure
I.2 Optical tweezers
I.2.1 The principle of optical trapping
I.2.2 Dual beam optical tweezers
I.3 Four molecular constructs
I.3.1 Preparation of dsDNA
I.3.2 Preparation of the RNA-DNA hybrid
I.3.3 Preparation of dsRNA
I.3.4 Preparation of DNA-RNA hybrid
I.3.5 Further treatment of the constructs
Supplementary Information for Paper 1
II Early steps of ribosome assembly 
IIIRésumé en Français 
III.1 Introduction
III.2 Comparaison entre quatre constructions moléculaires
III.3 Influence des protines au repliement de l’ARN formant le ribosome
A Appendix 
A.1 Preparation of the beads
A.2 Preparation of dsDNA
A.2.1 Polymerase chain reaction (PCR)
A.2.2 FseI restriction
A.2.3 Ligation of sur_DNAbiot_MB and sur_compDNA2biot_MB oligonu- cleotides
A.2.4 AflII restriction
A.2.5 Klenow Fragment (exo-) treatment
A.3 Preparation of RNA-DNA hybrid
A.3.1 In vitro transcription of RNA leading strand
A.3.2 Hybridization
A.3.3 Ligation of sur_RNAbiot_MB Ph
A.4 Preparation of dsRNA
A.4.1 PCR
A.4.2 In vitro transcription of RNA lagging strand
A.4.3 Hybridization of DNA and ligation of RNA oligonucleotides
A.4.4 Hybridization of two RNA strands
A.4.5 Ligation of sur_RNAbiot_MB Ph
A.5 Preparation of DNA-RNA hybrid
A.6 Preparation of 23S rRNA
A.6.1 Polymerase chain reaction (PCR)
A.6.2 In vitro transcription of 23S rRNA
Bibliography

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